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RESEARCH The aim of this article is to review the most up to date literature about CKI antibodies targeting PD-1/PD-L1 axis for the treatment of advanced solid tumors, particularly considering phase III randomized trials, starting from the first performed trials on the issue. Published papers were obtained from the Medline database. The search was implemented by reviewing the most important international scientific meetings abstract databases. In addition, indirect data on the topic were achieved by reading the most recent publications related to the use of CKI in different types of solid tumors. The ongoing trials were reached on the official website www.clincaltrials.gov, considering only randomized phase III studies. RESEARCH RESULTS Melanoma Treatment of advanced melanoma has been radically changed by the advent of CKI. After that the anti-CTLA4 antibody ipilimumab in the last years had become the backbone of this malignant tumor treatment, where traditional chemotherapy harvested very little success, the introduction of the anti-PD-1 antibodies nivolumab and pembrolizumab further improved the therapeutic armamentarium for melanoma.

o make trade-offs between attributes and attribute levels generating a unique set of values called part-worth utilities. A higher part-worth utility represents a higher level of value or importance individuals assign to that attribute. The design of CBC experiments is tailored based on the needs of an individual study. We used a systematic approach to designing the CBC study to elicit analgesic utilities reported in the present study. The procedures are detailed in a previously published manuscript[14]. Trade-offs were elicited on five analgesic attributes: (1) type of analgesic, (2) percentage pain relief with analgesics; (3) type of side-effects; (4) severity of side-effects; and (5) out-of-pocket cost of analgesics. In addition to the design components, we also investigated the internal, external predictive validity and temporal stability of the CBC experiment over the study period[14].

. After that the anti-CTLA4 antibody ipilimumab in the last years had become the backbone of this malignant tumor treatment, where traditional chemotherapy harvested very little success, the introduction of the anti-PD-1 antibodies nivolumab and pembrolizumab further improved the therapeutic armamentarium for melanoma. The first published phase III randomized study about PD-1/PD-L1 axis inhibition in this disease demonstrated, at the beginning of 2015, the advantage of nivolumab over chemotherapy with dacarbazine both in terms of overall survival (OS) and of progression free survival (PFS) among previously untreated patients with metastatic melanoma without BRAF mutation. Median PFS of 5.1 mo in the nivolumab group was more than doubled when compared to dacarbazine treated patients, with 2.2 mo [hazard ratio (HR) = 0.43, 95%CI: 0.34-0.56, P < 0.001]. OS was not reached in the nivolumab group, instead being 10.8 mo in the group treated with chemotherapy (HR = 0.42, 99%CI: 0.25-0.73, P < 0.001)[9]. An analogous comparison was made in patients who progressed after anti-CTLA4 treatment in the phase III randomized study CheckMate 037, reporting a response rate (RR) of 32% for nivolumab vs 11% with chemotherapy according to investigator’s choice. These findings have resulted in the inclusion of nivolumab in the new treatment options for a cancer with high unmet need[10].

ssed after anti-CTLA4 treatment in the phase III randomized study CheckMate 037, reporting a response rate (RR) of 32% for nivolumab vs 11% with chemotherapy according to investigator’s choice. These findings have resulted in the inclusion of nivolumab in the new treatment options for a cancer with high unmet need[10]. In parallel, pembrolizumab was compared with ipilimumab as the new standard of care for first line treatment of advanced melanoma in a phase III randomized trial, demonstrating to prolong PFS and OS with less toxicity respect to the CTLA4 inhibitor[11]. Nevertheless, the new frontier for untreated melanoma is currently represented by the combination of anti-CTLA4 and anti-PD-L1 antibodies: Larkin et al[12] demonstrated that the association of nivolumab and ipilimumab resulted in a significantly longer PFS than ipilimumab alone, despite 55% of treatment-related adverse events (AEs) of grade 3 or 4 (G3-4) vs 16% in the nivolumab group and 27% in the ipilimumab group. This three arms phase III randomized trial closed the matter of first line ipilimumab alone, otherwise confirming good effectiveness for nivolumab monotherapy in this setting[12].

Core tip: The onset of PD-1/PD-L1 targeted therapy in oncology has demonstrated the importance of this axis in the immune escape across almost all human cancers. A sort of revolution has been happening with the investigation of the new immune checkpoint inhibitors in the field of anticancer therapy. The aim of this article is to review the most up to date literature about the clinical effectiveness of the antibodies targeting PD-1/PD-L1 axis for the treatment of advanced solid tumors and to explore transversal challenges in the immune checkpoint blockade. INTRODUCTION After that the era of chemotherapy in the treatment of solid tumors have been overcome by the “translational era”, with the innovation introduced by targeted therapies, medical oncology is currently looking at the dawn of a new “immunotherapy era” with the advent of immune checkpoint inhibitors (CKI) antibodies. The strategy to maintain physiologic self-tolerance and to restore latent anti-tumor immunity is currently going through the whole oncology, gradually revolutionizing the standard of treatment of the most chemo-resistant tumors such as melanoma, lung and renal cancer. From the first class of antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), like ipilimumab and tremelimumab, burdened by significant autoimmune toxicity, the scenario is currently evolving in favor of the antibodies against programmed cell death protein 1 (PD-1) and its ligand PD-L1, in both cases inhibiting the PD-1/PD-L1 axis[1].

against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), like ipilimumab and tremelimumab, burdened by significant autoimmune toxicity, the scenario is currently evolving in favor of the antibodies against programmed cell death protein 1 (PD-1) and its ligand PD-L1, in both cases inhibiting the PD-1/PD-L1 axis[1]. The monoclonal antibodies nivolumab and pembrolizumab (anti-PD-1), atezolizumab, durvalumab and avelumab (anti-PD-L1), have been tested against multiple cancer types in the last years and are currently under investigation in several phase II and phase III clinical trials. Further similar antibodies are currently undergoing phase I experiences, in order to compete with the first arrivals on the clinical scenario[2-4]. All the antibodies cited in the text are reported in Table 1. Table 1 Immune-checkpoint inhibitors antibodies with their targets CKI Mechanism of action Nivolumab Anti-PD-1 Pembrolizumab Anti-PD-1 Atezolizumab Anti-PD-L1 Durvalumab Anti-PD-L1 Avelumab Anti-PD-L1 BMS936559 Anti-PD-L1 Pidilizumab Anti-PD-1 CKI: Checkpoint inhibitors. In all cases, the mechanism targets the inhibitory signal that contributes to the balance between co-stimulatory and inhibitory pathways in the regulation of T-cell response, starting from the antigen recognition by T-cell receptor. In fact, in contrast to other antibodies currently used for cancer therapy, CKI do not target tumor cells directly, but instead they target lymphocyte receptors or their ligands, with the aim to enhance endogenous antitumor response[5].

ation of T-cell response, starting from the antigen recognition by T-cell receptor. In fact, in contrast to other antibodies currently used for cancer therapy, CKI do not target tumor cells directly, but instead they target lymphocyte receptors or their ligands, with the aim to enhance endogenous antitumor response[5]. PD-1 belongs to the inhibitory B7-family molecules; it is upregulated and expressed by activated T-cells (but also B-cells, T regulatory and natural killer cells) and engaged through its ligands PD-L1 and PD-L2, expressed by the antigen presenting cells (APC) and by non-hematopoietic stem cells, aside from tumor cells. The role of PD-1 consists in the inhibition of the effector T-cells activity in peripheral tissues during the inflammatory response to infection and in the regulation and limitation of autoimmunity[6]. Within the tumor microenvironment, this endogenous mechanism favors immune resistance[7]. The major PD-1 ligand expressed on solid tumors cells is PD-L1, whose most important signal for induction is interferon-γ (IFN-γ), produced by T helper 1 (Th1) cells[8]. Most types of solid tumors have been demonstrated to express high levels of PD-L1 (melanoma, ovarian, lung cancer and genitourinary tumors among others), and more recently the importance of PD-L1 expression on the immune cells infiltrating the tumor also emerged, in particular on tumor-infiltrating lymphocytes (TILs). Nevertheless, the evidence about the prognostic and predictive role of these elements have not yet been clarified and it seems to be different basing on tumor type[5].

ntly the importance of PD-L1 expression on the immune cells infiltrating the tumor also emerged, in particular on tumor-infiltrating lymphocytes (TILs). Nevertheless, the evidence about the prognostic and predictive role of these elements have not yet been clarified and it seems to be different basing on tumor type[5]. Despite these unresolved issues, the findings described above provided the rationale for the capacity of the blockade of PD-1/PD-L1 axis to enhance intratumoral immune responses in a transversal way across different tumor types, firstly encouraged by preclinical evidence and then largely satisfied by the early results of several recent clinical studies. RESEARCH The aim of this article is to review the most up to date literature about CKI antibodies targeting PD-1/PD-L1 axis for the treatment of advanced solid tumors, particularly considering phase III randomized trials, starting from the first performed trials on the issue. Published papers were obtained from the Medline database. The search was implemented by reviewing the most important international scientific meetings abstract databases. In addition, indirect data on the topic were achieved by reading the most recent publications related to the use of CKI in different types of solid tumors. The ongoing trials were reached on the official website www.clincaltrials.gov, considering only randomized phase III studies.

despite 55% of treatment-related adverse events (AEs) of grade 3 or 4 (G3-4) vs 16% in the nivolumab group and 27% in the ipilimumab group. This three arms phase III randomized trial closed the matter of first line ipilimumab alone, otherwise confirming good effectiveness for nivolumab monotherapy in this setting[12]. Further phase III-IV trials are currently ongoing to test different dosing schedules of CKI[13], others to verify their efficacy in particular subgroups of patients like those with brain metastases[14], or to establish the correct duration of anti-PD-1 therapy in metastatic melanoma, especially in the case of long responders[15]. Again, more others are investigating alternative combinations[16,17] or treatment sequences, like ipilimumab plus nivolumab followed or preceded by dabrafenib and trametinib in BRAF mutated patients[18]. Moreover, after the Food and Drug Administration approval of ipilimumab for the adjuvant setting for melanoma[19], as discussed below, the PD-1 and PD-L1 inhibitors are currently under investigation for the adjuvant and neoadjuvant setting also in different tumor types in several clinical trials, which results are eagerly awaited, given the lower toxicity expected from this “second generation” of CKI (Table 2)[20-31]. Table 2 Phase III randomized clinical trials currently ongoing with PD-1/PD-L1 axis blockade in adjuvant setting for solid tumors

Moreover, after the Food and Drug Administration approval of ipilimumab for the adjuvant setting for melanoma[19], as discussed below, the PD-1 and PD-L1 inhibitors are currently under investigation for the adjuvant and neoadjuvant setting also in different tumor types in several clinical trials, which results are eagerly awaited, given the lower toxicity expected from this “second generation” of CKI (Table 2)[20-31]. Table 2 Phase III randomized clinical trials currently ongoing with PD-1/PD-L1 axis blockade in adjuvant setting for solid tumors Trial name/NCT Cancer type Immune checkpoint inhibitor Arms Primary endpoint Expected primary completion date No. of patients KEYNOTE-054[20] Melanoma Pembrolizumab Pembrolizumab vs placebo RFS 2018 900 NCT02506153[21] Melanoma Pembrolizumab Pembrolizumab vs high dose recombinant interferon-α-2B or ipilimumab OS 2020 1378 KEYNOTE-091 (PEARLS)[22] NSCLC Pembrolizumab Pembrolizumab vs placebo DFS 2021 1380 IMvigor010[23] Bladder cancer Atezolizumab Atezolizumab vs observation DFS 2021 440 IMpower010[24] NSCLC Atezolizumab Atezolizumab vs BSC after adjuvant CT1 DFS 2020 1127 NCT02768558[25] NSCLC (locally advanced) Nivolumab Nivolumab vs placebo (after CT1-RT) OS 2022 660 ANVIL[26] NSCLC Nivolumab Nivolumab vs observation DFS 2018 714 CheckMate 238[27] Melanoma Nivolumab Nivolumab + placebo vs ipilimumab + placebo RFS 2018 800 CheckMate 274[28] Urothelial cancers Nivolumab Nivolumab vs placebo DFS 2020 640 CheckMate 577[29] Esophageal or gastroesophageal junction cancer (locally advanced) Nivolumab Nivolumab vs placebo (after CT1-RT and surgery) DFS 2019 760 PACIFIC[30] NSCLC (locally advanced) Durvalumab Durvalumab vs placebo (after CT1-RT) OS 2017 702 NCT02273375[31] NSCLC Durvalumab Durvalumab vs placebo DFS 2025 1100 1 According to the standard of care and basing on the choice of the investigator. RFS: Recurrence free survival; NSCLC: Non-small cell lung cancer; DFS: Disease free survival; CT: Chemotherapy; OS: Overall survival; RT: Radiotherapy.

1-RT) OS 2017 702 NCT02273375[31] NSCLC Durvalumab Durvalumab vs placebo DFS 2025 1100 1 According to the standard of care and basing on the choice of the investigator. RFS: Recurrence free survival; NSCLC: Non-small cell lung cancer; DFS: Disease free survival; CT: Chemotherapy; OS: Overall survival; RT: Radiotherapy. Lung cancer Lung cancer immunotherapy have an historical background, but it has not shown significant survival benefit until the recent advent of CKI. Conversely to anti-CTLA4 antibodies, which demonstrated a certain efficacy only when combined with chemotherapy, the inhibition of PD-1/PD-L1 axis clearly works as single strategy in non-small cell lung cancer (NSCLC)[32]. The first step through immunotherapy for lung cancer in clinical practice was the approval of CKI monotherapy with nivolumab (and more recently with atezolizumab) for NSCLC patients pretreated with first line chemotherapy, on the basis of the first published randomized trials[33-35].

Conversely to anti-CTLA4 antibodies, which demonstrated a certain efficacy only when combined with chemotherapy, the inhibition of PD-1/PD-L1 axis clearly works as single strategy in non-small cell lung cancer (NSCLC)[32]. The first step through immunotherapy for lung cancer in clinical practice was the approval of CKI monotherapy with nivolumab (and more recently with atezolizumab) for NSCLC patients pretreated with first line chemotherapy, on the basis of the first published randomized trials[33-35]. Anti-PD1 antibodies are going to radically revolutionize lung cancer treatment regardless of the histology, especially after the recently published results of KEYNOTE 024 trial[36], providing the outstanding evidence of pembrolizumab superiority compared to chemotherapy as first line treatment for NSCLC, in terms of PFS (10.3 mo vs 6 mo, P < 0.001), OS (80% vs 72% at 6 mo, P = 0.005), RR (45% vs 28%) and safety among patients bearing strong PD-L1 expression on tumor cells (at least 50% was required for enrollment). This latter evidence, despite concerned to the 30% of overall NSCLC population, will provide the rationale to radically change the therapeutic paradigm for NSCLC, shifting CKI treatment option to first line in a great subgroup of patients. The selection of patients basing on a single biomarker, despite potentially harmful, has been demonstrated to be effective in this case, as proven by the recently announced failure of the analogue phase III trial with nivolumab, whose patients were enrolled independently from PD-L1 status[37].

eat subgroup of patients. The selection of patients basing on a single biomarker, despite potentially harmful, has been demonstrated to be effective in this case, as proven by the recently announced failure of the analogue phase III trial with nivolumab, whose patients were enrolled independently from PD-L1 status[37]. Several phase III studies are currently still ongoing in order to investigate further CKI antibodies in all treatment lines, in different treatment regimens and with alternative combinations targeting PD-1/PD-L1 axis in advanced NSCLC (Table 3)[37-96]. Table 3 Phase III randomized clinical trials currently ongoing with PD-1/PD-L1 axis blockade in advanced setting for solid tumors Trial name/NCT Cancer type Immune checkpoint inhibitor Arms Treatment line Primary endpoint Expected primary completion date No.

Several phase III studies are currently still ongoing in order to investigate further CKI antibodies in all treatment lines, in different treatment regimens and with alternative combinations targeting PD-1/PD-L1 axis in advanced NSCLC (Table 3)[37-96]. Table 3 Phase III randomized clinical trials currently ongoing with PD-1/PD-L1 axis blockade in advanced setting for solid tumors Trial name/NCT Cancer type Immune checkpoint inhibitor Arms Treatment line Primary endpoint Expected primary completion date No. of patients STOP-GAP[15] Melanoma PD-1 inhibitor (any) Intermittent vs continuous therapy Any OS 2025 550 NCT02752074[16] Melanoma Pembrolizumab Pembrolizumab + epacadostat vs pembrolizumab + placebo I line PFS 2018 600 MASTERKEY-265[17] Melanoma Pembrolizumab Pembrolizumab + talimogene laherparepvec vs pembrolizumab + placebo I line PFS 2018 660 KEYNOTE-048[82] HNSCC Pembrolizumab Pembrolizumab vs CT1 + pembrolizumab vs CT1 I line PFS 2018 780 KEYNOTE-040[38] HNSCC Pembrolizumab Pembrolizumab vs methotrexate or docetaxel or cetuximab From II line OS 2017 466 KEYNOTE-204[39] Hodgkin lymphoma Pembrolizumab Pembrolizumab vs brentuximab From II line PFS 2019 300 KEYNOTE-045[40] Urothelial cancers Pembrolizumab Pembrolizumab vs paclitaxel, docetaxel or vinflunine From II line OS 20172 470 NCT02811861[41] Renal cell carcinoma Pembrolizumab Pembrolizumab + lenvatinib vs lenvatinib + everolimus vs sunitinib I line PFS 2020 735 KEYNOTE-426[102] Renal cell carcinoma Pembrolizumab Pembrolizumab + axitinib vs sunitinib I line PFS, OS 2019 840 KEYNOTE-240[42] HCC Pembrolizumab Pembrolizumab vs BSC II line PFS 2019 408 KEYNOTE-189[43] NSqNSCLC Pembrolizumab Platinum and pemetrexed ± pembrolizumab I line PFS 2017 570 KEYNOTE-407[44] SqNSCLC Pembrolizumab CT1 ± pembrolizumab I line PFS 2018 560 KEYNOTE-042[45] NSCLC PD-L1-positive Pembrolizumab Pembrolizumab vs platinum based CT1 I line OS 2018 1240 KEYNOTE-010[46] NSCLC Pembrolizumab Pembrolizumab vs docetaxel From II line OS 2019 1034 KEYNOTE-119[47] Triple negative breast cancer Pembrolizumab Pembrolizumab vs monochemotherapy II-III line PFS 2017 600 KEYNOTE-355[48] Triple negative breast cancer Pembrolizumab CT1 + pembrolizumab vs CT1 + placebo I line PFS 2019 858 KEYNOTE-177[49] MSI-H or dMMR colorectal carcinoma Pembrolizumab Pembrolizumab vs CT1 I line PFS 2019 270 KEYNOTE-181[50] Esophageal/esophago-gastric junction carcinoma Pembrolizumab Pembrolizumab vs monochemotherapy1 II line PFS 2018 600 KEYNOTE-061[51] Esophageal/esophago-gastric junction adenocarcinoma Pembrolizumab Pembrolizumab vs paclitaxel II line PFS 2017 720 KEYNOTE-062[52] Esophageal/esophago-gastric junction carcinoma Pembrolizumab Pembrolizumab vs CT1

ic junction carcinoma Pembrolizumab Pembrolizumab vs monochemotherapy1 II line PFS 2018 600 KEYNOTE-061[51] Esophageal/esophago-gastric junction adenocarcinoma Pembrolizumab Pembrolizumab vs paclitaxel II line PFS 2017 720 KEYNOTE-062[52] Esophageal/esophago-gastric junction carcinoma Pembrolizumab Pembrolizumab vs CT1 + pembrolizumab vs CT1 I line PFS 2019 750 JAVELIN Ovarian 200[53] Ovarian cancer (platinum resistant) Avelumab Avelumab vs avelumab plus PLD vs PLD From II line OS 2018 550 JAVELIN Ovarian 100[54] Ovarian cancer Avelumab CT1 vs CT1 followed by avelumab maintenance vs CT1 + avelumab followed by avelumab maintenance I line PFS 2019 951 JAVELIN Renal 101[55] Renal cell cancer Avelumab Avelumab with axitinib vs sunitinib I line PFS 2018 583 JAVELIN Bladder 100[56] Urothelial cancer Avelumab Avelumab vs BSC (maintenance after CT1) I line maintenance OS 2019 668 JAVELIN Gastric 100[57] Adenocarcinoma of the stomach or of the gastro-esophageal junction Avelumab CT1 continuation vs avelumab in maintenance after CT1 I line OS 2018 666 JAVELIN Gastric 300[58] Adenocarcinoma of the stomach or of the gastro-esophageal junction Avelumab Avelumab + BSC vs CT1 + BSC vs BSC III line OS 2017 330 JAVELIN Lung 100[59] NSCLC (PD-L1 positive) Avelumab Avelumab vs platinum based CT1 I line PFS 2017 420 JAVELIN Lung 200[60] NSCLC (PD-L1 positive) Avelumab Avelumab vs docetaxel From II line OS 2017 650 OAK[61] NSqNSCLC Atezolizumab Atezolizumab vs docetaxel From II line OS 2017 1225 IMvigor211[62] Bladder cancer Atezolizumab Atezolizumab vs monochemotherapy II line OS 2017 932 IMvigor130[63] Urothelial carcinoma (ineligible for cisplatin) Atezolizumab Atezolizumab + CT1 vs placebo + CT1 I line PFS 2019 435 IMpower110[64] NSqNSCLC Atezolizumab Atezolizumab vs platin + pemetrexed I line PFS 2019 570 IMpower111[65] SqNSCLC Atezolizumab Atezolizumab vs gemcitabine + platin I line PFS 2017 ND IMpower131[66] SqNSCLC Atezolizumab Atezolizumab + nab-paclitaxel + carboplatin vs atezolizumab + paclitaxel + carboplatin vs nab-paclitaxel + carboplatin I line PFS 2023 1200 IMpower210[67] NSCLC Atezolizumab Atezolizumab vs docetaxel II line OS 2019 563 IMpower130[68] NSqNSCLC Atezolizumab Atezolizumab + nab-paclitaxel + carboplatin vs nab-paclitaxel + carboplatin I line PFS 2019 550 IMpower150[69] NSqNSCLC Atezolizumab Atezolizumab + carboplatin + paclitaxel ± bevacizumab vs carboplatin + paclitaxel + bevacizumab I line PFS 2017 1200 IMpassion130[70] Triple negative breast cancer Atezolizumab Atez

zumab Atezolizumab + nab-paclitaxel + carboplatin vs nab-paclitaxel + carboplatin I line PFS 2019 550 IMpower150[69] NSqNSCLC Atezolizumab Atezolizumab + carboplatin + paclitaxel ± bevacizumab vs carboplatin + paclitaxel + bevacizumab I line PFS 2017 1200 IMpassion130[70] Triple negative breast cancer Atezolizumab Atez olizumab + nab-paclitaxel vs placebo + nab paclitaxel I line PFS 2020 900 IMmotion151[71] Renal cell carcinoma Atezolizumab Atezolizumab + bevacizumab vs sunitinib I line PFS 2020 900 IMpower133[72] SCLC Atezolizumab Carboplatin and etoposide ± atezolizumab I line OS 2019 400 NCT02788279[73] Colorectal carcinoma Atezolizumab Atezolizumab + cobimetinib vs atezolizumab vs regorafenib From III line OS 2019 360 KESTREL[74] HNSCC Durvalumab Durvalumab vs durvalumab + tremelimumab vs SOC I line PFS 2017 628 MYSTIC[75] NSCLC Durvalumab Durvalumab vs durvalumab + tremelimumab vs SOC I line PFS 2017 1092 Danube[76] Bladder cancer Durvalumab Durvalumab vs durvalumab + tremelimumab vs SOC1 I line PFS 2017 525 Lung-MAP[77] SqNSCLC (biomarker-targeted) Durvalumab, nivolumab Docetaxel vs durvalumab vs erlotinib vs AZD4547 vs ipilimumab vs palbociclib vs rilotumumab vs taselisib Any PFS 2022 10000 CAURAL[78] NSCLC T790M mutation positive Durvalumab AZD9291 + durvalumab vs AZD9291 II-III line PFS 2018 350 NCT02369874[79] HNSCC Durvalumab Durvalumab vs durvalumab + tremelimumab vs SOC1 II line OS 2018 720 NEPTUNE[80] NSCLC Durvalumab Durvalumab + tremelimumab vs SOC1 I line OS 2018 800 ARCTIC[81] NSCLC Durvalumab Durvalumab vs durvalumab + tremelimumab vs SOC1 II-III line OS 2016 730 NCT02224781[18] Melanoma BRAFV600 mutated Nivolumab Dabrafenib + trametinib followed by ipilimumab + nivolumab vs ipilimumab + nivolumab followed by dabrafenib + trametinib I line OS 2019 300 NIBIT-M2[14] Melanoma brain metastases Nivolumab Fotemustine vs ipilimumab + fotemustine vs ipilimumab + nivolumab Any OS 2018 168 CheckMate 026[37] NSCLC PD-L1 positive (all) Nivolumab Nivolumab vs CT1 I line PFS 20162 535 CheckMate 651[83] H&N SCC Nivolumab Nivolumab + ipilimumab vs platinum + fluorouracil + cetuximab I line OS 2020 490 CheckMate 459[84] HCC Nivolumab Nivolumab vs sorafenib I line TTP 2017 726 NCT02267343[85] Gastric cancer Nivolumab Nivolumab vs placebo From II line OS 2017 480 NCT02569242[86] Esophageal cancer Nivolumab Nivolumab vs docetaxel/paclitaxel From II line OS 2019 390 CheckMate 214[87] Renal cell carcinoma Nivolumab Nivolumab + ipilimumab vs sunitinib I line PFS 2019 1070 CheckMat

ne TTP 2017 726 NCT02267343[85] Gastric cancer Nivolumab Nivolumab vs placebo From II line OS 2017 480 NCT02569242[86] Esophageal cancer Nivolumab Nivolumab vs docetaxel/paclitaxel From II line OS 2019 390 CheckMate 214[87] Renal cell carcinoma Nivolumab Nivolumab + ipilimumab vs sunitinib I line PFS 2019 1070 CheckMat e 143[88] Glioblastoma Nivolumab Nivolumab vs bevacizumab II line OS 2017 440 CheckMate 141[89] H&N SCC Nivolumab Nivolumab vs cetuximab/methotrexate/docetaxel monotherapy Any OS 2018 360 CheckMate 227[90] NSCLC Nivolumab Nivolumab vs nivolumab + ipilimumab vs nivolumab + platinum doublet CT1 I line OS 2018 1980 CheckMate 451[91] SCLC Nivolumab Nivolumab vs nivolumab + ipilimumab vs placebo after platinum based CT1 Maintenance after I line OS 2018 810 CheckMate 498[92] Glioblastoma (unmethylated MGMT) Nivolumab Nivolumab + RT vs temozolomide + RT I line PFS 2019 550 CheckMate 331[93] SCLC Nivolumab Nivolumab vs topotecan/amrubicin II line OS 2018 480 CheckMate 078[94] NSCLC Nivolumab Nivolumab vs docetaxel From II line OS 2018 500 NCT02339571[95] Melanoma Nivolumab Nivolumab + ipilimumab ± sargramostim I line OS 2021 400 CheckMate 401[96] Melanoma Nivolumab Nivolumab + ipilimumab vs nivolumab I line OS 2021 615 1 According to the standard of care and basing on the choice of the investigator;

olumab Nivolumab vs docetaxel From II line OS 2018 500 NCT02339571[95] Melanoma Nivolumab Nivolumab + ipilimumab ± sargramostim I line OS 2021 400 CheckMate 401[96] Melanoma Nivolumab Nivolumab + ipilimumab vs nivolumab I line OS 2021 615 1 According to the standard of care and basing on the choice of the investigator; 2 The trial has results but it is still unpublished. OS: Overall survival; PFS: Progression free survival; HNSCC: Head and neck squamous cell carcinoma; HCC: Hepatocarcinoma; NSqNSCLC: Non-squamous non-small cell lung cancer; SqNSCLC: Squamous non-small cell lung cancer; CT: Chemotherapy; NSCLC: Non-small cell lung cancer; MSI-H: High microsatellite instability; dMMR: Deficient mismatch repair; PLD: Pegylated liposomal doxorubicin; SCLC: Small cell lung cancer; TTP: Time to progression; ORR: Objective response rate. Also adjuvant paradigm has been pursued in lung cancer: Table 2 summarizes all the ongoing phase III studies in this field. Squamous cell lung cancer: Squamous cell histology had the first indication for CKI therapy, basing on the outstanding results of CheckMate 017 trial comparing nivolumab vs docetaxel in advanced squamous NSCLC (SqNSCLC) progressive to previous chemotherapy[33]. With a median OS of 9.2 mo vs 6 mo, nivolumab reduced the risk of death of 41%, with an HR of 0.59 (95%CI: 0.44-0.79), P < 0.001. The advantage was confirmed also for RR, PFS and safety profile, finally providing an unprecedented treatment option also in terms of tolerability.

progressive to previous chemotherapy[33]. With a median OS of 9.2 mo vs 6 mo, nivolumab reduced the risk of death of 41%, with an HR of 0.59 (95%CI: 0.44-0.79), P < 0.001. The advantage was confirmed also for RR, PFS and safety profile, finally providing an unprecedented treatment option also in terms of tolerability. Non-squamous cell lung cancer: With a slight delay and with not as brilliant but positive results, nivolumab was also approved for non-squamous NSCLC (non-SqNSCLC) treatment after failure of chemotherapy, on the basis of an analogous phase III randomized trial demonstrating an improvement of median OS from 9.4 mo with docetaxel to 12.2 mo (HR = 0.73, 95%CI: 0.59-0.89, P = 0.002)[34]. In this study, nivolumab was associated with better OS and RR but not with longer PFS compared to chemotherapy. A crossing of the PFS curves suggested a delay of the benefit with nivolumab, consistent with the results of previous immune system modulating agents, probably reflecting a pattern of response typical of immunotherapy and the use of inadequate response assessment measurements for this type of drug[97]. Other thoracic malignancies: Among other thoracic tumors, small cell lung cancer (SCLC), malignant pleural mesothelioma (MPM) and thymic epithelial tumors (TETs), under the thrust of true unmet medical needs, came across immunotherapy with CKI.

Non-squamous cell lung cancer: With a slight delay and with not as brilliant but positive results, nivolumab was also approved for non-squamous NSCLC (non-SqNSCLC) treatment after failure of chemotherapy, on the basis of an analogous phase III randomized trial demonstrating an improvement of median OS from 9.4 mo with docetaxel to 12.2 mo (HR = 0.73, 95%CI: 0.59-0.89, P = 0.002)[34]. In this study, nivolumab was associated with better OS and RR but not with longer PFS compared to chemotherapy. A crossing of the PFS curves suggested a delay of the benefit with nivolumab, consistent with the results of previous immune system modulating agents, probably reflecting a pattern of response typical of immunotherapy and the use of inadequate response assessment measurements for this type of drug[97]. Other thoracic malignancies: Among other thoracic tumors, small cell lung cancer (SCLC), malignant pleural mesothelioma (MPM) and thymic epithelial tumors (TETs), under the thrust of true unmet medical needs, came across immunotherapy with CKI. Preliminary data for PD-1/PD-L1 blockade in SCLC were encouraging and currently ongoing phase III studies are investigating CKI both in pretreated and untreated advanced SCLC patients[72,93] or as maintenance treatment after standard treatment either in extensive or in limited disease[91].

Other thoracic malignancies: Among other thoracic tumors, small cell lung cancer (SCLC), malignant pleural mesothelioma (MPM) and thymic epithelial tumors (TETs), under the thrust of true unmet medical needs, came across immunotherapy with CKI. Preliminary data for PD-1/PD-L1 blockade in SCLC were encouraging and currently ongoing phase III studies are investigating CKI both in pretreated and untreated advanced SCLC patients[72,93] or as maintenance treatment after standard treatment either in extensive or in limited disease[91]. Great expectations have been made for MPM, because of the known relationship between neoplastic and inflammatory counterpart in this tumor, recognized to have a T-cell inflamed phenotype. At the moment, only preliminary data have been published and CKI are currently under proposal for further investigations in this disease. Finally, early phases studies are ongoing to test CKI immunotherapy also in TETs[98]. Renal cancer After the pivotal trial Checkmate 025, nivolumab has vowed to became the cornerstone of previously treated metastatic renal cell carcinoma (mRCC) therapy, finally offering an OS improvement in a setting where targeted therapies have fallen short of expectation[99]. The median OS was 25 mo (95%CI: 21.8-not estimable) with nivolumab and 19.6 mo (95%CI: 17.6-23.1) with everolimus, with a HR of 0.73 and a RR of 25% vs 5% (P < 0.001). Also in terms of toxicity, nivolumab was superior to the standard treatment everolimus, with 19% vs 37% of AEs.

es have fallen short of expectation[99]. The median OS was 25 mo (95%CI: 21.8-not estimable) with nivolumab and 19.6 mo (95%CI: 17.6-23.1) with everolimus, with a HR of 0.73 and a RR of 25% vs 5% (P < 0.001). Also in terms of toxicity, nivolumab was superior to the standard treatment everolimus, with 19% vs 37% of AEs. In the light of these results, nivolumab currently represents a new standard of treatment for mRCC after disease progression to first line antiangiogenic therapy. On this auriferous vein other phase III randomized trials have been planned and their results are eagerly awaited. Worthy of note, a phase III randomized trial with an innovative design is comparing the combination of lenvatinib and everolimus (which recently achieved great results in phase II[100]) with the combination of lenvatinib and pembrolizumab vs the standard sunitinib. Such ambitious trials will probably provide the cornerstone of the future clinical practice in RCC[41,101].

innovative design is comparing the combination of lenvatinib and everolimus (which recently achieved great results in phase II[100]) with the combination of lenvatinib and pembrolizumab vs the standard sunitinib. Such ambitious trials will probably provide the cornerstone of the future clinical practice in RCC[41,101]. After reaching the indication for second line treatment, also first line setting has been investigated, with the planning of interesting trials currently still ongoing. In previously untreated RCC patients, atezolizumab in combination with bevacizumab is being compared to sunitinib[71]; the same standard of treatment is in turn compared to pembrolizumab combined with axitinib[102] and then to nivolumab plus ipilimumab[87]. Eventually, also avelumab plus axitinib is being investigated vs sunitinib[55]. In all cases, the control arm is represented by such a big standard of therapy (sunitinib) that, in case of positive results, the clinical practice for RCC will completely change, switching from angiogenesis inhibition to immune-checkpoint blockade. Urothelial cancers Since no significant improvements have been achieved in metastatic bladder cancer for long time, the impressive results of recent trials with CKI, in particular with the anti-PD-L1 atezolizumab, have given new hope to finally cure urothelial cancer[103,104].

After reaching the indication for second line treatment, also first line setting has been investigated, with the planning of interesting trials currently still ongoing. In previously untreated RCC patients, atezolizumab in combination with bevacizumab is being compared to sunitinib[71]; the same standard of treatment is in turn compared to pembrolizumab combined with axitinib[102] and then to nivolumab plus ipilimumab[87]. Eventually, also avelumab plus axitinib is being investigated vs sunitinib[55]. In all cases, the control arm is represented by such a big standard of therapy (sunitinib) that, in case of positive results, the clinical practice for RCC will completely change, switching from angiogenesis inhibition to immune-checkpoint blockade. Urothelial cancers Since no significant improvements have been achieved in metastatic bladder cancer for long time, the impressive results of recent trials with CKI, in particular with the anti-PD-L1 atezolizumab, have given new hope to finally cure urothelial cancer[103,104]. Atezolizumab is currently been approved for treatment of urothelial cancer on the basis of a randomized phase II trial comparing this anti-PD-L1 with standard treatment, demonstrating its advantage over chemotherapy in both platinum pretreated ineligible patients and in chemotherapy pretreated patients[105]. At the same time, phase III studies in second line setting are ongoing and both atezolizumab and pembrolizumab have been compared to different second line chemotherapeutic regimens in all urothelial cancers: The trial with pembrolizumab has been recently early stopped due to the meeting of the primary endpoint (OS)[40,62]. Also avelumab and durvalumab reached phase III investigation in bladder cancer, but in the first line setting; the latter combined with the anti-CTLA4 tremelimumab vs standard first line chemotherapy[56,76]. A further interesting study in metastatic urothelial cancer is recruiting naive patients ineligible to cisplatin to receive atezolizumab in combination with chemotherapy (gemcitabine and carboplatin) as first line treatment[63].

atter combined with the anti-CTLA4 tremelimumab vs standard first line chemotherapy[56,76]. A further interesting study in metastatic urothelial cancer is recruiting naive patients ineligible to cisplatin to receive atezolizumab in combination with chemotherapy (gemcitabine and carboplatin) as first line treatment[63]. Not less significant the promising evidence about the role of CKI in the adjuvant setting of urothelial cancer: Atezolizumab is under investigation vs only observation after cystectomy in PD-L1 positive high risk muscle-invasive bladder cancer[23] and also nivolumab is being tested in this setting[28]. Head and neck cancer Head and neck squamous cell carcinoma (HNSCC) undoubtedly a promising candidate for CKI because of the profound immune suppression from which is characterized. As the matter of fact, a phase III randomized study comparing nivolumab to the standard of treatment in pretreated HNSCC patients was early stopped after the clear demonstration of an improvement in terms of OS for nivolumab[89]. This trial provided very promising results in platinum refractory disease, encouraging the planning of further phase III studies, currently ongoing, also for pembrolizumab[38,82] and early phases trials with durvalumab and avelumab[106]. Despite an apparently not so favorable toxicity profile, also anti-CTLA4 antibodies are being tested in combination with anti-PD-1 or anti-PD-L1 agents in HNSCC. Phase III studies with this therapeutic strategy are currently ongoing both in pretreated patients and in first line setting[74,79].

Head and neck cancer Head and neck squamous cell carcinoma (HNSCC) undoubtedly a promising candidate for CKI because of the profound immune suppression from which is characterized. As the matter of fact, a phase III randomized study comparing nivolumab to the standard of treatment in pretreated HNSCC patients was early stopped after the clear demonstration of an improvement in terms of OS for nivolumab[89]. This trial provided very promising results in platinum refractory disease, encouraging the planning of further phase III studies, currently ongoing, also for pembrolizumab[38,82] and early phases trials with durvalumab and avelumab[106]. Despite an apparently not so favorable toxicity profile, also anti-CTLA4 antibodies are being tested in combination with anti-PD-1 or anti-PD-L1 agents in HNSCC. Phase III studies with this therapeutic strategy are currently ongoing both in pretreated patients and in first line setting[74,79]. Other tumors The PD-1/D-L1 axis has been targeted in other tumor types than those cited above, with an interesting rationale and supported by phase I-II experiences, despite still remaining in shadow waiting for phase III results. In ovarian cancer, despite several early phase studies currently ongoing with nivolumab, pembrolizumab, BMS936559 (an anti-PD-L1) and avelumab, the emerged response rates are relatively low, in front of a manageable safety profile[53,54,107].

Other tumors The PD-1/D-L1 axis has been targeted in other tumor types than those cited above, with an interesting rationale and supported by phase I-II experiences, despite still remaining in shadow waiting for phase III results. In ovarian cancer, despite several early phase studies currently ongoing with nivolumab, pembrolizumab, BMS936559 (an anti-PD-L1) and avelumab, the emerged response rates are relatively low, in front of a manageable safety profile[53,54,107]. Pembrolizumab, aside from early investigations in soft tissue and bone sarcomas[108], is currently under phase III investigation in hepatocellular carcinoma[42], in esophageal and gastric carcinoma[50-52], in Hodgkin and non-Hodgkin lymphoma[39]. In these latter malignancies also nivolumab and pidilizumab, anti-PD-1 antibodies, besides from atezolizumab and durvalumab, anti-PD-L1 antibodies, are being evaluated in early phases[109]. Furthermore, different treatment lines of advanced gastric cancer are being tested with avelumab[57,58]. Some initial encouraging data are emerging from ongoing studies in favor of the employment of CKI also in central nervous system (CNS) malignancies, such as glioblastoma, where unmet clinical needs are leading to new investigations[88,92]. Disappointing results were instead obtained for pancreatic cancer, despite a certain evidence for durvalumab[110].

sic, (2) percentage pain relief with analgesics; (3) type of side-effects; (4) severity of side-effects; and (5) out-of-pocket cost of analgesics. In addition to the design components, we also investigated the internal, external predictive validity and temporal stability of the CBC experiment over the study period[14]. Analgesic attitudes and barriers: Barriers Questionnaire-II[20] was used to assess patients’ attitudes and beliefs about the management of cancer pain. It is a 27-item measure which elicits patients’ pain management concerns in eight domains: (1) fear of addiction; (2) fear of tolerance; (3) fear of side effects; (4) fatalism about cancer pain; (5) desire to be a good patient; (6) fear of distracting health provider from treating cancer; (7) fear that the analgesics impair the immune system; and (8) concern that analgesics may mask ability to monitor illness symptoms. The response range is from 0 (do not agree) to 5 (agree very much). The scores are based on sums for items for the total scale and four subscales (physiological, fatalism, communication, and harmful effects). The internal consistency reliability of the scale is excellent at 0.89[20].

emerging from ongoing studies in favor of the employment of CKI also in central nervous system (CNS) malignancies, such as glioblastoma, where unmet clinical needs are leading to new investigations[88,92]. Disappointing results were instead obtained for pancreatic cancer, despite a certain evidence for durvalumab[110]. About colorectal cancer, despite the initial evidence to be not responsive to nivolumab, a subset of patients has been identified as potentially best responders to pembrolizumab, revealing that the mismatch repair (MMR) status can predict clinical benefit with enhanced responsiveness in tumors with microsatellite instability (MSI)[111]. With this rationale, phase III randomized studies have been initiated in order to compare standard therapy with pembrolizumab in MSI colorectal cancer patients[49]. Furthermore, atezolizumab is currently under investigation alone or in combination with cobimetinib (mitogen activate protein kinase-inhibitor) vs regorafenib (antiangiogenic multi-kinase inhibitor) in all advanced colorectal tumors[73]. Eventually, a great interest for PD-1/PD-L1 blockade is represented by triple negative breast cancer: Phase III trials are currently ongoing with pembrolizumab compared to chemotherapy and with atezolizumab combined with nab-paclitaxel both in neo-adjuvant and advanced setting[47,48,70,112].

About colorectal cancer, despite the initial evidence to be not responsive to nivolumab, a subset of patients has been identified as potentially best responders to pembrolizumab, revealing that the mismatch repair (MMR) status can predict clinical benefit with enhanced responsiveness in tumors with microsatellite instability (MSI)[111]. With this rationale, phase III randomized studies have been initiated in order to compare standard therapy with pembrolizumab in MSI colorectal cancer patients[49]. Furthermore, atezolizumab is currently under investigation alone or in combination with cobimetinib (mitogen activate protein kinase-inhibitor) vs regorafenib (antiangiogenic multi-kinase inhibitor) in all advanced colorectal tumors[73]. Eventually, a great interest for PD-1/PD-L1 blockade is represented by triple negative breast cancer: Phase III trials are currently ongoing with pembrolizumab compared to chemotherapy and with atezolizumab combined with nab-paclitaxel both in neo-adjuvant and advanced setting[47,48,70,112]. Transversal challenges Immune-related toxicity: The management of the “new toxicities” of CKI is transversal to all malignancies and to all cited antibodies, unavoidably involving other specialists beyond the oncologist, such as the endocrinologist and the immunologist in first line.

Eventually, a great interest for PD-1/PD-L1 blockade is represented by triple negative breast cancer: Phase III trials are currently ongoing with pembrolizumab compared to chemotherapy and with atezolizumab combined with nab-paclitaxel both in neo-adjuvant and advanced setting[47,48,70,112]. Transversal challenges Immune-related toxicity: The management of the “new toxicities” of CKI is transversal to all malignancies and to all cited antibodies, unavoidably involving other specialists beyond the oncologist, such as the endocrinologist and the immunologist in first line. These immune-related adverse events (irAEs) are due to the infiltration of tissues by activated T-lymphocytes responsible of autoimmunity. As a consequence, the block of the immune-checkpoint can amplify any immune response in all organs: Skin, gastrointestinal tract, endocrine glands, lung, CNS, liver, kidney, hematological cells, muscular-articular system, heart and eyes can all be affected. Nevertheless, most of these irAEs are rare and only fatigue, rash, pruritus, diarrhea, nausea and arthralgia occurs in > 10% of cases. On the other hand, despite being rare, interstitial pneumonitis is the main life-threatening toxicity for anti PD-1/PD-L1 agents[113]. Potentially predisposing conditions for irAEs development could be represented by personal or family history of autoimmune disease (genetic determinants), by underlying silent autoimmunity, chronic viral infections or other personal ecological factors (such as the microbiome in the case of enterocolitis)[114].

These immune-related adverse events (irAEs) are due to the infiltration of tissues by activated T-lymphocytes responsible of autoimmunity. As a consequence, the block of the immune-checkpoint can amplify any immune response in all organs: Skin, gastrointestinal tract, endocrine glands, lung, CNS, liver, kidney, hematological cells, muscular-articular system, heart and eyes can all be affected. Nevertheless, most of these irAEs are rare and only fatigue, rash, pruritus, diarrhea, nausea and arthralgia occurs in > 10% of cases. On the other hand, despite being rare, interstitial pneumonitis is the main life-threatening toxicity for anti PD-1/PD-L1 agents[113]. Potentially predisposing conditions for irAEs development could be represented by personal or family history of autoimmune disease (genetic determinants), by underlying silent autoimmunity, chronic viral infections or other personal ecological factors (such as the microbiome in the case of enterocolitis)[114]. The prevention, the anticipation, the detection and then the treatment (with multidisciplinary approach) and monitoring of irAEs are the principles of their correct clinical management. Depending on their severity, irAEs require temporary or permanent discontinuation of CKI therapy, use of high doses corticosteroids or, in severe cases, of anti-TNF treatment with infliximab. The current management guidelines are based on recent expert consensus recommendations published about the issue[115].

nagement. Depending on their severity, irAEs require temporary or permanent discontinuation of CKI therapy, use of high doses corticosteroids or, in severe cases, of anti-TNF treatment with infliximab. The current management guidelines are based on recent expert consensus recommendations published about the issue[115]. Response assessment: RECIST vs immune-related criteria: Based on survival analysis, traditional response evaluation criteria in solid tumors (RECIST) might underestimate the benefit of CKI[116]. The pattern of response of immunotherapy, radically different from those of standard chemotherapy and also of antiangiogenic agents, is frequently not captured by the conventional RECIST[117]. This led to the development of the immune-related response criteria (irRC)[118], assessing tumor burden as a continuous variable and evaluating percentage changes in several target lesions overtime. In this system, the appearance of new lesions does not mean progressive disease but it is considered and reassessed in the context of a dynamic evaluation. Moreover, the thresholds to determine progression or response (25% increase and 50% decrease) are higher than those of RECIST (20% increase and 30% decrease)[119]. Given the reported evidence, modified criteria are undoubtedly mandatory in the response assessment to the new immunotherapy, in order to prevent premature discontinuation of treatment.

etermine progression or response (25% increase and 50% decrease) are higher than those of RECIST (20% increase and 30% decrease)[119]. Given the reported evidence, modified criteria are undoubtedly mandatory in the response assessment to the new immunotherapy, in order to prevent premature discontinuation of treatment. PD-L1 expression as response predictor: In the context of solid tumors treated with PD-1/PD-L1 inhibitors, the predictive role of PD-L1 expression on tumor cells and, as more recently discovered, on immune infiltrating cells, represents an actual issue of great interest and constitutes a significant cue of discussion for clinical researchers[120]. Currently, on the basis of the state of art, the predictive value of PD-L1 on tumor cells is limited to NSCLC and melanoma, especially for anti-PD-1 antibodies, whilst a more predictive significance of PD-L1 expression on the immune cells infiltrating the tumor seems to emerge for urothelial cancers in the case of anti-PD-L1 antibodies[121,122]. Nevertheless, a great limit of such speculations is represented by the scarce reliance and reproducibility of the different methods used for the biomarker’s detection, with controversial results depending on the staining technique, on the different anti-PD-L1 antibodies and finally on the sample used for immune-histochemical assay (primary tumor vs metastases samples, with the challenge of heterogeneity). Moreover, confusing data emerged from the use (and the lack of validation) of different cut-off for PD-L1 expression, from 1%, to 5%, to 50% threshold in different trials[120].

odies and finally on the sample used for immune-histochemical assay (primary tumor vs metastases samples, with the challenge of heterogeneity). Moreover, confusing data emerged from the use (and the lack of validation) of different cut-off for PD-L1 expression, from 1%, to 5%, to 50% threshold in different trials[120]. Aside from PD-L1 expression, further multiple factors have been explored and are currently undergoing investigations as predictive elements for response to CKI: Among these, an increasing interest is being acquired by the micro-environmental features of the tumor, such as the infiltrating immune cells sub-populations and their biomarkers expression[123].

multiple factors have been explored and are currently undergoing investigations as predictive elements for response to CKI: Among these, an increasing interest is being acquired by the micro-environmental features of the tumor, such as the infiltrating immune cells sub-populations and their biomarkers expression[123]. Microsatellite instability and hyper-mutational status: The MSI phenotype, as a consequence of a defective DNA-MMR system, characterizes a subgroup of tumors harboring a large number of somatic mutations (high mutational load). Since these mutations have the potential to encode a great number of immunogenic neoantigens, a particular susceptibility of MSI-hyper-mutated cancers to PD-1/PD-L1 axis blockade have been hypothesized and more recently proven[124]. As the matter of fact, MSI tumors have a microenvironment characterized by abundant T-cell infiltrate, with activated CD8+ cytotoxic T lymphocyte (CTL) and activated Th1 producing IFN-γ, high expression of PD-L1 (in particular by TILs and myeloid cells infiltrating the tumor) and great overexpression of immune-checkpoint related proteins[125]. All these elements configure the elective candidate cancer for immune-checkpoint inhibition and suggest to investigate CKI in all cancer types with MMR defects.

high expression of PD-L1 (in particular by TILs and myeloid cells infiltrating the tumor) and great overexpression of immune-checkpoint related proteins[125]. All these elements configure the elective candidate cancer for immune-checkpoint inhibition and suggest to investigate CKI in all cancer types with MMR defects. Additionally, tumors with polymerase E (POLE) mutations, despite stable microsatellites, have been demonstrated to contain a high mutational load. Also these POLE-ultra-mutated cancers are characterized by an active Th1/CTL microenvironment and upregulated immune checkpoints, constituting an ideal target for CKI therapy as well as MSI tumors[126]. In conclusion, among apparently resistant cancer types (such as colon cancer), CKI have been proven to exert an effect in case of MMR defects and trials on this selected population are currently ongoing to investigate the efficacy of anti-PD-1 antibodies[49]. Immune system modulation with sequential or association strategies: Given the great benefit in terms of OS and the long lasting impact of CKI therapy on patients’ survival in the responding cases, probably due to immunological memory, two major issues remain to be addressed: The sensitization of non-responders and the disease control in patients initially pseudo-progressive. With these aims, combination strategies have been planned and investigated in the last years, either combining immunotherapy with chemotherapy, radiotherapy and targeted agents or associating different CKI[127].

essed: The sensitization of non-responders and the disease control in patients initially pseudo-progressive. With these aims, combination strategies have been planned and investigated in the last years, either combining immunotherapy with chemotherapy, radiotherapy and targeted agents or associating different CKI[127]. The strategy to increase the immunogenicity of tumors can be pursued through the enhancement of antigen presentation (boosting antigens release or stimulating APC function), the stimulation of major histocompatibility complex (MHC) class I expression, the down-regulation of the T-reg cells and the stimulation of the T-cells infiltration. Some of these mechanisms can be achieved with promising combination strategies. Chemotherapeutic agents are capable to induce immunogenic cancer death, generating a strong immune stimulation. Among these, cyclophosphamide have additionally been shown to reduce the number of circulating T-reg cells, removing a key element of immunosuppression, and moreover to sensitize tumor cells to T-cell mediated apoptosis, potentially boosting the effect of the immune checkpoint blockade[128-130]. Considering the criticism of a combination between CKI and chemotherapy, given expected short term immunosuppressive effect of the latter, in our opinion a sequential strategy could represent a good opportunity to take advantage of cell death and antigen release caused by an induction chemotherapy, in order to prepare a more immunogenic environment for the subsequent CKI[131].

chemotherapy, given expected short term immunosuppressive effect of the latter, in our opinion a sequential strategy could represent a good opportunity to take advantage of cell death and antigen release caused by an induction chemotherapy, in order to prepare a more immunogenic environment for the subsequent CKI[131]. A great interest for the potential stimulation of the immune-response through radiotherapy has been suggested by the evidence about the immune-mediated abscopal effect[132]. Aside from interesting case reports, clinical trials in this field are currently in early phases and eagerly awaited[133]. Targeted therapy combinations with immunotherapy are currently under investigation, in early phases, with interesting results[127]. The rationale of such strategies could be represented by the aim to obtain a more rapid RR and to boost PFS with the targeted agent, in expectation of the long-term effect on survival of the CKI. Finally, the combination of anti-PD-1 and anti-CTLA4 antibodies, despite the increased immune-related toxicity, has been shown to improve the outcomes in a phase III randomized trial in metastatic melanoma, early changing the standard of treatment a few years after the onset of the new immunotherapy with ipilimumab[134]. Several trials investigating such association of CKI are currently ongoing: The management of irAEs will probably represent the main criticism of such strategies[127].

omized trial in metastatic melanoma, early changing the standard of treatment a few years after the onset of the new immunotherapy with ipilimumab[134]. Several trials investigating such association of CKI are currently ongoing: The management of irAEs will probably represent the main criticism of such strategies[127]. Targeting PD-1/PD-L1 axis in adjuvant setting: The rationale for the PD-1/PD-L1 axis inhibition for adjuvant purposes is in the concept of “immunological memory”, generated by the cancer-immunity cycle, starting from the release of cancer cell antigens also in the early phases of tumorigenesis. After the APC migration in the lymph nodes and the presentation of antigens in the context of MHC-I molecules to CD8+ T cells, aside from effector T-lymphocytes capable of activation against cancer neo-antigens, memory T-cells are also generated. These quiescent lymphocytes are appointed to the subsequent immune-response and could contribute to avoid disease relapse[135].

sentation of antigens in the context of MHC-I molecules to CD8+ T cells, aside from effector T-lymphocytes capable of activation against cancer neo-antigens, memory T-cells are also generated. These quiescent lymphocytes are appointed to the subsequent immune-response and could contribute to avoid disease relapse[135]. Considering the widely acceptable toxicity profile of CKI, the proposal of using them as adjuvant therapy, to prevent relapses after surgery of early disease while maintaining a good quality of life, appears very favorable. In support of this, we have the approval of the CTLA4 inhibitor ipilimumab for adjuvant treatment in melanoma, on the basis of a recent pivotal trial[136]. For PD-1/PD-L1 axis inhibitors, nevertheless, the investigation in adjuvant setting is quite early, in spite of a more favorable safety management. A noteworthy issue about immune-adjuvant treatment with these compounds (unlike the case of ipilimumab) is the correct duration of therapy, ranging from one to more years in different planned trials. The currently ongoing studies are reported in Table 2.

g is quite early, in spite of a more favorable safety management. A noteworthy issue about immune-adjuvant treatment with these compounds (unlike the case of ipilimumab) is the correct duration of therapy, ranging from one to more years in different planned trials. The currently ongoing studies are reported in Table 2. PERSPECTIVES Considering the wide range of settings and combinations covered by the ongoing clinical trials with CKI treatment, we think that the future directions for immunotherapy are still to be written and they are probably different basing on cancer types. The reason of this latter statement, not so obvious as it may seem, is likely due to the other different therapies to whom immune-checkpoint blockade needs to be sequenced and alternated in each tumor, more than to a real difference in the target, which is always represented by the immune system and by its relationship with the tumor rather than by the tumor itself. From this point of view, a key issue could be represented by the immunomodulating potential of the current standard of treatment in each case, sometimes widely unknown and rarely explored before the “immunotherapy era”[137].

PERSPECTIVES Considering the wide range of settings and combinations covered by the ongoing clinical trials with CKI treatment, we think that the future directions for immunotherapy are still to be written and they are probably different basing on cancer types. The reason of this latter statement, not so obvious as it may seem, is likely due to the other different therapies to whom immune-checkpoint blockade needs to be sequenced and alternated in each tumor, more than to a real difference in the target, which is always represented by the immune system and by its relationship with the tumor rather than by the tumor itself. From this point of view, a key issue could be represented by the immunomodulating potential of the current standard of treatment in each case, sometimes widely unknown and rarely explored before the “immunotherapy era”[137]. The great advantage of anti-PD-1/PD-L1 agents is undoubtedly represented by their very favorable safety profile, with large tolerability in almost all patients. Combinations of CKI with standard chemotherapy or targeted therapies, despite possibly more effective, have the risk of became unsustainable both in terms of costs and of toxicity, significantly impacting on the final outcome. Nevertheless, alternating targeted and immunotherapy might permit to modulate tumor metabolism, inflammation and immune infiltration, allowing to modify the relationship between cancer and immune system.

e risk of became unsustainable both in terms of costs and of toxicity, significantly impacting on the final outcome. Nevertheless, alternating targeted and immunotherapy might permit to modulate tumor metabolism, inflammation and immune infiltration, allowing to modify the relationship between cancer and immune system. Thus, in order to fully take advantage of its potential, the winning strategy with immune-checkpoint blockade could be represented by an ingenious sequence, exploiting the immunomodulating properties of previous and subsequent drugs with the aim of boosting immune system activation against the tumor.

e risk of became unsustainable both in terms of costs and of toxicity, significantly impacting on the final outcome. Nevertheless, alternating targeted and immunotherapy might permit to modulate tumor metabolism, inflammation and immune infiltration, allowing to modify the relationship between cancer and immune system. Thus, in order to fully take advantage of its potential, the winning strategy with immune-checkpoint blockade could be represented by an ingenious sequence, exploiting the immunomodulating properties of previous and subsequent drugs with the aim of boosting immune system activation against the tumor. CONCLUSION The onset of PD-1/PD-L1 targeted therapy has demonstrated the importance of this axis in the immune escape across almost all human cancers. Despite being burdened by some issues not still addressed, such as the correct duration of therapy in the responsive patients, the new CKI allowed to significantly prolong survival and to generate durable response, demonstrating remarkable efficacy in a wide range of cancer types. However, such benefit is not extended to all patients, and some of them experienced immune escape despite therapy. The investigation about mechanisms leading to the development of primary or secondary immune escape must represent the key element of future studies in the whole immuno-oncology, with the aim of resensitize these patients to the immune checkpoint blockade. The future approach to the problem may be represented by a personalized cancer immunotherapy, allowed only by multiparameter biomarkers approaches, as interestingly suggested by Kim et al[138] in a recent review about the “step to success (or failure)” to PD-1/PD-L1 blockade. In their proposal, a hypothetical algorithm could provide the assessment of specific immune-related biomarkers in each patient’s tumor, allowing to create a personal mapping according to which characteristics the oncologist could chose (or exclude) the optimal immunotherapy or immunotherapeutic combination for each single case.

proposal, a hypothetical algorithm could provide the assessment of specific immune-related biomarkers in each patient’s tumor, allowing to create a personal mapping according to which characteristics the oncologist could chose (or exclude) the optimal immunotherapy or immunotherapeutic combination for each single case. Waiting for the possible realization of such sophistication of therapy, the immune checkpoint blockade in oncology is currently experiencing promising huge advances, shifting the classical paradigm of anticancer treatment from targeting the tumor to targeting the immune system and increasing our hopes to gain the immune control of oncological disease. Conflict-of-interest statement: No potential conflicts of interest. No financial support. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Italy Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B, B Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: August 18, 2016 First decision: September 28, 2016 Article in press: November 29, 2016 P- Reviewer: Qin JM, Tirumani SH, Tomizawa M, Tsikouras PPT, Zhang L S- Editor: Ji FF L- Editor: A E- Editor: Wu HL

Core tip: Breast cancer is the second leading cause of cancer deaths in women. Triple negative breast cancer is an aggressive subtype that lacks targeted therapy. Obesity is a risk factor for breast cancer and is associated with high leptin levels. Leptin induces the expression of cell cycle associated proteins advancing cell cycle progression. Leptin also increases breast cancer stem cell growth, which promotes chemotherapeutic resistance. We have developed a leptin antagonist linked to iron oxide nanoparticles (IONP-LPrA2) which significantly inhibits leptin-induced cell proliferation and survival of breast cancer cells treated with chemotherapeutics. IONP-LPrA2 can increase chemotherapeutic efficacy in breast cancer.

omotes chemotherapeutic resistance. We have developed a leptin antagonist linked to iron oxide nanoparticles (IONP-LPrA2) which significantly inhibits leptin-induced cell proliferation and survival of breast cancer cells treated with chemotherapeutics. IONP-LPrA2 can increase chemotherapeutic efficacy in breast cancer. INTRODUCTION The American Cancer Society estimates that there will be nearly 300000 new breast cancer cases diagnosed worldwide and approximately 50000 women will die from breast cancer in 2016[1]. Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer diagnoses. TNBC is a subtype of breast cancer characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor-2 (HER2)[1,2]. Due to the absence of receptor expression; this form of breast cancer, which predominantly affects younger, African American and Hispanic patients lacks targeted therapeutic options[3]. TNBC patients are commonly treated with chemotherapy; however these patients make up approximately 30% of breast cancer-related deaths annually[4]. This necessitates the development of targeted therapies for this more aggressive form of the disease.

American and Hispanic patients lacks targeted therapeutic options[3]. TNBC patients are commonly treated with chemotherapy; however these patients make up approximately 30% of breast cancer-related deaths annually[4]. This necessitates the development of targeted therapies for this more aggressive form of the disease. There are many factors that increase the risk of developing TNBC including environment, genetic susceptibility, and obesity[5]. Obesity has a negative impact on breast cancer patient survival and, like TNBC, is associated with an increased risk of recurrence[6]. Obesity is correlated to high levels of leptin, a cytokine produced by adipose tissue which regulates satiety. The leptin signaling pathway occurs in approximately 80% of breast cancers[7]. The binding of leptin to its receptor, Ob-R, leads to activation of pathways involved in cell proliferation, migration, and survival[8]. Leptin is a survival factor in breast cancer and may have the ability to limit the effectiveness of chemotherapy drugs by activating the JAK2/STAT3, MAPK/ERK, and PI3/Akt signaling pathways[9,10]. Therefore leptin signaling inhibition has become a promising therapeutic area for breast cancer, particularly in the case of TNBC for which there is no targeted therapy[11].

ay have the ability to limit the effectiveness of chemotherapy drugs by activating the JAK2/STAT3, MAPK/ERK, and PI3/Akt signaling pathways[9,10]. Therefore leptin signaling inhibition has become a promising therapeutic area for breast cancer, particularly in the case of TNBC for which there is no targeted therapy[11]. The binding of leptin to Ob-R upregulates Notch, interleukin 1 (IL-1), vascular endothelial growth factor (VEGF), and its receptor VEGFR2; which promote breast cancer cell survival and angiogenesis[12]. The harmful effects of leptin signaling on breast cancer onset and progression have been shown to be diminished by the leptin peptide receptor antagonist 2 (LPrA2)[13]. LPrA2 and the pegylated form (PEG-LPrA2) have been shown to cause a delay in cancer onset and progression as well as a reduction in 4T1-tumor growth in BALB/C mice[14]. Additionally, PEG-LPrA2 has been shown to decrease MDA-MB-231 and MCF-7-tumor growth in SCID mice[15]. In another study, diet-induced obese (DIO) C57BL/6J mice treated with the carcinogen 7, 12-Dimethylbenz (a) anthracene (DMBA) along with PEG-LPrA2 did not develop tumors[16]. The anti-tumor activity of LPrA2 provides mounting evidence for its usefulness in cancer therapy.

MB-231 and MCF-7-tumor growth in SCID mice[15]. In another study, diet-induced obese (DIO) C57BL/6J mice treated with the carcinogen 7, 12-Dimethylbenz (a) anthracene (DMBA) along with PEG-LPrA2 did not develop tumors[16]. The anti-tumor activity of LPrA2 provides mounting evidence for its usefulness in cancer therapy. The leptin signaling pathway plays a major role in breast cancer cell growth, angiogenesis, as well as metastasis and invasion[8]. Although the leptin antagonist LPrA2 attenuates leptin signaling, it is limited by its insolubility in water and short half-life of 1-2 h[14,17,18]. Here we describe the coupling of LPrA2 to a nanoparticle delivery system which uses iron oxide nanoparticles (IONPs) to capture multiple LPrA2 peptides. We assessed the conjugation of LPrA2 to IONPs (IONP-LPrA2) to determine the inhibitory effect on breast cancer cell growth and survival. Because LPrA2 decreases breast cancer tumor growth and chemotherapy is widely used in the treatment of breast cancer, we sought to assess if combining IONP-LPrA2 and chemotherapeutic drugs would allow for reduction of the effective dose. Thus, we evaluated the survival of human breast cancer cell lines with IONP-LPrA2 and a panel of anti-cancer drugs.

ncer tumor growth and chemotherapy is widely used in the treatment of breast cancer, we sought to assess if combining IONP-LPrA2 and chemotherapeutic drugs would allow for reduction of the effective dose. Thus, we evaluated the survival of human breast cancer cell lines with IONP-LPrA2 and a panel of anti-cancer drugs. MATERIALS AND METHODS Reagents and antibodies IONPs were obtained from Ocean Nanotech San Diego, CA. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), Sulfo-NHS, and other chemicals were purchased from Sigma Aldrich St. Louis, MO. Ob-R (sc-8325), Cyclin D1 (sc-246), pSTAT3 (sc-8059), STAT3 (sc-8019) antibodies were purchased from Santa Cruz Biotechnology Santa Cruz, CA. Anti-rabbit and anti-mouse conjugated to horseradish peroxidase were obtained from Bio-Rad Laboratories Hercules, CA. Dulbecco’s Modified Eagles Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMEM), Protease and Phosphatase Inhibitor cocktails, Penicillin/Streptomycin, Slide-a-lyzer dialysis cassette, and Western blotting chemiluminescence substrate were purchased from Thermo Fisher Scientific Rockford, IL. Mammocult complete medium was obtained from Stem Cell Technologies Vancouver, BC. Fetal bovine serum was obtained from Med Supply Partners Atlanta, GA. Leptin was purchased from R and D Systems Minneapolis, MN. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit was purchased from Molecular Probes Eugene, OR. Annexin V/fluorescein Isothiocyanate (FITC) and propidium iodide (PI) were obtained from Nexcelom Bioscience Boston, MA. Cisplatin (Cis) was purchased from Millipore Billerica, MA. Cyclophosphamide (CTX), paclitaxel (PTX), and doxorubicin (Dox) were obtained from SelleckChem Houston, TX.

om Molecular Probes Eugene, OR. Annexin V/fluorescein Isothiocyanate (FITC) and propidium iodide (PI) were obtained from Nexcelom Bioscience Boston, MA. Cisplatin (Cis) was purchased from Millipore Billerica, MA. Cyclophosphamide (CTX), paclitaxel (PTX), and doxorubicin (Dox) were obtained from SelleckChem Houston, TX. Nanoparticle conjugation LPrA2 was synthesized as described[8,19]. LPrA2 was de-salted using the slide-a-lyzer dialysis cassette (Thermo Fisher). LPrA2 was conjugated to IONPs (Ocean Nanotech) by the outlined method[20]. Western blot analysis IONP-LPrA2 was separated by SDS-PAGE. LPrA2 and LPrA2-Scramble (Sc) were used as positive and negative controls, respectively. The peptides were transferred onto nitrocellulose membranes (Bio-Rad). The membranes were probed with an LPrA2 antibody, purified from antigen injected rabbit bleeds. Anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used for further detection of the peptides. Chemiluminescent detection of the bands was displayed by Western blotting substrate (Thermo Fisher). Nanoparticle tracking analysis Dilutions of 1:10000 of the bound and unbound particles were sonicated for 30 min. The size and distribution of the conjugated and unconjugated IONPs were determined by the NanoSight (Malvern Instruments Ltd., Worcestershire, United Kingdom).

Western blot analysis IONP-LPrA2 was separated by SDS-PAGE. LPrA2 and LPrA2-Scramble (Sc) were used as positive and negative controls, respectively. The peptides were transferred onto nitrocellulose membranes (Bio-Rad). The membranes were probed with an LPrA2 antibody, purified from antigen injected rabbit bleeds. Anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used for further detection of the peptides. Chemiluminescent detection of the bands was displayed by Western blotting substrate (Thermo Fisher). Nanoparticle tracking analysis Dilutions of 1:10000 of the bound and unbound particles were sonicated for 30 min. The size and distribution of the conjugated and unconjugated IONPs were determined by the NanoSight (Malvern Instruments Ltd., Worcestershire, United Kingdom). Cell culture Human ER+ MCF-7 cells in addition to TNBC MDA-MB-231 and HCC1806 cells (American Type Culture Collection, ATCC, Manassas, VA) were cultured in DMEM (Thermo Fisher) with 10% FBS and 1% penicillin/streptomycin (Thermo Fisher) and maintained in an incubator at 37 °C with 5% CO2.

Nanoparticle tracking analysis Dilutions of 1:10000 of the bound and unbound particles were sonicated for 30 min. The size and distribution of the conjugated and unconjugated IONPs were determined by the NanoSight (Malvern Instruments Ltd., Worcestershire, United Kingdom). Cell culture Human ER+ MCF-7 cells in addition to TNBC MDA-MB-231 and HCC1806 cells (American Type Culture Collection, ATCC, Manassas, VA) were cultured in DMEM (Thermo Fisher) with 10% FBS and 1% penicillin/streptomycin (Thermo Fisher) and maintained in an incubator at 37 °C with 5% CO2. Cell lysis and immunoblotting analysis Cells were seeded at 2 × 105 in 6 well cell culture plates and grown to 70%-80% confluence. The cells were treated with leptin (1.2 nmol/L) (R and D Systems), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L) for 24-48 h. Basal cells served as untreated controls. The cells were lysed with RIPA buffer (Sigma) containing protease/phosphatase inhibitors (Thermo Fisher). Proteins were pulled down by Immunoprecipitation. Immunoblotting analysis was performed as described[21]. The membranes were incubated with Ob-R, cyclin D1, pSTAT3, and STAT3 (Santa Cruz Biotechnology) antibodies overnight at 4 °C. GAPDH (Sigma) was used as a protein loading control. Relative protein levels were determined by Image J software (National Institute of Health, NIH).

ing analysis was performed as described[21]. The membranes were incubated with Ob-R, cyclin D1, pSTAT3, and STAT3 (Santa Cruz Biotechnology) antibodies overnight at 4 °C. GAPDH (Sigma) was used as a protein loading control. Relative protein levels were determined by Image J software (National Institute of Health, NIH). Cell cycle analysis Cells were seeded at 2 × 105 in 6 well cell culture plates and grown to 70%-80% confluence. They were treated with IONP-LPrA2 at indicated (pmol/L) concentrations plus leptin (1.2 nmol/L for 24-48 h. Leptin and unconjugated LPrA2 served as controls. The cells were trypsinized, washed with 1 × PBS, and resuspended in cold 100% methanol (Sigma). The were stored at -20 °C prior to analysis (< 1 wk). Afterward, the cells were centrifuged to remove the methanol. They were resuspended in 50 μL PI (Nexcelom) and incubated at 37 °C for 40 min. The cells were centrifuged to remove the PI, resuspended in 1 × PBS, and analyzed by the Nexcelom Cellometer Vision® image based cytometer to determine the percentage of cells in the S phase of the cell cycle.

ifuged to remove the methanol. They were resuspended in 50 μL PI (Nexcelom) and incubated at 37 °C for 40 min. The cells were centrifuged to remove the PI, resuspended in 1 × PBS, and analyzed by the Nexcelom Cellometer Vision® image based cytometer to determine the percentage of cells in the S phase of the cell cycle. MTT assay Cells were seeded at 5 × 103 in 96 well cell culture plates and grown to 70%-80% confluence. The cells were treated with leptin (1.2 nmol/L), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L) for 24-48 h. Basal cells served as untreated controls. The media was removed from the cells, the wells were washed with 1 × PBS, and 200 μL of IMEM (Thermo Fisher) together with 10 μL of sterile 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5 mg/mL in PBS, (Molecular Probes) were added. The plates were incubated for 4 h at 37 °C. Following incubation the media was removed, 50 μL of Dimethyl sulfoxide was added to the wells, and the plates were incubated at 37 °C for 30 min. The absorbance was read at 540 nm using a microplate reader (Molecular Devices) to measure cell proliferation.

robes) were added. The plates were incubated for 4 h at 37 °C. Following incubation the media was removed, 50 μL of Dimethyl sulfoxide was added to the wells, and the plates were incubated at 37 °C for 30 min. The absorbance was read at 540 nm using a microplate reader (Molecular Devices) to measure cell proliferation. Tumorsphere formation MDA-MB-231 cells were seeded at 5 × 103-2 × 104 cells/mL in low attachment plates and grown for 1-2 wk in Mammocult complete medium (Stem Cell Technologies) supplemented with heparin and hydrocortisone and treated with leptin (1.2 nmol/L), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L). Basal tumorspheres served as untreated controls. The tumorspheres were visually assessed by light microscopy. The size of the tumorspheres were determined and the number of tumorspheres were counted manually in triplicate.

one and treated with leptin (1.2 nmol/L), or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L). Basal tumorspheres served as untreated controls. The tumorspheres were visually assessed by light microscopy. The size of the tumorspheres were determined and the number of tumorspheres were counted manually in triplicate. Apoptosis assay Cells were seeded at 2 × 105 in 6 well cell culture plates and grown to 70%-80% confluence. They were treated with the chemotherapeutic drugs: Cis (Millipore), CTX, PTX, and Dox (SelleckChem) in 5% FBS with or without IONP-LPrA2 for time periods ranging from 1-6 d. Before trypsinizing, the supernatants were transferred into microfuge tubes for subsequent analysis. The trypsinized cells were added to the supernatants and centrifuged. The pellets were washed with 1 × PBS and resuspended in Annexin V binding buffer (Nexcelom). Annexin V/FITC, and PI, 5 μL each (Nexcelom) were added with mixing. The samples were incubated in the dark at room temperature for 15 min. The cells were washed with 1 × PBS, centrifuged, and resuspended in Annexin V binding buffer to a concentration of 3 × 104 cells per 20 μL. The samples were analyzed by the Cellometer Vision. The viability was determined by multiplying the percentage of live cells by the total cell count. Statistical analysis All experiments were performed in triplicate. One-way ANOVA (SigmaPlot) was used to determine statistical significance among treatment groups and controls. Data presented as the average ± standard deviation (SD). P values of P < 0.05 were considered statistically significant.

Apoptosis assay Cells were seeded at 2 × 105 in 6 well cell culture plates and grown to 70%-80% confluence. They were treated with the chemotherapeutic drugs: Cis (Millipore), CTX, PTX, and Dox (SelleckChem) in 5% FBS with or without IONP-LPrA2 for time periods ranging from 1-6 d. Before trypsinizing, the supernatants were transferred into microfuge tubes for subsequent analysis. The trypsinized cells were added to the supernatants and centrifuged. The pellets were washed with 1 × PBS and resuspended in Annexin V binding buffer (Nexcelom). Annexin V/FITC, and PI, 5 μL each (Nexcelom) were added with mixing. The samples were incubated in the dark at room temperature for 15 min. The cells were washed with 1 × PBS, centrifuged, and resuspended in Annexin V binding buffer to a concentration of 3 × 104 cells per 20 μL. The samples were analyzed by the Cellometer Vision. The viability was determined by multiplying the percentage of live cells by the total cell count. Statistical analysis All experiments were performed in triplicate. One-way ANOVA (SigmaPlot) was used to determine statistical significance among treatment groups and controls. Data presented as the average ± standard deviation (SD). P values of P < 0.05 were considered statistically significant. Biostatistics statement The statistical review was performed by Ward Kirlin, PhD. The appropriate ANOVA of variance was performed on the data presented in this paper, and levels of statistical significance are based on the F-values and Tukey’s multiple comparisons between group means as determined using SigmaPlot (Systat Software, Inc.). Mean + SDs are indicated in the graphical analysis, based on replicates of densitometry analysis of Western blots, the percentage of cells in S-phase of the cell cycle, or percentage of proliferating cells as indicated in the figures.

omparisons between group means as determined using SigmaPlot (Systat Software, Inc.). Mean + SDs are indicated in the graphical analysis, based on replicates of densitometry analysis of Western blots, the percentage of cells in S-phase of the cell cycle, or percentage of proliferating cells as indicated in the figures. RESULTS Generation and characterization of IONP-LPrA2 The leptin antagonist, LPrA2, has been shown to inhibit breast cancer growth and progression in vitro as well as in vivo[2,22,23]. To increase its efficacy, LPrA2 was conjugated to IONPs. IONPs are amphiphilic and have a 10 nm core[20]. The binding of LPrA2 to IONPs was facilitated by EDAC, which activates the carboxyl group on the IONP surface and allows the formation of an amide bond with the amino group of LPrA2 (Figure 1A). To confirm the binding of the LPrA2 peptides to the nanoparticles, the conjugates were analyzed by SDS-PAGE and Western blot. With LPrA2 antibody incubation, bands were detected at approximately 100 kD, indicating conjugated LPrA2, and approximately 3 kD indicating unbound LPrA2. Unconjugated LPrA2 and LPrA2-Sc were used as positive and negative controls, respectively (Figure 1B). To further characterize IONP-LPrA2, 1:10000 dilutions of the conjugated and unconjugated IONPs were measured by NanoSight nanoparticle tracking analysis (Malvern); in which the left and right Y-axes show particle number and percent distribution, and the X-axis displays particle size. The size of the unconjugated IONP was found to be 14 nm while conjugated IONP-LPrA2 measured 20 nm. This data suggests that the conjugation of LPrA2 to IONPs was successful.

cking analysis (Malvern); in which the left and right Y-axes show particle number and percent distribution, and the X-axis displays particle size. The size of the unconjugated IONP was found to be 14 nm while conjugated IONP-LPrA2 measured 20 nm. This data suggests that the conjugation of LPrA2 to IONPs was successful. Figure 1 Generation and characterization of IONP-LPrA2. A: Conjugation of IONP-LPrA2. LPrA2 was conjugated to IONPs via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), which activates the carboxyl group on the IONP surface allowing it to form a covalent bond with the amino group of LPrA2 (displayed by TEM, transmission electron microscopy, Ocean Nanotech); B: Western blot confirmation of IONP-LPrA2 conjugation. Conjugated IONP-LPrA2 (100 kD) was detected by Western blot with an LPrA2 antibody, purified from antigen injected rabbit bleeds. Unconjugated LPrA2 (3 kD) and the scrambled peptide LPrA2-Sc (3 kD) served as positive and negative controls, respectively; C: NanoSight analysis of unconjugated and conjugated IONPs. The particle size of unconjugated IONP (14 nm) shown in black and the conjugated IONP-LPrA2 (20 nm) shown in red were determined by nanoparticle tracking analysis. The hyperbolic curve shows that the particles are 100% homogeneous.

tive controls, respectively; C: NanoSight analysis of unconjugated and conjugated IONPs. The particle size of unconjugated IONP (14 nm) shown in black and the conjugated IONP-LPrA2 (20 nm) shown in red were determined by nanoparticle tracking analysis. The hyperbolic curve shows that the particles are 100% homogeneous. Ob-R expression and effect of IONP-LPrA2 on leptin-induced pSTAT3 and cyclin D1 levels in human breast cancer cells In order to determine the effects of IONP-LPrA2 on leptin signaling inhibition, we first had to confirm expression of the leptin receptor, Ob-R, in the human breast cancer cell lines. Immunoprecipitation and subsequent Western blot analysis showed Ob-R expression in MDA-MB-231, HCC1806, and MCF-7 cells (Figure 2A). Leptin signaling activates the JAK2/STAT3, MAPK/ERK, and PI3/Akt signaling pathways, which are implicated in its anti-apoptotic activity[9]. For this reason, we aimed to determine the effect of IONP-LPrA2 treatment on active/phosphorylated, pSTAT3. Leptin significantly increased the level of pSTAT3 in MDA-MB-231 and HCC1806 cells. IONP-LPrA2 significantly inhibited the effect of leptin on pSTAT3 levels in HCC1806 cells. No significant changes occurred in pSTAT3 levels in MCF-7 cells treated with leptin and IONP-LPrA2 (Figure 2B and C). Because leptin has been shown to increase cyclin D1 levels in breast cancer cells[14,15], we sought to determine the effect of IONP-LPrA2 treatment on cyclin D1 expression in MDA-MB-231, HCC1806, and MCF-7 breast cancer cells. Leptin significantly induced cyclin D1 expression in all cell lines (Figure 2B and C). The addition of IONP-LPrA2 significantly inhibited the effect of leptin on cyclin D1 expression in all cell lines (Figure 2B and C). These results suggest that IONP-LPrA2 abrogates the effect of leptin on leptin-induced signaling pathways.

significantly induced cyclin D1 expression in all cell lines (Figure 2B and C). The addition of IONP-LPrA2 significantly inhibited the effect of leptin on cyclin D1 expression in all cell lines (Figure 2B and C). These results suggest that IONP-LPrA2 abrogates the effect of leptin on leptin-induced signaling pathways. Figure 2 Ob-R expression and effect of IONP-LPrA2 on leptin-induced pSTAT3 and cyclin D1 levels in human breast cancer cells. A: Detection of Ob-R expression. The expression of Ob-R was detected by Western blot in MDA-MB-231, HCC1806, and MCF-7 cells; B: IONP-LPrA2 inhibition of leptin-induced pSTAT3 and cyclin D1 levels. Lysates were obtained from MDA-MB-231, HCC1806, and MCF-7 cells treated with leptin (1.2 nmol/L) or IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L) for 24-48 h. pSTAT3 and cyclin D1 levels were detected by Western blot. STAT3 served as a loading control for pSTAT3. GAPDH served as a loading control for cyclin D1; C: Densitometric analysis of pSTAT3 and cyclin D1 levels. Graphs represent quantitative analysis of pSTAT3 and cyclin D1 levels in MDA-MB-231, HCC1806, and MCF-7 cells with Image J software. Relative protein level was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative protein level in cells pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05.

C1806, and MCF-7 cells with Image J software. Relative protein level was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative protein level in cells pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05. IONP-LPrA2 inhibits leptin-induced cell cycle progression of human breast cancer cell lines Leptin has been shown to increase expression of the cell cycle associated protein, cyclin D1[14,15]. To illustrate the effect of leptin on cell cycle progression, the number of cells in the S phase was determined by cell cycle analysis with the Cellometer Vision (Nexcelom). MDA-MB-231, HCC1806, and MCF-7 human breast cancer cells lines were treated with leptin (1.2 nmol/L) and IONP-LPrA2 plus leptin in order to determine its antagonistic effect. The cells were treated with IONP-LPrA2 concentrations ranging from 0.0018-0.036 pmol/L. MDA-MB-231 and HCC1806 TNBC cell lines were treated for 24 h while the ER+ MCF-7 cells were treated for 48 h to produce an effect. Treatment with leptin caused a significant increase in cell cycle progression in HCC1806 (Figure 3B) and MCF-7 (Figure 3C), but had no significant effect on MDA-MB-231 cells (Figure 3A). Treatment with IONP-LPrA2 plus leptin abrogated leptin-induced cell cycle progression at 0.0018-0.0036 pmol/L in MDA-MB-231, at 0.0018-0.036 pmol/L in HCC1806, and at 0.0018-0.0072 in MCF-7 cells (Figure 3). This data elucidated the effective dilution of IONP-LPrA2 for abrogation of leptin-induced cell cycle progression in each of the cell lines.

us leptin abrogated leptin-induced cell cycle progression at 0.0018-0.0036 pmol/L in MDA-MB-231, at 0.0018-0.036 pmol/L in HCC1806, and at 0.0018-0.0072 in MCF-7 cells (Figure 3). This data elucidated the effective dilution of IONP-LPrA2 for abrogation of leptin-induced cell cycle progression in each of the cell lines. Figure 3 IONP-LPrA2 inhibits leptin-induced cell cycle progression of human breast cancer cell lines. IONP-LPrA2 inhibits S phase progression in breast cancer cells. A: MDA-MB-231; B: HCC1806; C: MCF-7. The cells were seeded in 6 well plates and treated with leptin (1.2 nmol/L), LPrA2 (1.2 nmol/L) plus leptin (1.2 nmol/L), or IONP-LPrA2 at indicated concentrations plus leptin (1.2 nmol/L) for 24-48 h. The percentage of cells in S phase was determined by cell cycle analysis, a measure of propidium iodide (PI) fluorescence. Relative percentage of cells in S phase was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative percentage of cells in S phase pretreated with leptin antagonists and then leptin differed significantly from those treated with leptin alone, cP < 0.05.

luorescence. Relative percentage of cells in S phase was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative percentage of cells in S phase pretreated with leptin antagonists and then leptin differed significantly from those treated with leptin alone, cP < 0.05. IONP-LPrA2 inhibits leptin-induced cell proliferation in human breast cancer cells Leptin signaling stimulates breast cancer cell survival and proliferation[8]. To ascertain the manner in which IONP-LPrA2 affects cell proliferation, an MTT assay was performed. MDA-MB-231, HCC1806, and MCF-7 cell were treated with leptin (1.2 nmol/L) and IONP-LPrA2 (0.0036) plus leptin (1.2 nmol/L). Leptin treatment significantly increased cell proliferation and IONP-LPrA2 significantly diminished the effect of leptin in all of the cell lines (Figure 4). This data indicates that IONP-LPrA2 prevents leptin induction of cell proliferation. Figure 4 IONP-LPrA2 inhibits leptin-induced cell proliferation in human breast cancer cells. MDA-MB-231, HCC1806, and MCF-7 cells were seeded in 96 well plates and treated with leptin (1.2 nmol/L) and IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L) for 24-48 h. Cell proliferation was determined by MTT assay. Relative percentage of proliferating cells was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative percentage of proliferating cells pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05.

by MTT assay. Relative percentage of proliferating cells was significantly increased in leptin treated cell lines compared to basal (untreated) cells, aP < 0.05. Relative percentage of proliferating cells pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05. IONP-LPrA2 decreases MDA-MB-231 tumorsphere formation Self-renewal is a hallmark of cancer. Leptin has been shown to increase self-renewal and breast cancer stem cell (BCSC) growth[24]. To learn how IONP-LPrA2 affects BCSC growth, tumorsphere formation was assessed. MDA-MB-231 TNBC cells were treated with leptin (1.2 nmol/L) and IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L). Untreated, basal, MDA-MB-231 cells developed few small and medium tumorspheres (100-200 μm), cells treated with leptin showed a significant increase in the development of medium (200 μm) and large tumorspheres (> 200 μm) in comparison to basal. Cells treated with IONP-LPrA2 plus leptin displayed a significant decrease in medium tumorsphere growth relative to the leptin treated (Figure 5). This data shows that IONP-LPrA2 treatment may decrease BCSC growth.

increase in the development of medium (200 μm) and large tumorspheres (> 200 μm) in comparison to basal. Cells treated with IONP-LPrA2 plus leptin displayed a significant decrease in medium tumorsphere growth relative to the leptin treated (Figure 5). This data shows that IONP-LPrA2 treatment may decrease BCSC growth. Figure 5 IONP-LPrA2 decreases MDA-MB-231 tumorsphere formation. A: IONP-LPrA2 attenuation of leptin-induced tumorsphere formation. MDA-MB-231 cells were grown in low attachment plates with mammocult medium for 1-2 wk, under treatment with leptin (1.2 nmol/L) and IONP-LPrA2 (0.0036 pmol/L) plus leptin (1.2 nmol/L). Tumorspheres were counted. Tumorspheres were grouped according to size: Small (< 100 μm), medium (100-200 μm) and large (> 200 μm); B: Effect of leptin and IONP-LPrA2 on number and size of tumorspheres. Graph represents quantitative analysis of small, medium, and large tumorspheres in response to leptin and IONP-LPrA2 treatment. The number of colonies was significantly increased in leptin treated cells compared to basal (untreated) cells, aP < 0.05. The number of colonies pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05.

umorspheres in response to leptin and IONP-LPrA2 treatment. The number of colonies was significantly increased in leptin treated cells compared to basal (untreated) cells, aP < 0.05. The number of colonies pretreated with IONP-LPrA2 and then leptin differed significantly from those treated with leptin alone, cP < 0.05. The effect of chemotherapeutics on survival of breast cancer cell lines Chemotherapy is among the most common treatments for breast cancer in addition to radiation and surgery[25]. To determine the effective dose of chemotherapeutics, cells were treated with a panel of anti-cancer drugs and viability was tested by the Annexin V FITC/PI Assay (Nexcelom). MDA-MB-231, HCC1806, and MCF-7 cells were treated with Cis (0.001-1.1 μmol/L), CTX (0.01-100 μmol/L), Dox (0.01-50 μmol/L), and PTX (0.05-1.0 μmol/L) for time periods ranging from 1-6 d to determine an effective dose to reduce cell viability (Figure 6). Cis and Dox reduced cell viability in 24 h while CTX and PTX treated cells required up to 6 d to produce an effect. All cell lines displayed a similar response to Cis and PTX (Figure 6A and D). MDA-MB-231 cells appeared to be more sensitive to CTX and Dox (Figure 6B and C).

ive dose to reduce cell viability (Figure 6). Cis and Dox reduced cell viability in 24 h while CTX and PTX treated cells required up to 6 d to produce an effect. All cell lines displayed a similar response to Cis and PTX (Figure 6A and D). MDA-MB-231 cells appeared to be more sensitive to CTX and Dox (Figure 6B and C). Figure 6 The effect of chemotherapeutics on survival of breast cancer cell lines. The effective dose of the chemotherapeutics. A: Cisplatin (Cis); B: Cyclophosphamide (CTX); C: Doxorubicin (Dox); D: Paclitaxel (PTX) were determined in MDA-MB-231, HCC1806, and MCF-7 cells. The cells were seeded in 6 well plates and treated with Cis (0.001-1.1 μmol/L), CTX (0.01-100 μmol/L), Dox (0.01-50 μmol/L), and PTX (0.05-1.0 μmol/L) for 1-6 d. Percent survival was determined by the Annexin V/FITC and PI assay. The relative survival was determined by multiplying the percentage of live cells by the total cell count.

seeded in 6 well plates and treated with Cis (0.001-1.1 μmol/L), CTX (0.01-100 μmol/L), Dox (0.01-50 μmol/L), and PTX (0.05-1.0 μmol/L) for 1-6 d. Percent survival was determined by the Annexin V/FITC and PI assay. The relative survival was determined by multiplying the percentage of live cells by the total cell count. Determination of the effect of IONP-LPrA2 on survival of breast cancer cells treated with chemotherapeutics Chemotherapy has many detrimental side effects; because of this it is advantageous to utilize adjuvant therapies in order to reduce the effective dose. To determine the adjuvant potential of IONP-LPrA2, cells were treated with chemotherapeutics combined with IONP-LPrA2 and analyzed for viability by the Annexin V FITC/PI Assay (Nexcelom). MDA-MB-231, HCC1806, and MCF-7 cells were treated with chemotherapeutics at concentrations determined in Figure 6 in media containing 5% FBS to mimic physiological leptin levels, in addition to IONP-LPrA2 (0.0036 pmol/L) for time periods ranging from 1-6 d. The treatment concentrations were MDA-MB-231 (Cis 0.001 μmol/L, CTX 0.5 μmol/L, Dox 0.4 μmol/L, PTX 0.5 μmol/L); HCC1806 (Cis 0.036 μmol/L, CTX 1 μmol/L, Dox 10 μmol/L, PTX 0.5 μmol/L); and MCF-7 (Cis 0.036 μmol/L, CTX 5 μmol/L, Dox 0.01 μmol/L, PTX 1 μmol/L). MDA-MB-231 TNBC cells treated with IONP-LPrA2 displayed a significant decrease in viable cells when dosed with Cis and CTX (Figure 7A and B). HCC1806 TNBC cells treated with IONP-LPrA2 showed a significant reduction in viable cells when dosed with Cis and Dox (Figure 7A and C). ER+ MCF-7 cells treated with IONP-LPrA2 did not show a significant decrease in viable cells when treated with chemotherapeutics (Figure 7). Although cells were treated with PTX for up to 6 d to reduce cell viability, IONP-LPrA2 showed no additional decrease in viability when combined with PTX (Figure 7D). PTX is an anti-microtubule agent which acts on the M phase of the cell cycle while the other chemotherapeutics act on DNA which affects the S phase[2]. This data suggests that IONP-LPrA2 increases the potency of chemotherapeutics on TNBC cells, particularly anti-cancer drugs which target DNA.

ined with PTX (Figure 7D). PTX is an anti-microtubule agent which acts on the M phase of the cell cycle while the other chemotherapeutics act on DNA which affects the S phase[2]. This data suggests that IONP-LPrA2 increases the potency of chemotherapeutics on TNBC cells, particularly anti-cancer drugs which target DNA. Figure 7 Determination of the effect of IONP-LPrA2 on survival of breast cancer cells treated with chemotherapeutics. MDA-MB-231, HCC1806, and MCF-7 cells were treated with an effective dose of the chemotherapeutics. A: Cisplatin (Cis); B: Cyclophosphamide (CTX); C: Doxorubicin (Dox); D: Paclitaxel (PTX) plus IONP-LPrA2 (0.0036 pmol/L). The cells were seeded in 6 well plates and treated with chemotherapeutics at effective concentrations determined in Figure 6 for 1-6 d. The treatment concentrations were MDA-MB-231 (Cis 0.001 μmol/L, CTX 0.5 μmol/L, Dox 0.4 μmol/L, PTX 0.5 μmol/L); HCC1806 (Cis 0.036 μmol/L, CTX 1 μmol/L, Dox 10 μmol/L, PTX 0.5 μmol/L); and MCF-7 (Cis 0.036 μmol/L, CTX 5 μmol/L, Dox 0.01 μmol/L, PTX 1 μmol/L). Percent of survival was determined by the Annexin V/FITC and PI assay. The relative survival was determined by multiplying the percentage of live cells by the total cell count. Percent viability was significantly decreased in cells treated with chemotherapeutic compared to basal (untreated) cells, aP < 0.05. Cells treated with chemotherapeutic and IONP-LPrA2 differed significantly from those treated with chemotherapeutic alone, cP < 0.05.

the percentage of live cells by the total cell count. Percent viability was significantly decreased in cells treated with chemotherapeutic compared to basal (untreated) cells, aP < 0.05. Cells treated with chemotherapeutic and IONP-LPrA2 differed significantly from those treated with chemotherapeutic alone, cP < 0.05. DISCUSSION In spite of methods for early detection of breast cancer, it remains the second leading cause of cancer deaths in women in the United States[1]. TNBC is a subtype of breast cancer characterized by the lack of hormone receptor expression. The absence of hormone receptors makes this more aggressive form of breast cancer even more difficult to treat. Obesity is often associated with poorer outcomes in individuals with breast cancer, particularly those with TNBC[25]. Obesity is characterized by an excess of the inflammatory cytokine, leptin. Elevated leptin levels display a significant correlation with metastasis and lower breast cancer patient survival[26]. The leptin antagonist, LPrA2 has been shown to inhibit leptin signaling in breast and other cancer types, but the actions of LPrA2 are restricted by its low MW of < 3 kD, short half-life, and insolubility in water[8,27]. IONP-LPrA2 was developed to circumvent these limitations. IONPs conjugated to other peptides, such as the amino terminal fragment of urokinase type plasminogen activator (ATF-uPA) are stable for more than 48 h in in vivo imaging experiments[20]. IONPs are amphiphilic, small (10 nm core size), and uniformly sized to facilitate delivery which prevents phagocytosis[28]. The characteristics of IONPs make them an ideal delivery system for LPrA2 to target and treat breast cancer. In the present study, IONP-LPrA2 was used to evaluate its ability to inhibit leptin signaling in human breast cancer cells. The data indicates that IONP-LPrA2 abrogates cell cycle progression and acts as an adjuvant when administered with chemotherapeutics.

l delivery system for LPrA2 to target and treat breast cancer. In the present study, IONP-LPrA2 was used to evaluate its ability to inhibit leptin signaling in human breast cancer cells. The data indicates that IONP-LPrA2 abrogates cell cycle progression and acts as an adjuvant when administered with chemotherapeutics. Decreased levels of pSTAT3 and cyclin D1 with IONP-LPrA2 treatment were shown by Western blot. Cyclin D1 is a cell cycle regulatory gene. STAT3 is a transcription factor responsible for the regulation of cyclin D1[10]. Decreased levels of pSTAT3 with IONP-LPrA2 treatment were seen at time points as early as 5-15 min post treatment. Previous studies have shown that leptin is mitogenic and increases cyclin D1 in ER+ MCF-7 breast cancer cells[14,15]. Because leptin increases cyclin D1 and IONP-LPrA2 inhibits the effect of leptin, utilizing agents that target cyclin D1 may be a plausible method to treat breast cancer. In this study, we have shown that IONP-LPrA2 decreases pSTAT3 and cyclin D1. The decreased levels of these leptin-induced targets may inhibit cell cycle progression in ER+ MCF-7 cells as well as MDA-MB-231 and HCC1806 TNBC cells.

, utilizing agents that target cyclin D1 may be a plausible method to treat breast cancer. In this study, we have shown that IONP-LPrA2 decreases pSTAT3 and cyclin D1. The decreased levels of these leptin-induced targets may inhibit cell cycle progression in ER+ MCF-7 cells as well as MDA-MB-231 and HCC1806 TNBC cells. Inhibition of cell cycle progression by IONP-LPrA2 was displayed by image based cytometry. Leptin has been shown to increase levels of cyclin D1[14,15]. In this study, we show that IONP-LPrA2 decreases cyclin D1 expression, but the effect on cell cycle progression was yet to be determined. Here we show that IONP-LPrA2 treatment decreases the percentage of cells in the S phase of the cell cycle, where DNA is synthesized, as or more effectively than LPrA2 alone. Interestingly, the greatest decrease in the percentage of cells in S phase with IONP-LPrA2 treatment was seen in HCC1806 TNBC cells derived from a non-metastatic squamous cell carcinoma in contrast to MCF-7 and MDA-MB-231 cells derived from metastatic adenocarcinomas. This data suggests that IONP-LPrA2 inhibition of cell cycle progression may reduce the advancement of breast cancer, and may be particularly beneficial in the treatment of non-metastatic and squamous cell carcinomas.

amous cell carcinoma in contrast to MCF-7 and MDA-MB-231 cells derived from metastatic adenocarcinomas. This data suggests that IONP-LPrA2 inhibition of cell cycle progression may reduce the advancement of breast cancer, and may be particularly beneficial in the treatment of non-metastatic and squamous cell carcinomas. Chemotherapy is the first line of treatment for TNBC. Although TNBC is generally more responsive to chemotherapy than other forms of breast cancer, there is an increased risk of developing drug resistance[29]. BCSC growth and self-renewal play an important role in breast cancer drug resistance and leptin increases the risk[24]. These cells express molecular markers for breast cancer, CD44+CD24-/ALDH+[10]. We have demonstrated that leptin induces in vitro BCSC, tumorsphere, formation and treatment with IONP-LPrA2 attenuates the effect of leptin in MDA-MB-231 TNBC cells. These results indicate that IONP-LPrA2 prevents BCSC formation and may decrease chemoresistance in TNBC.

markers for breast cancer, CD44+CD24-/ALDH+[10]. We have demonstrated that leptin induces in vitro BCSC, tumorsphere, formation and treatment with IONP-LPrA2 attenuates the effect of leptin in MDA-MB-231 TNBC cells. These results indicate that IONP-LPrA2 prevents BCSC formation and may decrease chemoresistance in TNBC. Chemotherapeutic treatment of breast cancer is plagued with high toxicity. Toxic side effects and the development of drug resistance are cause for the development of adjuvant therapies. The need for adjuvant therapies is exacerbated in TNBC patients who often experience relapse and develop resistance to chemotherapy[29]. TNBC is commonly treated with combination chemotherapy[25]. Here, we treated breast cancer cells with a panel of commonly used chemotherapeutics (Cis, CTX, Dox and PTX) in addition to IONP-LPrA2 to test its ability to decrease cell viability more than the drugs alone. We demonstrated that TNBC cells, MDA-MB-231 displayed a significant decrease in viability with Cis and CTX plus IONP-LPrA2; and HCC1806 showed a significant reduction in live cells when treated with Cis and Dox plus IONP-LPrA2. ER+ MCF-7 cells treated with chemotherapeutics plus IONP-LPrA2 did not show a significant decrease in viable cells. Also, there was no significant decrease in viability in the cells treated with PTX plus IONP-LPrA2. This may be due, in part, to PTX’s anti-microtubule action, which affects the M phase of the cell cycle[25]. Cis, CTX, and Dox act on DNA which affects the S phase[25]. These drugs may work synergistically with IONP-LPrA2, which also appears to act on the S phase. These data indicate that IONP-LPrA2 may act as a chemotherapeutic adjuvant by decreasing viability, thereby decreasing the effective dose in TNBC.

e cell cycle[25]. Cis, CTX, and Dox act on DNA which affects the S phase[25]. These drugs may work synergistically with IONP-LPrA2, which also appears to act on the S phase. These data indicate that IONP-LPrA2 may act as a chemotherapeutic adjuvant by decreasing viability, thereby decreasing the effective dose in TNBC. In conclusion, IONP-LPrA2 was found to decrease the level of leptin-induced targets pSTAT3 and cyclin D1. IONP-LPrA2 decreased DNA synthesis during the S phase of the cell cycle and reduced proliferation in both ER+ and TNBC cells. When combined with chemotherapeutics, particularly drugs targeting the S phase, IONP-LPrA2 showed an additive effect on the reduction of live breast cancer cells. These findings indicate that IONP-LPrA2 may be useful in the prevention of tumor cell growth and proliferation in breast cancer. Further, treatment with IONP-LPrA2 may allow for lower chemotherapeutic dosing. These results are potentially beneficial for obese patients with elevated leptin levels, whom have a higher incidence and thus poorer outcome of TNBC. Taken together, the present data provides confirmation of our hypothesis that IONP-LPrA2 treatment may be useful in impairing tumor growth and when given in combination with the indicated chemotherapeutics has the potential to increase drug effectiveness. These data indicate that there is a synergistic effect with IONP-LPrA2 and chemotherapeutics which affect the S phase of the cell cycle in vitro.

P-LPrA2 treatment may be useful in impairing tumor growth and when given in combination with the indicated chemotherapeutics has the potential to increase drug effectiveness. These data indicate that there is a synergistic effect with IONP-LPrA2 and chemotherapeutics which affect the S phase of the cell cycle in vitro. ACKNOWLEDGMENTS The authors warmly thank Dr. Ming Bo Huang for facilitating the nanoparticle tracking analysis. We also thank Mr. Patrick Abramson and Ms. Aria Armstrong for aesthetic assistance with diagrams and figures. COMMENTS Background Obesity and high leptin levels are strongly associated with breast cancer relapse, drug resistance, and poorer patient outcomes. Overexpression of leptin and its receptor, Ob-R, induce breast cancer cell growth and proliferation. Triple negative breast cancer (TNBC) is a subtype of breast cancer which comprises approximately 15% of cases and is an aggressive form of the disease with no targeted therapy. TNBC chemotherapeutic treatment often leads to chemoresistance and shows several undesirable side effects. Leptin is proliferative and is a survival factor for breast cancer treated with chemotherapeutics. Therefore, the authors have developed a leptin peptide receptor antagonist coupled to IONP-LPrA2, which successfully inhibits leptin signaling as well as increases chemotherapeutic effectiveness in breast cancer and is particularly promising for TNBC treatment.

l factor for breast cancer treated with chemotherapeutics. Therefore, the authors have developed a leptin peptide receptor antagonist coupled to IONP-LPrA2, which successfully inhibits leptin signaling as well as increases chemotherapeutic effectiveness in breast cancer and is particularly promising for TNBC treatment. Research frontiers IONP-LPrA2 could be a new and effective biological for blocking pro-oncogenic and drug resistance effects of leptin in breast cancer, especially in obese patients suffering from TNBC that are treated with chemotherapeutics. Innovations and breakthroughs This study describes for the first time the production and characterization of a new biological bound to nanoparticles that can effectively block leptin signaling inducing proliferation and survival in breast cancer cells treated with chemotherapeutics. Applications In recent years, IONPs have become an important tool for biomedical applications. The use IONPs has been employed in vaccinations, drug delivery, MRI, and molecular imaging. The authors’ data suggests combining IONPs with the leptin antagonist, LPrA2, prevents the growth of breast cancer cells and acts as a chemotherapeutic adjuvant by reducing the effective dose.

t tool for biomedical applications. The use IONPs has been employed in vaccinations, drug delivery, MRI, and molecular imaging. The authors’ data suggests combining IONPs with the leptin antagonist, LPrA2, prevents the growth of breast cancer cells and acts as a chemotherapeutic adjuvant by reducing the effective dose. Terminology Leptin signaling occurs when the hormone is secreted by the adipose tissue and binds to its receptor, Ob-R. Breast cancer, particularly in obese individuals, is associated with high levels of leptin. Leptin signaling leads to increased breast cancer cell growth, proliferation and drug resistance. The inhibition of leptin signaling with the nanoparticle-linked leptin antagonist, IONP-LPrA2, provides a promising new way to improve breast cancer chemotherapy. Peer-review This manuscript provides useful information to the medical students, clinicians, and researchers in this field, therefore, is acceptable for publication. Supported by The National Cancer Institute at the National Institutes of Health (1R41 CA183399-01A1 to Ruben R Gonzalez-Perez; 5U54 CA118638, S21 MD000101, 5G12 MD0076021, G12 RR026250-03, NIH RR03034 and 1C06 RR18386 to Morehouse School of Medicine); the National Institute of General Medical Sciences, Research Initiative for Scientific Enhancement Program (RISE 5R25 GM058268 to Tia Harmon); and the Congressionally Directed Medical Research Programs-Department of Defense (CDMRP DOD W81XWH-13-1-0382 to Ruben R Gonzalez-Perez). Informed consent statement: N/A.

Supported by The National Cancer Institute at the National Institutes of Health (1R41 CA183399-01A1 to Ruben R Gonzalez-Perez; 5U54 CA118638, S21 MD000101, 5G12 MD0076021, G12 RR026250-03, NIH RR03034 and 1C06 RR18386 to Morehouse School of Medicine); the National Institute of General Medical Sciences, Research Initiative for Scientific Enhancement Program (RISE 5R25 GM058268 to Tia Harmon); and the Congressionally Directed Medical Research Programs-Department of Defense (CDMRP DOD W81XWH-13-1-0382 to Ruben R Gonzalez-Perez). Informed consent statement: N/A. Conflict-of-interest statement: The authors of this manuscript indicate that there are no known conflicts of interest. Data sharing statement: None. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: August 29, 2016 First decision: November 14, 2016 Article in press: December 28, 2016 P- Reviewer: Sonoda K, Zhang XQ S- Editor: Ji FF L- Editor: A E- Editor: Wu HL

Core tip: Lack of adherence to analgesia for cancer pain is a prevalent clinical problem. The 2016 Centers for Disease Control and Prevention guidelines provide recommendations to clinicians for opioid prescription. However, this focus will be incomplete without understanding what concerns anchor patients’ decisions to use analgesia for cancer pain. We used a trade-off analysis technique and novel adaptive methods to first show that unique clusters of patients exist based on the main concerns that anchor their preferences for analgesia for cancer pain. We then identified factors that predict membership in each preference cluster. We found that socioeconomic factors, including education, health literacy, income (rather than attitudes and beliefs about analgesics) played a role in predicting three out of four clusters. Most analgesic beliefs and concerns, including the widely indicated addiction concerns, did not predict cluster membership.

ster. We found that socioeconomic factors, including education, health literacy, income (rather than attitudes and beliefs about analgesics) played a role in predicting three out of four clusters. Most analgesic beliefs and concerns, including the widely indicated addiction concerns, did not predict cluster membership. INTRODUCTION In the early part of 2016, the Centers for Disease Control and Prevention (CDC) released guidelines for prescribing opioids in chronic pain, including cancer pain beyond active cancer treatment[1]. While the guidelines are shaping a conversation and debate among professionals and policy makers on opioid prescription[2-4], little is known about the other side of the coin-patients’ preferences that shape their analgesic taking behaviors. Cancer pain in the United States is mainly managed using analgesics[5]. Non-pharmacological pain treatment approaches are either not consistently offered to patients by their clinicians/covered by health insurance or lack data on clinical effectiveness[6-10]. For the treatments that have demonstrated clinical effectiveness, the cost burden for the patients may be excessive[11,12]. Thus, clinicians and oncologists rely on analgesics as well as opioid medications to help patients whose daily lives and function are affected by significant pain[11]. Unfortunately, patients with unrelieved chronic pain have some of the lowest quality of life observed for any medical condition[13].

e excessive[11,12]. Thus, clinicians and oncologists rely on analgesics as well as opioid medications to help patients whose daily lives and function are affected by significant pain[11]. Unfortunately, patients with unrelieved chronic pain have some of the lowest quality of life observed for any medical condition[13]. Despite widespread use of analgesics in managing cancer pain, there is serious paucity of literature to understand the heuristics cancer patients may employ in making decisions to use analgesics. The few extant studies had methodological aims, that is to investigate the predictive validity of a trade-off analysis technique in eliciting analgesic preferences with diverse subgroups of patients with cancer pain[14]. Others investigating analgesic trade-offs included patients with cancer as part of the broader category of chronic pain sufferers[15]. Also, to our knowledge, no studies have investigated the sociodemographic and clinical predictors of patients’ analgesic preferences. Thus, the purpose of this study was to investigate if unique clusters exist with regard to cancer patients’ preference to use analgesics for cancer pain and factors predicting cluster membership.

our knowledge, no studies have investigated the sociodemographic and clinical predictors of patients’ analgesic preferences. Thus, the purpose of this study was to investigate if unique clusters exist with regard to cancer patients’ preference to use analgesics for cancer pain and factors predicting cluster membership. MATERIALS AND METHODS This was a prospective study conducted with a cohort of adult (18 years or older) patients who were diagnosed with solid tumors or multiple myelomas and had at least one prescription of around-the-clock pain medication for cancer or cancer-treatment-related pain. Patients were self-identified African-Americans and Whites and were recruited from two outpatient medical oncology clinics within a large health system in Philadelphia, United States. Data were collected at baseline and at 3-mo. This study was approved by the Institutional Review Board of the University of Pennsylvania. All patients provided written informed consent.

nd Whites and were recruited from two outpatient medical oncology clinics within a large health system in Philadelphia, United States. Data were collected at baseline and at 3-mo. This study was approved by the Institutional Review Board of the University of Pennsylvania. All patients provided written informed consent. Measures Analgesic concern: Analgesic preferences (utilities) for cancer pain was derived from a choice-based conjoint (CBC) analysis experiment, which is a valuation technique based on the Random Utility Theory[16] and mathematical psychology[17]. The goal of CBC is to elicit what people value and what really drives them to choose one set of alternatives over another when facing competing choices[18]. CBC proposes that the overall utility or desirability of any good can be described based on the value of its separate, but, conjoined parts[19], which are termed “attributes”. Each attribute may have multiple levels. Individuals are asked to make trade-offs between attributes and attribute levels generating a unique set of values called part-worth utilities. A higher part-worth utility represents a higher level of value or importance individuals assign to that attribute. The design of CBC experiments is tailored based on the needs of an individual study.

bility to monitor illness symptoms. The response range is from 0 (do not agree) to 5 (agree very much). The scores are based on sums for items for the total scale and four subscales (physiological, fatalism, communication, and harmful effects). The internal consistency reliability of the scale is excellent at 0.89[20]. Analgesic side-effects: Side-effects resulting from taking analgesics were assessed using the Medication Side-effects Checklist (MSEC). MSEC elicits information on the presence and severity of eight common analgesic side-effects (i.e., constipation, drowsiness, nausea, vomiting, confusion, dry mouth, stomach irritation, itching) on a scale of 0-10 (no severity-extreme severity). The internal consistency reliability is 0.80[21]. Pain severity and pain-related function: The Brief Pain Inventory (BPI) was used to assess pain severity. The BPI has two subscales; pain intensity (4-items) and pain-related functional interference (7-items: General activity, mood, walking ability, normal work, relationships, sleep and enjoyment of life)[22]. Each item is scored on a 0-10 scale (0 = no pain and 10 = pain as bad as you can imagine; and 0 = no interference and 10). The psychometric properties of the BPI are well-established with cancer patients with a Cronbach’s alpha that ranges from 0.77 to 0.91[23,24].

work, relationships, sleep and enjoyment of life)[22]. Each item is scored on a 0-10 scale (0 = no pain and 10 = pain as bad as you can imagine; and 0 = no interference and 10). The psychometric properties of the BPI are well-established with cancer patients with a Cronbach’s alpha that ranges from 0.77 to 0.91[23,24]. Pain management index: Pain management index (PMI) is a measure of adequacy of pain treatment based on the World Health Organization’s (WHO) guidelines for managing cancer-related pain[25,26]. The measure takes into account the most potent analgesic prescribed to patients relative to the level of their reported pain. PMI is calculated by subtracting patient’s “pain worst” score (from BPI coded as mild, moderate, or severe) from the most potent analgesia prescribed based on the 3-step WHO analgesic ladder. A negative PMI means inadequate analgesic prescription relative to the pain level. Social support questionnaire: A 6-item instrument was used to measure participants’ perceptions of social support and satisfaction with social support[27]. The first part of the question asks participants to list individuals who provide social support and the second part asks them to indicate the level of satisfaction with this support. This questionnaire is an abridged version of the original 27-item Social Support Questionnaire[27].

satisfaction with social support[27]. The first part of the question asks participants to list individuals who provide social support and the second part asks them to indicate the level of satisfaction with this support. This questionnaire is an abridged version of the original 27-item Social Support Questionnaire[27]. Prescribed analgesics: Prescribed analgesics were coded according to the WHO analgesic ladder[25,26]. This included step 1 (non-opioid analgesics); step 2 (weak opioid analgesics such as codeine); and step 3 (strong opioids such as morphine, oxycodone, methadone). Sociodemographic and clinical variables: Sociodemographic data were gathered on age, gender, self-identified race, marital status, education, health insurance, household income, job status and health literacy. Health literacy was assessed using three brief screening questions that were previously validated[28] and performs well against the widely used Test of Functional Health Literacy in Adults[28]. The brief questions were also found to be effective in identifying inadequate health literacy (areas under the receiver operating characteristic curve of 0.87, 0.80 and 0.76, respectively for the three questions).

validated[28] and performs well against the widely used Test of Functional Health Literacy in Adults[28]. The brief questions were also found to be effective in identifying inadequate health literacy (areas under the receiver operating characteristic curve of 0.87, 0.80 and 0.76, respectively for the three questions). Clinical variables (collected from patients’ medical records) included stage of cancer, time since cancer diagnosis, past history of drug or substance abuse, comorbidities to compute the Charlson Comorbidity Index[29], presence of chronic kidney disease, and presence of depression. Pain and treatment related variables included total number and types of analgesics and co-analgesics, most potent analgesic prescribed, hours pain medications are effective, and pain relief with analgesics.

es to compute the Charlson Comorbidity Index[29], presence of chronic kidney disease, and presence of depression. Pain and treatment related variables included total number and types of analgesics and co-analgesics, most potent analgesic prescribed, hours pain medications are effective, and pain relief with analgesics. Statistical analysis Descriptive statistics were generated for available baseline variables. A wide variety of variables were considered within the four categories of sociodemographic; illness; pain, function and pain treatment; and analgesic attitudes and barriers. Patients were clustered on their responses to the five analgesic attributes determined by the CBC analysis using the adaptive statistical methods of Knafl et al[30]. A variety of clustering procedures and numbers of clusters were considered, but restricted to alternatives with each cluster containing at least 10% of the patients, thereby avoiding sparse clusters. A clustering alternative was selected using likelihood cross-validation (LCV) scores with likelihoods based on mixtures of multivariate normal distributions as commonly used in cluster analysis. Models were evaluated and compared using 10-fold LCV scores. These were computed by first randomly partitioning the data into 10 disjoint subsets, called folds. Likelihoods were then computed for the data in each fold using parameter estimates computed from the data in the other folds. These deleted fold likelihoods were combined over all the folds into a LCV score.

LCV scores. These were computed by first randomly partitioning the data into 10 disjoint subsets, called folds. Likelihoods were then computed for the data in each fold using parameter estimates computed from the data in the other folds. These deleted fold likelihoods were combined over all the folds into a LCV score. A larger LCV score indicates a better model for the data but not necessarily a distinctly better model. This issue was addressed using LCV ratio tests, based on the χ2 distribution (and so analogous to standard likelihood ratio tests). These tests were expressed in terms of a threshold for a distinct (or substantial or significant) percent change in the LCV scores. A percent decrease larger than the threshold indicates that the model with the larger LCV score provides a distinct improvement over the model with the smaller score. Otherwise, the model with the smaller score is a competitive alternative, and if also simpler then preferable as a parsimonious, competitive alternative. The threshold changes with the sample size.

indicates that the model with the larger LCV score provides a distinct improvement over the model with the smaller score. Otherwise, the model with the smaller score is a competitive alternative, and if also simpler then preferable as a parsimonious, competitive alternative. The threshold changes with the sample size. The indicators for being in each of the CBC clusters were modeled separately using logistic regression. This approach allows for identification of a different set of predictors for each cluster and so was considered preferable to multinomial regression modeling of membership in all four clusters combined since that would use the same predictors for all clusters. Each available baseline variable was used to adaptively identify an associated binary characteristic for predicting being in a CBC cluster by dichotomizing the associated variable’s values and choosing the dichotomization that maximized the LCV score (with likelihoods based on the Bernouilli distribution as appropriate for logistic regression). Only dichotomizations with both sets of values having at least 10% of the data were considered to avoid sparse cases. The binary characteristic was defined using the indicator variable with value 1 for the set of values generating an odds ratio (OR) > 1. This indicator was conservatively set to 0 for missing variable values if there were any. The total BQ-II along with each of its subscales and items were considered as predictors to provide a broad assessment of the impact of analgesic attributes and barriers on the analgesic preferences (CBC types or clusters).

) > 1. This indicator was conservatively set to 0 for missing variable values if there were any. The total BQ-II along with each of its subscales and items were considered as predictors to provide a broad assessment of the impact of analgesic attributes and barriers on the analgesic preferences (CBC types or clusters). Dichotomization can sometimes result in loss of predictive capability compared to using the associated variable as an unadjusted predictor. This can be assessed for ordinal and continuous variables by comparing LCV scores for models based on those variables to the models based on the associate binary characteristics, but only when there are no missing values. LCV ratio tests can be used to assess whether binary characteristics provide a distinct improvement or not by comparing their LCV scores to the score for the constant model (i.e., with only an intercept).

e variables to the models based on the associate binary characteristics, but only when there are no missing values. LCV ratio tests can be used to assess whether binary characteristics provide a distinct improvement or not by comparing their LCV scores to the score for the constant model (i.e., with only an intercept). An adaptive multiple binary characteristics model was generated for each CBC-cluster indicator based on the binary characteristics that were individually significantly (P < 0.05) related to it in bivariate models using standard Wald χ2 tests. The adaptive modeling process[31] is based on a heuristic search guided by LCV scores through alternative models. First, the model is systematically expanded adding in predictors, in this case binary characteristics, to the model. The expanded model is then contracted to remove extraneous predictors. LCV ratio tests are used to decide when to stop the contraction, leaving the adaptively generated model. This modeling process is implemented in a SAS® (SAS Institute Inc., Cary, NC) macro available upon request from G. Knafl. All results were computed in SAS Version 9.4. Biostatistics statement The statistical methods of this study were reviewed by Dr. George Knafl, Biostatistician and Professor in the School of Nursing at the University of North Carolina at Chapel Hill.

An adaptive multiple binary characteristics model was generated for each CBC-cluster indicator based on the binary characteristics that were individually significantly (P < 0.05) related to it in bivariate models using standard Wald χ2 tests. The adaptive modeling process[31] is based on a heuristic search guided by LCV scores through alternative models. First, the model is systematically expanded adding in predictors, in this case binary characteristics, to the model. The expanded model is then contracted to remove extraneous predictors. LCV ratio tests are used to decide when to stop the contraction, leaving the adaptively generated model. This modeling process is implemented in a SAS® (SAS Institute Inc., Cary, NC) macro available upon request from G. Knafl. All results were computed in SAS Version 9.4. Biostatistics statement The statistical methods of this study were reviewed by Dr. George Knafl, Biostatistician and Professor in the School of Nursing at the University of North Carolina at Chapel Hill. RESULTS Complete data were available for 207 patients (Figure 1). The baseline demographic and illness related data are presented in Tables 1 and 2, respectively. The mean age of the respondents was 54 years (SD = 11). More than half were married (53%) and had college or more than college education (64%). About one-third (35%) reported a household income of less than $30000 year. None of the patients had any missing CBC analgesic attribute values. Only three of all these variables had any missing values. The threshold for a distinct percent change in LCV score for data with 207 observations is 0.92% (in contrast, the percent decrease is 2.00% for 95 observations and 1.00% for 190 observations).

None of the patients had any missing CBC analgesic attribute values. Only three of all these variables had any missing values. The threshold for a distinct percent change in LCV score for data with 207 observations is 0.92% (in contrast, the percent decrease is 2.00% for 95 observations and 1.00% for 190 observations). Table 1 Baseline sociodemographic variables (n = 207) Variable Range n (%)1 Mean (SD) Age 23-75 53.8 (11.1) Education Elementary 3 (1.5) High school 70 (33.8) College/Trade school 101 (48.8) More than college 33 (15.9) Employment status Employed outside home (full-time) 43 (20.8) Employed outside home (part-time) 12 (5.8) Employed at home (full-time) 4 (1.9) Employed at home (part-time) 4 (1.8) Retired 44 (21.3) Unemployed 25 (12.1) Other 75 (36.2) Health literacy 3-15 13.1 (2.6) Income < $10000 28 (13.5) $10000-$20000 26 (12.6) $20000-$30000 19 (9.2) $30000-$50000 36 (17.4) $50000-$70000 37 (17.9) $70000-$90000 24 (11.6) > $90000 37 (17.9) Primary insurance (1 missing) Private 107 (51.9) Medicare 41 (19.9) Medicaid 27 (13.1) Multiple 25 (12.1) VA/other 6 (2.9) Marital status Married 110 (53.1) Separated/Divorced 48 (23.2) Widowed 8 (3.9) Never married 41 (19.8) Race Black/African American 86 (41.5) White/Caucasian 121 (58.5) Social support 0.17-9.00 3.7 (2.1) 1 No missing values unless otherwise indicated. SD: Standard deviation; VA: Veterans Administration. Figure 1 Participant recruitment flow diagram. MEMS: Medication Event Monitoring; ATC: Around-the-clock. Table 2 Baseline illness and pain variables (n = 207)

Variable Range n (%)1 Mean (SD) Age 23-75 53.8 (11.1) Education Elementary 3 (1.5) High school 70 (33.8) College/Trade school 101 (48.8) More than college 33 (15.9) Employment status Employed outside home (full-time) 43 (20.8) Employed outside home (part-time) 12 (5.8) Employed at home (full-time) 4 (1.9) Employed at home (part-time) 4 (1.8) Retired 44 (21.3) Unemployed 25 (12.1) Other 75 (36.2) Health literacy 3-15 13.1 (2.6) Income < $10000 28 (13.5) $10000-$20000 26 (12.6) $20000-$30000 19 (9.2) $30000-$50000 36 (17.4) $50000-$70000 37 (17.9) $70000-$90000 24 (11.6) > $90000 37 (17.9) Primary insurance (1 missing) Private 107 (51.9) Medicare 41 (19.9) Medicaid 27 (13.1) Multiple 25 (12.1) VA/other 6 (2.9) Marital status Married 110 (53.1) Separated/Divorced 48 (23.2) Widowed 8 (3.9) Never married 41 (19.8) Race Black/African American 86 (41.5) White/Caucasian 121 (58.5) Social support 0.17-9.00 3.7 (2.1) 1 No missing values unless otherwise indicated. SD: Standard deviation; VA: Veterans Administration. Figure 1 Participant recruitment flow diagram. MEMS: Medication Event Monitoring; ATC: Around-the-clock. Table 2 Baseline illness and pain variables (n = 207) Variable Range n (%)1 Mean (SD) Cancer stage I 20 (9.7) II 33 (15.9) III 37 (17.9) IV 64 (30.9) Unknown or unsure 53 (25.6) Time since cancer diagnosis 1-120 mo 36.7 (35.5) Charlson comorbidity index 0-13 4.3 (2.6) General health Excellent 9 (4.3) Very good 23 (11.1) Good 63 (30.4) Fair 77 (37.2) Poor 35 (16.9) Physical health not good (number of days within last 30 d) 0-30 14.7 (10.7) Mental health not good (number of days within last 30 d) 0-30 9.5 (10.7) Past history of substance abuse No 172 (83.1) Yes 35 (16.9) Presence of depression No 120 (58.0) Yes 87 (42.0) Worst pain (last week) 0-10 (no pain - pain as bad as you can imagine) 6.9 (2.4) Average pain (last week) 0-10 (no pain - pain as bad as you can imagine) 4.9 (2.1) Least pain (last week) 0-10 (no pain - pain as bad as you can imagine) 3.4 (2.0) Pain-related functional interference score 7-70 (does not interfere-completely interferes) 35.2 (15.9) Pain relief with medications (last week) 1-10 (10%-100%) 7.2 (2.1) Pain management index -2 5 (2.4) -1 13 (6.3) 0 92 (44.4) 1 63 (30.4) 2 31 (15.0) 3 3 (1.4) Number of analgesic side effects (MSEC) 0-8 3.8 (2.4) Severity of analgesic side effects (MSEC) 8-80 (not severe-extremely severe) 25.2 (15.0) BQ-II analgesic barriers (total) 0-96 39.8 (20.1) No. of complementary alternative modalities used 0-8 2.1 (1.7) 1 No missing values unless otherwise indicated. BQ-II: Barriers questionnaire; MSEC: Medication Side-effects Checklist; SD: Standard deviation.

e effects (MSEC) 8-80 (not severe-extremely severe) 25.2 (15.0) BQ-II analgesic barriers (total) 0-96 39.8 (20.1) No. of complementary alternative modalities used 0-8 2.1 (1.7) 1 No missing values unless otherwise indicated. BQ-II: Barriers questionnaire; MSEC: Medication Side-effects Checklist; SD: Standard deviation. Unique analgesic preference clusters Using methods described (see data analysis), a 4-cluster solution was chosen. Figure 2 contains plots of the four cluster centroids, that is, the vectors with entries equal to averages of the five CBC analgesic attributes for patients in the clusters. Based on these plots, the clusters were characterized in terms of the more strongly rated analgesic attributes (Table 3). Figure 2 Choice-based conjoint analgesic attribute types. CBC: Choice-based conjoint. Table 3 Description of analgesic preference clusters (n = 207)

Unique analgesic preference clusters Using methods described (see data analysis), a 4-cluster solution was chosen. Figure 2 contains plots of the four cluster centroids, that is, the vectors with entries equal to averages of the five CBC analgesic attributes for patients in the clusters. Based on these plots, the clusters were characterized in terms of the more strongly rated analgesic attributes (Table 3). Figure 2 Choice-based conjoint analgesic attribute types. CBC: Choice-based conjoint. Table 3 Description of analgesic preference clusters (n = 207) Cluster n (%) Salient concern(s) 1 84 (40.6) Pain relief 2 23 (11.1) Type of analgesic 3 57 (27.5) Pain relief, type of side-effects and severity of side-effects 4 43 (20.8) Type of side-effects Cluster 1 (pain relief) For less than half the patients (41%) in this study, expectation of pain relief was the main anchor in making analgesic related trade-offs for cancer pain. A total of 16 individually significant binary characteristics were identified for patients in this cluster (Supplemental Table 1). Patients in cluster 1 were more likely be White/Caucasians, carried a private health insurance, had higher education and health literacy, and reported less analgesic-related barriers in general. The strongest of these predictors, that is, the one generating the best (largest) LCV score, was lower endorsement of the belief that pain medicine can mask changes in your health with LCV score 0.51908 (LCV scores not reported).

er education and health literacy, and reported less analgesic-related barriers in general. The strongest of these predictors, that is, the one generating the best (largest) LCV score, was lower endorsement of the belief that pain medicine can mask changes in your health with LCV score 0.51908 (LCV scores not reported). The individually significant binary characteristics were adaptively combined into a multiple logistic regression model (Table 4). The three factors that remained in the multiple risk factor model and predicted membership in cluster 1 included, higher education, poor physical health and a lower endorsement of the belief that pain medications can mask changes in health. The most important of these (i.e., the one whose removal generated the lowest LCV score) was BQ-II item, pain medicine can mask changes in your health. The LCV score was 0.53503, and so this model provided a distinct improvement over the best individual binary characteristic model with percent decrease 2.98% (since this was larger than the threshold of 0.92%). Table 4 Multiple binary characteristics model for cluster 1 (pain relief)

The individually significant binary characteristics were adaptively combined into a multiple logistic regression model (Table 4). The three factors that remained in the multiple risk factor model and predicted membership in cluster 1 included, higher education, poor physical health and a lower endorsement of the belief that pain medications can mask changes in health. The most important of these (i.e., the one whose removal generated the lowest LCV score) was BQ-II item, pain medicine can mask changes in your health. The LCV score was 0.53503, and so this model provided a distinct improvement over the best individual binary characteristic model with percent decrease 2.98% (since this was larger than the threshold of 0.92%). Table 4 Multiple binary characteristics model for cluster 1 (pain relief) Variable domain Variable Characteristic n (% out of 207) P value OR 95%CI Sociodemographic Education College/trade school or more than college vs Elementary or High school 134 (64.7) 0.001 3.88 1.75-8.59 Illness Physical health not good (number of days within last 30 d) ≥ 22 vs < 22 59 (28.5) 0.002 2.81 1.47-5.38 Pain, function and pain treatment NS Analgesic attitudes and barriers BQ-II item - pain medicine can mask changes in your health ≤ 3 vs > 3 158 (76.3) 0.016 2.26 1.17-4.36 BQ-II: Barriers questionnaire II; CI: Confidence interval; OR: Odds ratio; NS: None significant.

t 30 d) ≥ 22 vs < 22 59 (28.5) 0.002 2.81 1.47-5.38 Pain, function and pain treatment NS Analgesic attitudes and barriers BQ-II item - pain medicine can mask changes in your health ≤ 3 vs > 3 158 (76.3) 0.016 2.26 1.17-4.36 BQ-II: Barriers questionnaire II; CI: Confidence interval; OR: Odds ratio; NS: None significant. Cluster 2 (type of analgesic) For only 11% of patients in this study, the main anchor for analgesic trade-offs was “type of analgesic”. A total of 15 individually significant binary characteristics were identified for patients in cluster type 2 (Supplemental Table 2). Patients in cluster 2 were more likely to have lower income, lower social support, greater burden of comorbidities and pain, and lower relief from taking pain medications. Patients in this cluster were more likely to hold beliefs such as pain medications can harm immune system, or make you addicted. However, the strongest of these predictors was lower (≤ $50000) income with LCV score 0.71212 (LCV scores not reported). In the multiple logistic regression model, lower social support, health literacy and income levels were predictive of membership in this cluster (Table 5). The most important of these was health literacy (LCV score was 0.72894), and so this model provided a distinct improvement over the best individual binary characteristic model with percent decrease 2.31%. Table 5 Multiple binary characteristics model for cluster 2 (type of analgesic)

In the multiple logistic regression model, lower social support, health literacy and income levels were predictive of membership in this cluster (Table 5). The most important of these was health literacy (LCV score was 0.72894), and so this model provided a distinct improvement over the best individual binary characteristic model with percent decrease 2.31%. Table 5 Multiple binary characteristics model for cluster 2 (type of analgesic) Variable domain Variable Characteristic n (% out of 207) P value OR 95%CI Sociodemographic Health literacy = 15 vs < 15 93 (44.9) 0.006 3.86 1.46-10.2 Income ≤ $50000 vs < $50000 109 (52.7) 0.017 3.64 1.26-10.5 Social support ≤ 4.17 vs > 4.17 137 (66.2) 0.027 4.25 1.18-15.4 Illness NS Pain, function and pain treatment NS Analgesic attitudes and barriers NS CI: Confidence interval; OR: Odds ratio; NS: None significant.

eracy = 15 vs < 15 93 (44.9) 0.006 3.86 1.46-10.2 Income ≤ $50000 vs < $50000 109 (52.7) 0.017 3.64 1.26-10.5 Social support ≤ 4.17 vs > 4.17 137 (66.2) 0.027 4.25 1.18-15.4 Illness NS Pain, function and pain treatment NS Analgesic attitudes and barriers NS CI: Confidence interval; OR: Odds ratio; NS: None significant. Cluster 3 (pain relief, type of side-effects and severity of side-effects) More than one in four patients (28%) made trade-offs based on multiple factors including expectation of pain relief, type of side-effects, and severity of side-effects. A total of 18 individually significant binary characteristics were identified for patients in cluster 3 (Supplemental Table 3). Patients in this cluster were more likely to be married, had greater social support, reported lower pain and pain related functional impairment, and greater pain relief with analgesics. They were less likely to report analgesic side-effects and had lower endorsement for BQ items indicating lower attitudinal barriers. The strongest of these predictors was lower average pain (≤ 6) in the last week with LCV score 0.56530 (LCV scores not reported). In the multiple logistic regression model, being married, having greater social support, having lower average pain, lower side-effects predicted membership in cluster 3 (Table 6). Table 6 Multiple binary characteristics model for cluster 3 (pain relief, type of side-effects and severity of side-effects)

Cluster 3 (pain relief, type of side-effects and severity of side-effects) More than one in four patients (28%) made trade-offs based on multiple factors including expectation of pain relief, type of side-effects, and severity of side-effects. A total of 18 individually significant binary characteristics were identified for patients in cluster 3 (Supplemental Table 3). Patients in this cluster were more likely to be married, had greater social support, reported lower pain and pain related functional impairment, and greater pain relief with analgesics. They were less likely to report analgesic side-effects and had lower endorsement for BQ items indicating lower attitudinal barriers. The strongest of these predictors was lower average pain (≤ 6) in the last week with LCV score 0.56530 (LCV scores not reported). In the multiple logistic regression model, being married, having greater social support, having lower average pain, lower side-effects predicted membership in cluster 3 (Table 6). Table 6 Multiple binary characteristics model for cluster 3 (pain relief, type of side-effects and severity of side-effects) Variable domain Variable Characteristic n (% out of 207) P value OR 95%CI Sociodemographic Marital status Married vs Separated, Divorced, Widowed or Never married 110 (53.1) 0.023 2.26 1.12-4.56 Social support ≥ 1.83 vs < 1.83 177 (85.5) 0.022 4.55 1.24-16.7 Illness Mental health not good (number of days within last 30 d) ≥ 2 vs < 2 140 (67.6) 0.002 3.46 1.55-772 Pain, function and pain treatment Average pain (last week) ≤ 6 vs > 6 163 (78.7) 0.01 4.41 1.42-6.86 Severity of analgesic side effects (MSEC) ≤ 28 vs > 28 133 (64.3) 0.005 3.11 1.41-6.86 Analgesic attitudes and barriers NS CI: Confidence interval; OR: Odds ratio; MSEC: Medication Side-effects Checklist; NS: None significant.

unction and pain treatment Average pain (last week) ≤ 6 vs > 6 163 (78.7) 0.01 4.41 1.42-6.86 Severity of analgesic side effects (MSEC) ≤ 28 vs > 28 133 (64.3) 0.005 3.11 1.41-6.86 Analgesic attitudes and barriers NS CI: Confidence interval; OR: Odds ratio; MSEC: Medication Side-effects Checklist; NS: None significant. Cluster 4 (type of side-effects) For one in five patients (21%), type of side-effects experienced was the main factor driving analgesic trade-offs. A total of 21 individually significant binary characteristics were identified for patients in cluster type 4 (Supplemental Table 4). Patients in this cluster had lower education and health literacy, were more likely to be Blacks/African Americans, reported lower relief with medications and reported shorter duration of relief with pain medications. Patients in this cluster were more likely to report greater severity of analgesic side-effects and past history of substance abuse but fewer number of days when mental health was not good. Patients in this cluster had the highest number of BQ barriers than any other cluster. In the multiple logistic regression model, four factors including, lower health literacy, mental health, more analgesic side effects, and belief that pain medications keep you from knowing what is going on in your body predicted membership in this cluster (Table 7). Table 7 Multiple binary characteristics model for cluster 4 (type of side-effects)

In the multiple logistic regression model, four factors including, lower health literacy, mental health, more analgesic side effects, and belief that pain medications keep you from knowing what is going on in your body predicted membership in this cluster (Table 7). Table 7 Multiple binary characteristics model for cluster 4 (type of side-effects) Variable domain Variable Characteristic n (% out of 207) P value OR 95%CI Sociodemographic Health literacy ≤ 13 vs > 13 84 (40.6) 0.004 3.11 1.43-6.76 Illness Mental health not good (number of days within last 30 d) ≤ 12 vs > 12 144 (69.6) 0.001 6.18 2.06-18.5 Pain, function and pain treatment Severity of analgesic side effects (MSEC) ≥ 40 vs < 40 37 (17.9) 0.002 4.19 1.68-10.5 Analgesic attitudes and barriers BQ-II item - pain medicine can keep you from knowing what’s going on in your body ≥ 4 vs < 4 42 (20.3) < 0.001 5.25 2.32-11.9 BQ-II: Barriers questionnaire; MSEC: Medication Side-effects Checklist; CI: Confidence interval; OR: Odds ratio.

SEC) ≥ 40 vs < 40 37 (17.9) 0.002 4.19 1.68-10.5 Analgesic attitudes and barriers BQ-II item - pain medicine can keep you from knowing what’s going on in your body ≥ 4 vs < 4 42 (20.3) < 0.001 5.25 2.32-11.9 BQ-II: Barriers questionnaire; MSEC: Medication Side-effects Checklist; CI: Confidence interval; OR: Odds ratio. DISCUSSION This is the first study to identify the sociodemographic and clinical predictors of unique clusters based on what may drive patients’ preference for analgesic treatment for cancer pain. Lack of adherence to analgesia for cancer pain is a prevalent clinical problem[32-35]. Studies in cancer[35] and non-cancer[36-43] pain settings suggest that patterns of analgesic adherence are consequential in explaining clinical and health services outcomes. The 2016 CDC guidelines provide recommendations to clinicians for opioid prescription[1]. However, this focus will be incomplete without an understanding of how patients take prescribed analgesics and what salient concerns anchor their decisions. Previous studies have documented correlates of non-adherence to analgesia for cancer pain[44-47]. These studies do not allow discerning how risk factors and predictors may be distributed dissimilarly across subgroups of cancer patients. Using a well-established trade-off analysis technique (CBC) and more novel adaptive methods, we first showed that unique clusters of patients exist based on the main concern(s) anchoring their preferences for analgesia for cancer pain. We then identified sociodemographic and clinical factors that predict membership in each preference cluster.

trade-off analysis technique (CBC) and more novel adaptive methods, we first showed that unique clusters of patients exist based on the main concern(s) anchoring their preferences for analgesia for cancer pain. We then identified sociodemographic and clinical factors that predict membership in each preference cluster. Importantly, for an overwhelming majority in this study, analgesic preference for cancer pain was driven by a single salient underlying concern (see cluster 1, 2 and 4). In multivariable analysis to identify predictors of these clusters, “clinical” and “socioeconomic factors” (rather than attitudes and beliefs) were found important. Of note, at least one socioeconomic factor (including education, health literacy, income) played a role in predicting three out of four preference clusters. Furthermore, most analgesic beliefs and concerns, including the widely implicated addiction concerns, did not play a role as predictors of cluster membership. Only the belief that pain medications can mask changes in health or keep you from knowing what is going on in your body was found significant in predicting two of the four clusters. This is a common clinical concern among cancer patients and relates to the fear of disease progression[48-50].

tors of cluster membership. Only the belief that pain medications can mask changes in health or keep you from knowing what is going on in your body was found significant in predicting two of the four clusters. This is a common clinical concern among cancer patients and relates to the fear of disease progression[48-50]. An interesting finding was the contrast between cluster 1 and 4. Unlike cluster 1 (pain relief), those in the side-effects cluster (cluster 4) had lower health literacy and greater analgesic barriers using BQ-II questionnaire. Patients in this cluster were more likely to report greater burden of analgesic side-effects. Of note, there is a stark difference in the identified correlates of these two clusters. The correlates of cluster 1 included being white/Caucasian and having higher education, income and health literacy and lower analgesic barriers. Cluster 4, however was predicted by being African Americans and having lower education, literacy, and more analgesic barriers. Another interesting noteworthy contrast between the two clusters (1 and 4) was that in the multiple logistic regression models, individuals in cluster 1 (pain relief) were less likely to believe that pain medications can mask changes in your health whereas patients in cluster 4 were more likely to endorse pain can keep you from knowing what is going on in your body. Thus, literacy and analgesic beliefs appear to be at play in different ways in the two clusters.

uster 1 (pain relief) were less likely to believe that pain medications can mask changes in your health whereas patients in cluster 4 were more likely to endorse pain can keep you from knowing what is going on in your body. Thus, literacy and analgesic beliefs appear to be at play in different ways in the two clusters. Previous studies have investigated and found racial and socioeconomic disparities in pain management in general, including cancer pain management[51-55]. Our findings indicate that analgesic side-effects are also poorly treated in cancer patients with lower health literacy. These patients will benefit from meticulous assessment of pain and symptoms and accessible interventions that promote self-advocacy and negotiation of pain and side-effects management with their clinicians and oncologists.

hat analgesic side-effects are also poorly treated in cancer patients with lower health literacy. These patients will benefit from meticulous assessment of pain and symptoms and accessible interventions that promote self-advocacy and negotiation of pain and side-effects management with their clinicians and oncologists. In the last few decades, significant resources have been devoted towards psychoeducational interventions that have a major focus on dismantling analgesic beliefs and barriers[56]. Unfortunately, a number of systematic reviews show that these interventions do not improve adherence to analgesia for cancer pain or cancer pain outcomes[57,58]. Our findings imply that meticulous assessment of clinical factors such as pain levels, analgesic side-effects, and addressing SES factors (such as health literacy) may play a role in improving cancer pain outcomes. Also, the finding that decision-making for most patients was driven by single salient underlying factor raises an exciting possibility of designing two-part interventions focused on eliciting real-time trade-offs and linking real-time preferences sequentially to brief, tailored, and patient-centered clinical interventions.

, the finding that decision-making for most patients was driven by single salient underlying factor raises an exciting possibility of designing two-part interventions focused on eliciting real-time trade-offs and linking real-time preferences sequentially to brief, tailored, and patient-centered clinical interventions. Study limitations The clusters identified in this study are based on the CBC design. While CBC is a well-established method and we previously tested the validity of the CBC utilities used in this study, there is a notable consideration. About 1 in 3 patients used lexicographic decision rules (i.e., unwillingness to trade more or less of one attribute in favor or detriment of the other)[14]. These processes may represent patients’ actual preferences or mental shortcuts to get through the CBC exercise, potentially compromising the clinical validity of the data. Our confidence that the clusters represent actual preferences is enhanced by the study findings. For instance, patients in cluster 4 (side effects) were more likely to report greater burden of analgesic side-effects, which remained significant in the multivariable model. Similarly, patients in cluster 3 weighed multiple factors similarly (pain relief, type and severity of side-effects) possibly because of their experience of lower pain severity and lower burden of side-effects (e.g., MSEC < 28 in cluster 3 vs > 40 in cluster 4). These findings increase confidence that the clusters identified in this study represent actual preferences rather than mental shortcuts. Also, we restricted our analysis to those patients who completed the study to avoid having missing data that may have affected the conclusions of the study. Excluded patients were with advanced illness who died or were too sick to complete the study (Figure 1), thus we caution against generalizing the findings to those with advanced illness. Nevertheless, our findings inform a scarce body of literature on what anchors cancer patients’ preferences in using analgesia for cancer pain and a potential new path to brief, tailored, and accessible interventions to improve pain and functional outcomes among cancer patients.

findings to those with advanced illness. Nevertheless, our findings inform a scarce body of literature on what anchors cancer patients’ preferences in using analgesia for cancer pain and a potential new path to brief, tailored, and accessible interventions to improve pain and functional outcomes among cancer patients. COMMENTS Background The purpose of this study was to investigate if unique clusters exist with regard to patients’ concerns in using analgesics for cancer pain and factors predicting cluster membership. Research frontiers The new Centers for Disease Control and Prevention opioid guidelines are shaping a national conversation among professionals and policy makers on opioid prescription. Little is known about the other side of the coin, i.e., cancer patients’ concerns in using analgesia and factors shaping these concerns and preferences that may relate to their analgesic taking patterns. This study fills this important gap.

onal conversation among professionals and policy makers on opioid prescription. Little is known about the other side of the coin, i.e., cancer patients’ concerns in using analgesia and factors shaping these concerns and preferences that may relate to their analgesic taking patterns. This study fills this important gap. Innovations and breakthroughs The authors employed novel statistical methods to understand unique subgroups of patients based on their concerns in using analgesics for cancer pain and identified sociodemographic and clinical correlates of these unique clusters. In recent decades, significant resources have been committed to psychoeducational interventions that have a major focus on dismantling analgesic beliefs and barriers. However, recent systematic reviews show that psychoeducational interventions do not consistently improve adherence to analgesia for cancer pain or cancer pain outcomes. The authors’ findings suggest that careful assessment of clinical factors such as analgesic side-effects and addressing social determinants, such as patients’ health literacy, may play a role in improving cancer pain outcomes. Applications The authors’ study finding that decision-making for most patients was driven by single salient underlying factor raise an exciting possibility of designing two-part interventions focused on eliciting real-time trade-offs and linking real-time preferences sequentially to brief, tailored, and patient-centered clinical interventions.

study finding that decision-making for most patients was driven by single salient underlying factor raise an exciting possibility of designing two-part interventions focused on eliciting real-time trade-offs and linking real-time preferences sequentially to brief, tailored, and patient-centered clinical interventions. Terminology Analgesic concerns and preferences in this study were elicited using choice-based conjoint (CBC) analysis, which is a trade-off analysis technique. Individuals are asked to make trade-offs between attributes (e.g., pain relief, side-effects) and attribute levels (e.g., percent pain relief, severity of side-effects) generating a unique set of values called part-worth utilities. A higher part-worth utility represents a higher level of value or importance an individual assign to that attribute. Peer-review The paper contributes important information. Supported by National Institutes of Health/National Institute of Nursing Research, No. NIH/NINR RC1-NR011591. Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of the University of Pennsylvania (Philadelphia). Informed consent statement: All study participants provided informed written consent prior to study data collection. Conflict-of-interest statement: There are no conflicts of interest to report. Data sharing statement: No additional data are available. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0

Conflict-of-interest statement: There are no conflicts of interest to report. Data sharing statement: No additional data are available. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: August 23, 2016 First decision: October 21, 2016 Article in press: December 14, 2016 P- Reviewer: Fassoulaki A, Noll-Hussong M S- Editor: Ji FF L- Editor: A E- Editor: Wu HL

Core tip: We wrote an interesting case report about a pancreatic plasmacytoma for which diagnosis, including endoscopic diagnosis, was a challenge. In a second part, a systematic pubmed search was performed from 1950 to June 2016, reporting characteristics and route to diagnosis of 63 similar cases reports! Strengths of our paper are the original route to diagnosis (by a simple ultrasound guided paracentesis, after failed of the endoscopic route) and our literature search which is particularly exhaustive: we are first to identify more 20 case similar reports (63!!) and their characteristics. INTRODUCTION Here we describe the case of a pancreatic plasmacytoma and difficulties to establish the diagnosis. Characteristics of patients and routes to diagnosis in this condition will be identified through a systematic literature search, in a second part.

Core tip: We wrote an interesting case report about a pancreatic plasmacytoma for which diagnosis, including endoscopic diagnosis, was a challenge. In a second part, a systematic pubmed search was performed from 1950 to June 2016, reporting characteristics and route to diagnosis of 63 similar cases reports! Strengths of our paper are the original route to diagnosis (by a simple ultrasound guided paracentesis, after failed of the endoscopic route) and our literature search which is particularly exhaustive: we are first to identify more 20 case similar reports (63!!) and their characteristics. INTRODUCTION Here we describe the case of a pancreatic plasmacytoma and difficulties to establish the diagnosis. Characteristics of patients and routes to diagnosis in this condition will be identified through a systematic literature search, in a second part. CASE REPORT A 71-year-old man was hospitalized for a clinical and biological presentation of acute pancreatitis. Pain occurred suddenly and was associated with an increased level of lipase above 2000 UI/L, a cholestatic icterus (bilirubin: 103 µmol/L) and a hepatic cytolysis (ALT: 154 UI/L; AST: 131 UI/L). An initial computerized tomography (CT) scan showed a significant but unspecific infiltration around the pancreas head, without dilatation of biliary ducts. A first endoscopic ultrasound (EUS) (Pentax, EG 3670 URK, France) showed similar data. The hypoechoic infiltration of the pancreas head was heterogeneous and extended to the hepatic hilum, in contact with portal vein. There was no biliary lithiasis, nor context of alcohol consumption during the last days before the admission. However, the patient was treated with Lenalidomide plus dexamethasone for a Immunoglobulin A (IgA) plasmacytoma diagnosed 3 years ago [t(4;14) positive, del(17p) negative; at baseline: LDH: 173 UI/L, monoclonal immunoglobulin peak: 40.5 g/L, Kappa and Lambda serum free light chain: 11.7 and 18.6 mg/L, respectively], without hypercalcemia nor kidney failure. He relapsed dramatically one year ago, with an extramedullar localization (L4 lumbar spine). Based on hematotoxicity (platelets: 41000 G/mm3) and lake of specific radiologic features, the initial diagnosis suspected was a dual hepatic and pancreatic toxicity of Lenalidomide. Indeed, acute pancreatitis and hepatitis had been occasionally reported as a side effect of Lenalidomide[1,2]. Common hepatitis viral serologies were tested before carrying out a transjugular hepatic biopsy which showed a histological aspect compatible with the diagnosis of drug hepatitis or hepatitis related to a biliary obstruction (centrilobular and portal infiltrate of polymorphs inflammatory cells including eosinophils). Although an empirical treatment with 500 mg intravenous methylprednisolone daily was started, bilirubin level increased at 345.8 μmol/L within the following ten days. Hence, a new CT-scan was performed and showed the occurrence of a mild to moderate dilatation of biliary ducts and a low abundance ascites.

eosinophils). Although an empirical treatment with 500 mg intravenous methylprednisolone daily was started, bilirubin level increased at 345.8 μmol/L within the following ten days. Hence, a new CT-scan was performed and showed the occurrence of a mild to moderate dilatation of biliary ducts and a low abundance ascites. At the moment of admission, the infiltration of the pancreas head significantly resembled a tumor (Figure 1) and the diagnosis of a pancreatic localization of the plasmacytoma was suspected. After platelets support, EUS (Pentax, EG 3670UTK, France) guided fine needle aspiration (FNA) was carried out with a 22-gauge needle. Tumor infiltration appeared to be growing due to portal vein invasion. Linear EUS passage through the pylorus was drastically limited, so that FNA was performed from the gastric antrum. Then, an endoscopic retrograde cholangiopancreatography was attempted to place a biliary stent for palliative treatment, but the cannulation of the bile duct had failed due to a major parietal oedema of the duodenum which was easily bleeding due to the contact of the sphincterotome. A percutaneous biliary drainage was considered, but an ultrasound-guided paracentesis was preferred, taking into account technical difficulties of the biliary drainage. Cytology of the FNA was not contributory (epithelial cells of pancreas without malignity signs) while the analysis of ascites showed plasmacytosis with severe atypia enabling the diagnosis of pancreatic plasmacytoma (Figure 2). Bone marrow was exempted from dystrophic plasma cells, proving an extramedullar relapse. The increase of the monoclonal spike (from 2.3 g/L to 8.1 g/L within 4 mo) and LDH (259 UI/L) was compatible with this diagnosis. Kappa and Lambda free light chain, at this time of the disease, were 0.4 mg/L and 24.8 mg/L, respectively, without hypercalcemia, Bence Jones proteinuria, nor kidney failure. Hence, after contacting the referral hematologist of the patient, a cure of 40 mg dexamethasone daily was started inciting a drastic decrease of bilirubin level within the next three days (183.1 μmol/L). Then, a second line of chemotherapy (Bortezomib + Cyclophosphamide) was started with a good short-term safety. Although a biological response, especially for monoclonal peak (2.1 g/L), at one month, the patient died 4 mo after the diagnosis of pancreatic plasmacytoma.

in level within the next three days (183.1 μmol/L). Then, a second line of chemotherapy (Bortezomib + Cyclophosphamide) was started with a good short-term safety. Although a biological response, especially for monoclonal peak (2.1 g/L), at one month, the patient died 4 mo after the diagnosis of pancreatic plasmacytoma. Figure 1 Abdominal computerized tomography scan showing a head pancreas mass extended to the hepatic hilum with mild to moderate dilatation of biliary ducts and a low abundance ascites. Figure 2 Peritoneal fluid Cytology, May-Grünwald-Giemsa stain. A: An almost pure population of myeloma cells (× 40); B: Malignant plasma cells exhibiting severe atypia (× 100). DISCUSSION Extramedullary plasmacytoma involvement is not an uncommon presentation, occurring in 10 %-15 % of patients[3]. They are commonly identified after the diagnosis of multiple myeloma. The most commonly involved organs are those located around skeletal lesions, and less frequently, skin, liver, kidney, or central nervous system. Regarding the digestive system, liver and spleen are classically the organs which could be damaged by disease through deposits of amyloid proteins[4]. Extramedullary plasmacytomas involving the pancreas is a very rare condition with a prevalence rate estimated at 2.3%, based on autopsy studies[5].

ntral nervous system. Regarding the digestive system, liver and spleen are classically the organs which could be damaged by disease through deposits of amyloid proteins[4]. Extramedullary plasmacytomas involving the pancreas is a very rare condition with a prevalence rate estimated at 2.3%, based on autopsy studies[5]. After conducting a systematic Pubmed search, we identified 63 case reports of pancreatic plasmacytoma and collected a set of clinical and diagnostic data which were reported in Table 1. About half of them were male, with a median age of 58.5 years old, and presented jaundice in 70.0% with (36%) or without pain. About 2/3 of patients (68.4%) had a known history of plasmacytoma since 1 year (0-13) (median, interquartile ranges 25%-75%), before the involvement of the pancreas head. Only two cases involved the body or the tail of the pancreas[6,7]. Only 1/3 of patients (32.6%) were diagnosed by EUS-guided FNA vs 1/5 (20.9%) by CT-guided percutaneous FNA. About ¼ of patients (25.6%) have needed for a surgical biopsy, including situation involving bowel obstruction. A direct biopsy of the mass was possible in 16.3% during an upper gastrointestinal endoscopy. Most of patients were treated with chemotherapy (56.0%) and/or radiotherapy (52.0%), providing a 100% tumor response rate. A biliary stent was placed in half of patients with jaundice (46.7%). Table 1 Main characteristics of the 63 patients who had been reported to date with a pancreas plasmacytoma: Results of a PubMed search from 1950 to June 2016

After conducting a systematic Pubmed search, we identified 63 case reports of pancreatic plasmacytoma and collected a set of clinical and diagnostic data which were reported in Table 1. About half of them were male, with a median age of 58.5 years old, and presented jaundice in 70.0% with (36%) or without pain. About 2/3 of patients (68.4%) had a known history of plasmacytoma since 1 year (0-13) (median, interquartile ranges 25%-75%), before the involvement of the pancreas head. Only two cases involved the body or the tail of the pancreas[6,7]. Only 1/3 of patients (32.6%) were diagnosed by EUS-guided FNA vs 1/5 (20.9%) by CT-guided percutaneous FNA. About ¼ of patients (25.6%) have needed for a surgical biopsy, including situation involving bowel obstruction. A direct biopsy of the mass was possible in 16.3% during an upper gastrointestinal endoscopy. Most of patients were treated with chemotherapy (56.0%) and/or radiotherapy (52.0%), providing a 100% tumor response rate. A biliary stent was placed in half of patients with jaundice (46.7%). Table 1 Main characteristics of the 63 patients who had been reported to date with a pancreas plasmacytoma: Results of a PubMed search from 1950 to June 2016 Demographic characteristics n (%) Male 22 (56.4) Age (years, median, IQR) 58.5 [51.2-82] Symptom(s) at diagnosis Jaundice 35 (70.0) Pain 18 (36.0) Myeloma Known history of myeloma 26 (41.3) Disease duration at diagnosis of pancreas plasmacytoma (years, median, IQR) 1 [0-13] Type Kappa 13 (71.4) Immunoglobulin A (36%), G (52%), M (12%) Diagnosis process of the pancreas plasmacytoma Endoscopic ultrasound FNA 14 (32.6) Percutaneous FNA 9 (20.9) Endoscopic biopsy 7 (16.3) Surgical biopsy 11 (25.6) Paracentesis 0 (0.0) Postmortem biopsy 3 (7.0) Management of the pancreas plasmacytoma Chemotherapy 14 (56.0) Radiotherapy 13 (52.0) Biliary stent in patients with jaundice 10 (40.0) Surgery 8 (32.0) Biliodigestive derivation 3 (37.5) Duodenopancreatectomy cephalic 2 (25.0) FNA: Fine needle aspiration; IQR: Interquartile range.

sis 0 (0.0) Postmortem biopsy 3 (7.0) Management of the pancreas plasmacytoma Chemotherapy 14 (56.0) Radiotherapy 13 (52.0) Biliary stent in patients with jaundice 10 (40.0) Surgery 8 (32.0) Biliodigestive derivation 3 (37.5) Duodenopancreatectomy cephalic 2 (25.0) FNA: Fine needle aspiration; IQR: Interquartile range. Hence, to the best our knowledge, this is the first case report of a pancreatic plasmacytoma which was diagnosed by ascites analysis. Diagnosis by noninvasive procedures and rapid response to conservative therapy were important in this patient’s care. It is very difficult to radiologically differentiate extramedullary plasmacytoma of the pancreas from other pancreatic tumors. EUS guided FNA provides the easiest and most safe route to diagnosis of pancreatic plasmacytoma. Studies have shown that the overall accuracy of EUS-guided FNA ranges between 71% and 90% in case of pancreatic tumor[8]. However, there is no corresponding data in case of pancreatic plasmacytoma. In our case, the missed diagnosis of pancreas plasmacytoma through EUS-guided FNA may be due to a sampling bias. Furthermore, we made only one diagnostic EUS attempt while in few cases reported, authors specified the need for repeating EUS-guided FNA[9-13].

Hence, to the best our knowledge, this is the first case report of a pancreatic plasmacytoma which was diagnosed by ascites analysis. Diagnosis by noninvasive procedures and rapid response to conservative therapy were important in this patient’s care. It is very difficult to radiologically differentiate extramedullary plasmacytoma of the pancreas from other pancreatic tumors. EUS guided FNA provides the easiest and most safe route to diagnosis of pancreatic plasmacytoma. Studies have shown that the overall accuracy of EUS-guided FNA ranges between 71% and 90% in case of pancreatic tumor[8]. However, there is no corresponding data in case of pancreatic plasmacytoma. In our case, the missed diagnosis of pancreas plasmacytoma through EUS-guided FNA may be due to a sampling bias. Furthermore, we made only one diagnostic EUS attempt while in few cases reported, authors specified the need for repeating EUS-guided FNA[9-13]. This case highlights that a pancreatic mass in patients with plasmacytoma should be systematically considered as an extramedullary extension of the disease until proven otherwise. Ascites analysis could be a simple route to diagnosis, even in low abundance. Finally, in case of jaundice, excluding angiocholitis, potential risks of biliary stenting should be taken into account, regarding safety and the drastic efficacy of radiotherapy or medical treatment (dexamethasone and chemotherapy). COMMENTS Case characteristics A 71-year-old man with history of plasmacytoma in relapse since one year, and treated with Lenalidomide.

This case highlights that a pancreatic mass in patients with plasmacytoma should be systematically considered as an extramedullary extension of the disease until proven otherwise. Ascites analysis could be a simple route to diagnosis, even in low abundance. Finally, in case of jaundice, excluding angiocholitis, potential risks of biliary stenting should be taken into account, regarding safety and the drastic efficacy of radiotherapy or medical treatment (dexamethasone and chemotherapy). COMMENTS Case characteristics A 71-year-old man with history of plasmacytoma in relapse since one year, and treated with Lenalidomide. Clinical diagnosis The initial diagnosis suspected was a dual hepatic and pancreatic toxicity of Lenalidomide. Differential diagnosis An adenocarcinoma of the pancreas, or other less frequent pancreatic tumor such as a non Hodgkin’s lymphoma, or endocrine tumor. Laboratory diagnosis An increased level of lipase above 2000 UI/L, a cholestatic icterus and a hepatic cytolysis. Imaging diagnosis Computerized tomography showed a significant but unspecific infiltration around the pancreas head, without dilatation of biliary ducts, extended to the hepatic hilum, and evolving as a pseudotumor within few days. Cytological diagnosis A (pancreatic) plasmacytoma. Treatment An empirical corticotherapy followed by a second line of chemotherapy (Bortezomib + Cyclophosphamide). Related reports Cytology of the mass was not contributory in contrast with the very low abundance ascites located around the liver.

Imaging diagnosis Computerized tomography showed a significant but unspecific infiltration around the pancreas head, without dilatation of biliary ducts, extended to the hepatic hilum, and evolving as a pseudotumor within few days. Cytological diagnosis A (pancreatic) plasmacytoma. Treatment An empirical corticotherapy followed by a second line of chemotherapy (Bortezomib + Cyclophosphamide). Related reports Cytology of the mass was not contributory in contrast with the very low abundance ascites located around the liver. Terms explanation Extramedullary plasmacytoma involvement is not an uncommon presentation, and occurres preferentially in located around skeletal lesions, or less frequently in, skin, liver, kidney, or central nervous system. Experiences and lessons A pancreatic mass occurring in a patient with history of plasmacytoma and with an uncommon presentation should make suspecting an extramedullar site of the disease. No diagnostic way should be forgot, even a simple analysis of an ascites sample. Peer-review This is an interesting case about pancreas involvement in a case with relapsed myeloma. Institutional review board statement: This case report was exempt from the internal Review Board standards of the Hepato-gastroenterology department managed by Pr Jean-Marc Phelip, at University of Saint-Etienne in Saint-Priest en Jarez. Informed consent statement: The patient who is involved in the present case report gave his verbal informed consent before his death, authorizing use and disclosure of his protected health information. Conflict-of-interest statement: None.

Institutional review board statement: This case report was exempt from the internal Review Board standards of the Hepato-gastroenterology department managed by Pr Jean-Marc Phelip, at University of Saint-Etienne in Saint-Priest en Jarez. Informed consent statement: The patient who is involved in the present case report gave his verbal informed consent before his death, authorizing use and disclosure of his protected health information. Conflict-of-interest statement: None. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: France Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C, C Grade D (Fair): D Grade E (Poor): 0 Peer-review started: October 11, 2016 First decision: November 30, 2016 Article in press: December 28, 2016 P- Reviewer: Bramhall S, Kyrtsonis MC, Mezalek ZT, Paydas S S- Editor: Ji FF L- Editor: A E- Editor: Wu HL

Core tip: The landscape of renal cell carcinoma has dramatically changed in the last decade. Today, at least 6 agents are approved after failure with cytokines, sunitinib or pazopanib in first line treatment. Lack of reliable biomarkers to select the best treatment in daily practice is somewhat frustrating. Therefore, our decisions in real practice are based on safety profiles, patient’ co-morbidities and physician experience or preference. Here we debate the pros and cons of the tumor-growth rate as a tool to select second line systemic treatment after failure to a prior tyrosine kinase-inhibitor in patients with advanced renal cell carcinoma.

s in real practice are based on safety profiles, patient’ co-morbidities and physician experience or preference. Here we debate the pros and cons of the tumor-growth rate as a tool to select second line systemic treatment after failure to a prior tyrosine kinase-inhibitor in patients with advanced renal cell carcinoma. INTRODUCTION The increased knowledge about the underlying pathogenesis of the metastatic renal cell carcinoma (mRCC) has led to the development of new therapeutic drugs that have completely changed patient prognosis. These drugs are targeting the vascular endothelial growth factor receptor (VEGFR) axis, the mammalian target of rapamycin (mTOR) pathway or the immune system and tumor cell interactions (PD1/PDL1). The number of patients that are candidates for a second line therapy after progressing on a first line varies from 43% to 79%[1]. The second line treatment is determinant in mRCC as patients can also benefit from an improvement in overall survival (OS) already achieved with first line choice and expand their chances for a longer therapeutic sequence. In this regard, a large registry-based experience in the United Kingdom has shown that those patients who received a second line treatment lived longer (33 mo; ranging from 30.8-35.2) than those who did not receive further treatment after first line (20.9 mo; ranging from 16.4-25.3)[2]. Fortunately, options for second line therapy have multiplied with the recent approval of nivolumab, cabozantinib and the combination of everolimus with lenvatinib[3-6]. However, there are no head-to-head comparisons between them and no predictive biomarker has been validated for the second line treatment decision making[7]. Besides, the uncertainty regarding the optimal therapeutic sequence, there is an urgent need for developing prognostic and predictive variables, in order to select patients who will benefit from a specific second line treatment[8].

redictive biomarker has been validated for the second line treatment decision making[7]. Besides, the uncertainty regarding the optimal therapeutic sequence, there is an urgent need for developing prognostic and predictive variables, in order to select patients who will benefit from a specific second line treatment[8]. There are some clinical and economic-derived factors coming from the pivotal trials of each agent that could be considered at the time of second line treatment decisions (Table 1). The patient’s tumor burden has been suggested from retrospective data as being strongly correlated with the progression free survival (PFS) and OS in patients with mRCC[9-12]. The expected response rate from the approved drugs has been reported to be different between cabozantinib, nivolumab and axitinib that achieve an overall response rate (ORR) of 17% to 22%, unlike the combination of everolimus with lenvatinib that has been reported to be of 35% in the phase II pivotal trial[3-6]. Moreover, the time required to achieve a tumor response is a major concern for heavily symptomatic patients that need an early tumor control. Prior tolerance and duration of response to first line treatment may identify those patients harboring a kidney tumor that greatly benefits from the angiogenic blockade (angiogenesis addiction), but may limit the decision in primary refractory patients[13,14]. Finally, we also propose the assessment of the tumor-growth rate (TGR), as a novel outcome measure that could help in the therapeutic sequence decision in the mRCC setting.

umor that greatly benefits from the angiogenic blockade (angiogenesis addiction), but may limit the decision in primary refractory patients[13,14]. Finally, we also propose the assessment of the tumor-growth rate (TGR), as a novel outcome measure that could help in the therapeutic sequence decision in the mRCC setting. Table 1 Phase III clinical trials evaluating approved drugs in second and subsequent treatment lines for metastatic renal cell carcinoma Axitinib Cabozantinib Lenvatinib + Everolimus Nivolumab Trial design Phase III Phase III Phase II Phase III Size 361 330 51 410 Patient population 2nd Line (100%) 2L- 71% 2nd Line (100%) 2L- 72% 3L- 29% 3L- 28% MSKCC risk % (Good/int/poor) 28/37/33 45/42/12 24/37/39 35/49/16 Comparator Sorafenib Everolimus Everolimus Everolimus ORR% (ICR) 19% 17% 35% 22% Progression disease (%) 22% 12% 4% 35% PFS (m) 6.7 (HR 0.66) 7.4 (HR 0.51) 12.8 (HR 0.40) 4.6 (HR 0.88) PFS (m) in pts with bone mets NR 7.4 (HR 0.33) NR NR OS (m) 20.1 (HR 0.96) 21.4 (HR 0.66) 25.5 (HR 0.59) 25.0 (HR 0.73) Dose reductions 30% 60% 71% N/A Discontinuations due to AEs 7% 9% 25% 8% Toxicity G3/4 (%) 56% 68% 71% 19% Average monthly cost (US basis) 9580$ 10229$ 22461$ 12435$ MSKCC: Memorial Sloan Kettering Cancer Center Criteria; ORR: Overall response rate; OS: Overall survival; PFS: Progression free survival; AE: Adverse events.

e reductions 30% 60% 71% N/A Discontinuations due to AEs 7% 9% 25% 8% Toxicity G3/4 (%) 56% 68% 71% 19% Average monthly cost (US basis) 9580$ 10229$ 22461$ 12435$ MSKCC: Memorial Sloan Kettering Cancer Center Criteria; ORR: Overall response rate; OS: Overall survival; PFS: Progression free survival; AE: Adverse events. Several authors have discussed that the Response Evaluation Criteria in Solid Tumors (RECIST) may be inadequate to completely evaluate the response of targeted therapies in mRCC as often induce long-lasting stable disease rather than tumor shrinkage[15-18]. In addition, these criteria do not take into account tumor growth kinetics, and might not be relevant in slow-growing diseases[19,20]. Therefore, alternate modalities to assess the drug response have been proposed to overcome the limitations of the RECIST criteria, such as Choi, SACT, MASS, ETPIC or iRECIST. These approaches include the tumor perfusion evaluation, via the use of CT response assessment combining reduction in both, size and arterial phase density, changes in tumor CT texture or metabolism or the immune component evaluation. However, none of them appear to be an adequate surrogate of response or clinical outcome for its application in routine clinical practice[16,18,21,22].

se of CT response assessment combining reduction in both, size and arterial phase density, changes in tumor CT texture or metabolism or the immune component evaluation. However, none of them appear to be an adequate surrogate of response or clinical outcome for its application in routine clinical practice[16,18,21,22]. TGR provides a dynamic and quantitative evaluation of tumor kinetics; it estimates the percentage of change in the tumor volume over one month. TGR is usually defined as the ratio between the slope of tumor growth before the initiation of treatment and the slope of tumor growth during treatment, and between the nadir and disease progression[9,23]. We can calculate TGR according to the formula shown in Figure 1[24]. The tumor size is defined using the sum of the longest diameters (SLD) of target lesions only, without considering non-target and new lesions. However, the assessment of the TGR in clinical practice is easier as there are internet tools available (http://ec2-54-218-32-173.us-west-2.compute.amazonaws.com:3838/tgrShiny/ or http://www.gustaveroussy.fr/doc/tgr_calculator/index_en.html). Figure 1 Tumor growth rate calculation formula. TGR: Tumor-growth rate.

TGR provides a dynamic and quantitative evaluation of tumor kinetics; it estimates the percentage of change in the tumor volume over one month. TGR is usually defined as the ratio between the slope of tumor growth before the initiation of treatment and the slope of tumor growth during treatment, and between the nadir and disease progression[9,23]. We can calculate TGR according to the formula shown in Figure 1[24]. The tumor size is defined using the sum of the longest diameters (SLD) of target lesions only, without considering non-target and new lesions. However, the assessment of the TGR in clinical practice is easier as there are internet tools available (http://ec2-54-218-32-173.us-west-2.compute.amazonaws.com:3838/tgrShiny/ or http://www.gustaveroussy.fr/doc/tgr_calculator/index_en.html). Figure 1 Tumor growth rate calculation formula. TGR: Tumor-growth rate. Current evidence from phase I studies in solid tumors and from phase III studies in mRCC (TARGET and RECORD trials) and metastatic neuroendocrine tumors (NETs) (CLARINET trial), although retrospective, show a significant association between prior TGR before the onset of the second line approach with the expected PFS and OS with the later systemic treatment administered[9,24-28]. Moreover, TGR could be an important tool in the evaluation of prognosis during treatment and after the discontinuation of VEGFR targeted agents. Iacovelli et al[29] showed that those patients with a higher than median TGR during treatment had a significantly shorter OS and, indeed, those patients with lower than the median TGR after discontinuation had longer OS, as compared to TGR after discontinuation greater than or equal to the median. Therefore, it would be possible to use TGR as a possible surrogate for tumor aggressiveness and survival in mRCC patients while on VEGFR-directed TKI in the first line. In the post hoc analysis from the CLARINET trial, TGR seemed to provide more precise information to predict pretreatment progression regarding actively growing tumors, but considered as stable disease by RECIST criteria, and more sensitive to detect early antitumor activity from treatment compared with RECIST criteria[28]. We consider that the addition of TGR in the assessment of individual patients undergoing targeted therapies may help clinicians to know if a given agent is modifying or not the course of the disease and guide the decision of which agent would be preferred in the subsequent line. However, for the use of TGR in the clinical setting, a prospective clinical trial for its validation would be needed[23].

ergoing targeted therapies may help clinicians to know if a given agent is modifying or not the course of the disease and guide the decision of which agent would be preferred in the subsequent line. However, for the use of TGR in the clinical setting, a prospective clinical trial for its validation would be needed[23]. Considering all aspects previously discussed, patients with mRCC that are candidate for a second line treatment could be differentiated into four main subgroups (Figure 2). Patients with florid symptoms, high tumor burden, short time to response to the first line (PFS less than 6 mo, so called, early progressors) and high TGR, in which we would need an early and high response, the combination of everolimus with lenvatinib should be considered, as we will target several mechanisms of action (VEGFR, fibroblast growing factor receptor, FGFR, and m-TOR pathways). In such patients, the expected benefit outweighs the increased toxicity of the combination therapy. In those patients with a long response to first antiangiogenic drug (PFS more than 18 mo, so called angiogenesis addicts) and low or intermediate TGR, the use of cabozantinib may be considered. Regarding those patients that are not responding radiographically but are stable for the advanced disease for a long period with a very low TGR (increase of less than 4% in the sum of the longest diameters per month) and have an adequate tolerability, we propose that axitinib could be a reliable option to prolong the clinical benefit. Finally, for patients with an interval free of progression with first line treatment between 6 and 18 mo, as considered intermediate-progressors, nivolumab may be the treatment of choice as an inhibitor of an actionable immune target by introducing a different mechanism of action against tumor growth.

clinical benefit. Finally, for patients with an interval free of progression with first line treatment between 6 and 18 mo, as considered intermediate-progressors, nivolumab may be the treatment of choice as an inhibitor of an actionable immune target by introducing a different mechanism of action against tumor growth. Figure 2 Hypothetical representation of different groups of patients and their patterns of response to first line treatment: Primary refractory patients with early progression and high tumor growth rate, intermediate progressors with intermediate tumor growth rate, very slow progressors with low tumor growth rate and late progressors with high tumor growth rate. TGR: Tumor-growth rate. Lastly, we highlight the upcoming availability of novel immune agents such as ipilimumab, atezolizumab, pembrolizumab either as single agent or in combination that might impact in the first line setting of patients with advanced RCC. Therefore, it is very likely that second line landscape of metastatic RCC may change shortly. Adaptation to the clinic of the amount of new data that are expected in a short term promises to be challenge.

either as single agent or in combination that might impact in the first line setting of patients with advanced RCC. Therefore, it is very likely that second line landscape of metastatic RCC may change shortly. Adaptation to the clinic of the amount of new data that are expected in a short term promises to be challenge. In conclusion, patients with mRCC receiving a second line treatment achieve a median OS of more than 2 years with novel agents. Thus, the optimal treatment selection in this setting allows us to provide the maximal clinical benefit to our patients, but with no definitive biomarker to guide our decision. In this setting, we have considered some relevant clinical parameters before choosing a certain agent such as the patient’s tumor burden, the expected response rate to the different drugs and the time to achieve this response, the prior response to previous VEGFR-TKIs, the toxicity profile of each agent and the patient preference. Thus, we propose the employment of the TGR as a new tool that could provide useful information in the management of mRCC patients in addition to clinical features that could better fit with one of the therapeutic alternatives (Figure 3). TGR may represent a surrogate of tumor aggressiveness, a relevant parameter before choosing a treatment and an early biomarker for treatment response and evaluation of the ability to interfere in the natural history of the tumor growth. TGR could be a valuable endpoint for clinical use in treatment decision-making favoring patients with mRCC, with more reliable information about prognosis and evaluation of response to molecular targeted agents.

or treatment response and evaluation of the ability to interfere in the natural history of the tumor growth. TGR could be a valuable endpoint for clinical use in treatment decision-making favoring patients with mRCC, with more reliable information about prognosis and evaluation of response to molecular targeted agents. Figure 3 Adapting the study data to our clinic. A proposed algorithm to treat second line metastatic renal cell carcinoma patients according to tumor growth rate and patients’ characteristics. TGR: Tumor-growth rate; TKI: Tyrosine kinase inhibitor; LEN: Lenvatinib; EVE: Everolimus. Conflict-of-interest statement: Grande E has served as advisor and delivered lectures for Pfizer, IPSEN, and Eisai; Martínez-Sáez O, Gajate-Borau P and Alonso-Gordoa T declares no conflict of interest related to this publication. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Spain Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: December 7, 2016 First decision: February 15, 2017 Article in press: March 13, 2017 P- Reviewer: Desai DJ, Iqbal M S- Editor: Song XX L- Editor: A E- Editor: Lu YJ

Core tip: Obesity and its main mediator leptin, are implicated in many protumorigenic processes, with emerging evidence from both the literature and our work pointing to a significant role in the development of resistance to chemotherapies. Chemoresistance is a major concern in the field of cancer therapy, as some cancers have no targeted therapies available. As obesity reaches epidemic proportions around the world, its impact on diseases like cancer and its treatment becomes more relevant. In this paper, we will discuss the current state of the literature regarding the influence of obesity and leptin on cancer treatment and the development of chemoresistance.

es available. As obesity reaches epidemic proportions around the world, its impact on diseases like cancer and its treatment becomes more relevant. In this paper, we will discuss the current state of the literature regarding the influence of obesity and leptin on cancer treatment and the development of chemoresistance. INTRODUCTION Obesity is the state of having excessive adipose tissue reserves, commonly defined as having a body mass index (BMI) of 30 or more. The global prevalence of obesity is high, with 37% of men and 38% of women being either overweight or obese[1]. There are significant health consequences for being overweight or obese. Obesity is closely associated to high rates of morbidity and mortality. It is considered responsible for an estimated 3.4 million deaths and 4% of years spent with a disability. There is a well-documented increased risk in obese and overweight people for numerous cancers, including thyroid, esophageal, kidney, colon, rectal, melanoma, leukemia, endometrial, gallbladder, pancreas and breast cancer[2-6]. In addition, weight gain before age 50 has been associated with greater risk of breast cancer, especially estrogen negative breast cancer[7-9]. A contributing factor could be complications related to therapy, as obesity is correlated with breast cancer recurrence, with increasing BMI being correlated with increased risk of breast cancer relapse. Obesity impacts on life expectancy, with premenopausal and postmenopausal obese women being 1.75 and 1.34 times, respectively, at increased risk of death from breast cancer[10].

py, as obesity is correlated with breast cancer recurrence, with increasing BMI being correlated with increased risk of breast cancer relapse. Obesity impacts on life expectancy, with premenopausal and postmenopausal obese women being 1.75 and 1.34 times, respectively, at increased risk of death from breast cancer[10]. A distinctive characteristic of obesity and overweight conditions is the high serum level of the main adipokine, leptin secreted by adipose tissue. Leptin, from the Greek “leptos”, thin, is a 16 kDa protein, composed of 167 aminoacids; its gene, Ob, is in humans on chromosome 7q32. Ob gene is composed by three exons and 2 introns, spanning 20 kb. Leptin is the first discovered adipokine, a cytokine secreted by adipocytes, both from the white adipose tissue and brown adipose tissue. Placenta, ovaries, skeletal muscle, bone marrow, stomach, pituitary gland, and mammary epithelial cells have been shown to express leptin[11]. Several cancer cell types and tumor stroma also express leptin[12].

ed adipokine, a cytokine secreted by adipocytes, both from the white adipose tissue and brown adipose tissue. Placenta, ovaries, skeletal muscle, bone marrow, stomach, pituitary gland, and mammary epithelial cells have been shown to express leptin[11]. Several cancer cell types and tumor stroma also express leptin[12]. OBESITY, LEPTIN/OB-R AND CANCER The main role of leptin is to regulate energy balance by inhibiting hunger. Leptin levels correlate to adiposity. Under physiological conditions leptin binds and activates receptors in the arcuate nucleus of the hypothalamus, which regulate appetite[13]. In obese people, a decreased sensitivity to leptin was observed, leading to a decreased capacity to feel satiety[14]. A consequence of this resistance is overeating that results in obesity and the concomitant high serum levels of leptin. In obese individuals serum leptin levels are 10 times higher (i.e., 40 ng/mL) than normal weight people (i.e., 4 ng/mL)[15]. The upregulation of leptin has an important role in carcinogenesis[16].

4]. A consequence of this resistance is overeating that results in obesity and the concomitant high serum levels of leptin. In obese individuals serum leptin levels are 10 times higher (i.e., 40 ng/mL) than normal weight people (i.e., 4 ng/mL)[15]. The upregulation of leptin has an important role in carcinogenesis[16]. Leptin receptor (Ob-R) is predominantly found in the hypothalamus[17], but is expressed at lower level in the whole body, including pancreas[18] and mammary epithelial cells[19]. Remarkably, cancer cells overexpress Ob-R, which enable them to respond to leptin that is more prominent in obese individuals showing high levels of the adipokine. Ob-R belongs to Class I super-family cytokine receptors. It is a transmembrane protein composed by an extra-cellular domain, responsible for binding leptin, a transmembrane domain and a cytoplasmic domain for signaling[20]. Currently six different isoforms of the leptin receptor have been identified, Ob-R a-f, generated by mRNA splicing or proteolytic processing. Ob-R isoforms are divided in three classes, short and long (which are bound to the cellular membrane) and secreted (a soluble protein that binds leptin in blood). The long isoform Ob-Rb (or l) is the predominant one, expressed at high levels in different cell types. Ob-Rb has full signaling capabilities in contrast to short Ob-R isoforms. It is generally accepted that leptin binding to Ob-R provokes the formation of a homodimer that is responsible for leptin-mediated signals. Leptin and Ob-R have absolute specificity for binding. Once leptin binds to Ob-Rb, it activates several signaling pathways. Because Class I cytokine receptors lack auto phosphorylation function they need auxiliary kinases to initiate signaling upon ligand binding. The first signaling event after leptin binding to Ob-Rb is the activation of janus kinase/signaling transducer and activation of transcription factor pathway (JAK/STAT)[21]. JAK2 recruitment to Ob-R intracytoplasmatic tail leads to the phosphorylation of the kinase, subsequent phosphorylation of Ob-R in several intracytoplasmatic sites and recruitment and phosphorylation of tyrosine residue on STATs. Phosphorylated STATs, then form hetero or homodimers and translocate to the nucleus to induce the transcription of specific genes[22].

tail leads to the phosphorylation of the kinase, subsequent phosphorylation of Ob-R in several intracytoplasmatic sites and recruitment and phosphorylation of tyrosine residue on STATs. Phosphorylated STATs, then form hetero or homodimers and translocate to the nucleus to induce the transcription of specific genes[22]. Leptin plays roles in other physiological functions, as indicated by the presence of its receptor in different organs and tissues types besides the hypothalamus[23]. Leptin is involved in immunity, proliferation, differentiation, apoptosis, angiogenesis, inflammation, fertility and oncogenesis[12,16,22]. Leptin is known to inhibit bone formation[24]. It can also regulate the ovulatory cycle by stimulating GnRH from the hypothalamus[25,26] and is an important factor in embryo implantation[27-29]. Leptin is involved in the onset of puberty[30], regulates glucose homeostasis[31], hematopoiesis[32], and modulate immunity like T cell activity in response to atherosclerosis[33]. Leptin has been speculated to be an inflammatory marker that responds specifically to adipose-derived inflammatory cytokines[34]. Obesity is a significant risk factor for cancer incidence and mortality. The effects of obesity on cancer could be due in part to leptin’s elevated levels and Ob-R over expression in cancer cells, which enable leptin-deregulated pleiotropic signals in cancer. Leptin has been shown to have several pro-tumorigenic effects, such as increasing cancer cell proliferation, anti-apoptosis, angiogenesis, self-renewal and possibly resistance to chemotherapeutic treatment[12,16].

s and Ob-R over expression in cancer cells, which enable leptin-deregulated pleiotropic signals in cancer. Leptin has been shown to have several pro-tumorigenic effects, such as increasing cancer cell proliferation, anti-apoptosis, angiogenesis, self-renewal and possibly resistance to chemotherapeutic treatment[12,16]. Several studies have linked the effects of leptin to the proliferation of cancer both in vivo and in vitro experimental models, and from patient data. Leptin signaling has been consistently linked to the development of breast, endometrial, pancreatic, colon, prostatic, hepatic, skin, brain, oesophagus, stomach, thyroid gland, and ovarian cancers, and leukemia and chondrosarcoma[35-43].

on of cancer both in vivo and in vitro experimental models, and from patient data. Leptin signaling has been consistently linked to the development of breast, endometrial, pancreatic, colon, prostatic, hepatic, skin, brain, oesophagus, stomach, thyroid gland, and ovarian cancers, and leukemia and chondrosarcoma[35-43]. Leptin induces breast cancer cell growth in vitro and in vivo. Several leptin-induced signaling pathways and factors have been linked to the proliferation of breast, endometrial and pancreatic cancer cells[12,16,36,37]. Leptin induced tumor cell growth and inhibited apoptosis in papillary thyroid cancer (PTC) cells. Serum levels of leptin were shown to be higher in patients with PTC than in negative controls[42]. An increase in the expression of leptin receptor Ob-R was observed in PTC specimens[44]. Leptin can induce the development of non-alcoholic fatty liver disease (NAFLD), one of the major cause of hepatocellular carcinoma, by promoting insulin resistance, steatosis and hepatic inflammation by increasing transforming growth factor beta (TGF-β) expression[43]. Leptin is overexpressed in colon cancer, Ob-R mRNA was found in cancer cell lines and colon tumors[45] and Ob-R protein expression was confirmed by western blot[46]. Serum leptin levels were significantly high in patients with lung cancer, compared to healthy individuals. Lung cancer tissues showed higher expression of leptin compared to normal lung tissue[47]. Leptin was shown to stimulate the proliferation of human myeloid leukemia cell lines[48], and it might play a role in the development of prostate cancer, it can increase growth and survival of prostate cancer cells and Ob-R mRNAs has been found in prostate cancer cells through RT-PCR[49]. Epithelial ovarian cancer (EOC) is one of the principal cause of death in gynecological malignancies, but the role of leptin in this disease still needs further investigation as Ob-R mRNA was found in several immortalized EOC cell lines[50]. Limited data suggested also a link between leptin and adrenal cancer[51].

49]. Epithelial ovarian cancer (EOC) is one of the principal cause of death in gynecological malignancies, but the role of leptin in this disease still needs further investigation as Ob-R mRNA was found in several immortalized EOC cell lines[50]. Limited data suggested also a link between leptin and adrenal cancer[51]. Leptin induced pleiotropic effects in cancer cells. Leptin increased breast cancer cell proliferation, which was linked to the up regulation of cyclin D[52] and increased expression of anti-apoptotic proteins like Bcl-2 in breast cancer[53]. Additionally, leptin can down regulate pro-apoptotic Bax[54]. Leptin induces tumor angiogenesis that has a pivotal role in solid tumor growth and metastasis. Leptin not only promotes the expression of angiogenic factors like vascular endothelial growth factor (VEGF)[55], VEGFR-2[52,56], and fibroblast growth factor 2 (FGF-2), but also itself induces vascular endothelial cell proliferation in vitro with similar effects than VEGF[57]. Moreover, in the absence of VEGF, leptin induced Notch signaling pathway in endothelial cells that was linked to leptin-induced transphosphorylation of VEGFR-1 and VEGFR-2[58]. Leptin induces two angiogenic factors: Interleukin (IL)-1[59] and Notch[60] that can increase VEGF expression. Moreover, leptin induces the secretion and synthesis of proteases and adhesion molecules needed for the development of angiogenesis. Leptin induces expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) that are involved in tissue remodeling, specifically the breakdown of extracellular matrix proteins[61,62]. Additionally, leptin induces the expression of avB3 integrin that is also involved in angiogenesis[37,63]. Leptin induces production of inflammatory cytokines like IL-1, IL-6 and tumor necrosis factor (TNF)-α, which like leptin can induce the expression of metalloproteinases, promoting tumor invasion and metastasis. TNF-α acts on adipocytes increasing leptin expression[34].

grin that is also involved in angiogenesis[37,63]. Leptin induces production of inflammatory cytokines like IL-1, IL-6 and tumor necrosis factor (TNF)-α, which like leptin can induce the expression of metalloproteinases, promoting tumor invasion and metastasis. TNF-α acts on adipocytes increasing leptin expression[34]. LEPTIN-INDUCED NOTCH AND RBP-JK AFFECT CANCER PROGRESSION Gonzalez-Perez’s lab earlier reported that leptin signaling crosstalk to Notch in breast cancer[60]. Notch signaling is an embryonic conserved pathway involved in proliferation, angiogenesis, cell fate and development. Notch system is composed by transmembrane proteins: Receptors (Notch1-4) and ligands expressed in adjacent cells (Delta-like, Dll1-3, and Jagged-like, JAG1-2), and molecular targets hairy enhancer of split (Hes1-7), hairy/enhancer-of-split related with YRPW motif subfamilies (Hey1, Hey2, HeyL, HesL/HelT, Dec1/BHLHB2, Dec2/BHLHB3) and survivin. Notch receptors are all composed of an extracellular domain (NECD) where ligands bind, a transmembrane domain (TM) and an intracellular domain (NICD). Notch is activated upon binding to a ligand that triggers a proteolytic cascade producing activated NICD, which is transported to the nucleus where it binds to a tumor repressor, DNA-binding protein, recombination signal binding protein for immunoglobulin kappa J (RBP-Jk) or CBF1/Su(H)/Lag-1 (CSL) family of transcription factors[64].

ted upon binding to a ligand that triggers a proteolytic cascade producing activated NICD, which is transported to the nucleus where it binds to a tumor repressor, DNA-binding protein, recombination signal binding protein for immunoglobulin kappa J (RBP-Jk) or CBF1/Su(H)/Lag-1 (CSL) family of transcription factors[64]. RBP-Jk is a DNA binding factor, which mediate either transcriptional repression or transcriptional activation. RBP-Jk binds to the ubiquitous corepressor proteins (Co-R: Silencing mediator of retinoid and thyroid hormone receptors, SMRT and Ski-interacting protein, SKIP)[65], histone deacetylases (HDACs), CBF1 interacting corepressors (CIR), and SAP30 (a linker between CBF1 and the HDAC complex)[66], which repress transcription of some genes. Thus, RBP-Jk is a transcription factor that acts as a repressor in complex with SMRT and SKIP when it is not associated with Notch. In contrast, activated NICD-RBP-Jk complex displaces co-repressors and recruits coactivator (Co-A). When RBP-Jk is associated with NICD it acts as a transcriptional activator in complex with mastermind-like proteins, MAML[67]. This process is required for Notch-induced canonical signals that increase the transcription of target genes such as Hes, Hey, nuclear factor-kappa B (NF-κB), cyclin D, c-Myc and others[64]. Additionally, Notch signaling is linked to expansion of cancer stem cell populations (CSC), which show self-renewal capabilities and can recapitulate tumor heterogeneity and are believed to be responsible for recurrence and drug resistance[68,69].

es, Hey, nuclear factor-kappa B (NF-κB), cyclin D, c-Myc and others[64]. Additionally, Notch signaling is linked to expansion of cancer stem cell populations (CSC), which show self-renewal capabilities and can recapitulate tumor heterogeneity and are believed to be responsible for recurrence and drug resistance[68,69]. Notch signaling is deregulated in many cancers. Indeed, deregulation of Notch signaling is a hallmark of breast cancer[64]. In breast and pancreatic cancer cells leptin upregulates Notch receptors, ligands and targets[16,60]. Moreover, latest reports show a positive correlation between leptin, Ob-R and Notch components in endometrial cancer tissues from obese patients[70]. Leptin induces RBP-Jk and Notch that impacts on CSC and self-renewal[16,60,71]. Moreover, a novel crosstalk between Notch, IL-1 and leptin (NILCO) was found in breast cancer[53,60,72]. NILCO induces proliferation/migration and upregulation of VEGF/VEGFR-2, and could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin effects in breast cancer[60]. Paradoxically, low expression of RBP-Jk has been reported in several solid tumors that was associated with increase aggressiveness[73]. Our preliminary data indicate that knockdown of RBP-Jk in breast cancer cells induces a dramatic increase of Notch 3 and Notch 4 expression, CSC population (CD24-/CD44+) and N-cadherin (epithelial-mesenchymal-transformation marker)[74]. These data may suggest that tumor repressor activities of RBP-Jk could overcome the oncogenic actions of NICD-RBP-Jk complex upon activation of Notch, thus, cancer cells downregulate RBP-Jk expression in order to proliferate and develop tumors. However, this topic deserves follow up and more deep mechanistic investigation.

e data may suggest that tumor repressor activities of RBP-Jk could overcome the oncogenic actions of NICD-RBP-Jk complex upon activation of Notch, thus, cancer cells downregulate RBP-Jk expression in order to proliferate and develop tumors. However, this topic deserves follow up and more deep mechanistic investigation. LEPTIN SIGNALING INDUCES BREAST CANCER PROGRESSION Leptin and Ob-R are low expressed in human mammary glands, yet they play a role in the normal development[75]. In contrast, leptin and Ob-R expression is upregulated in breast cancer[76]. Obese patients with breast cancer show tumoral leptin overexpression that correlated to larger and more advanced tumors[77]. The molecular mechanisms involved in obesity-related breast carcinogenesis are not very clear. The binding of leptin to its receptor on breast cancer cells induces the activation of multiple oncogenic pathways, including Jak/STAT3, ERK1/2, and phosphoinositide 3-kinase (PI-3K) pathways, cyclin D1 expression and retinoblastoma protein hyperphosphorylation[78]. Triple negative breast cancer (TNBC) showed high level of molecules correlated with metastasis and lower patients survival (i.e., IL-1, Notch and VEGF/VEGFR2). Notch, IL-1 and leptin crosstalk outcome (NILCO) seems to play essential roles in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer[64].

molecules correlated with metastasis and lower patients survival (i.e., IL-1, Notch and VEGF/VEGFR2). Notch, IL-1 and leptin crosstalk outcome (NILCO) seems to play essential roles in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer[64]. Breast adipose tissue is a source of estrogen, which is involved in tumorigenesis. Estrogens promote cell proliferation by inhibiting apoptosis and inducing angiogenesis[79]. Therefore, these molecules are breast cancer markers and therapeutic targets. A functional crosstalk between estrogen and leptin exists and may act to promote tumorigenesis[80]. The aromatization of androstenedione in adipose tissue is the main source of estrogen[81], a reaction catalysed by the enzyme aromatase, whose expression is increased by leptin in ER positive breast cancer cells[82]. Leptin has been shown to induce resistance in ER positive cancer cells to Faslodex[83] and Tamoxifen[84]. Leptin binding to ObR was also shown to transactivate HER2/neu[85], which is an important oncogenic protein involved in breast cancer growth. All these data indicate that leptin is involved in the development of breast cancer. Therefore, the use of leptin-signaling targeting drugs could be a novel strategy in breast cancer management.

o ObR was also shown to transactivate HER2/neu[85], which is an important oncogenic protein involved in breast cancer growth. All these data indicate that leptin is involved in the development of breast cancer. Therefore, the use of leptin-signaling targeting drugs could be a novel strategy in breast cancer management. LEPTIN SIGNALING PROMOTES THE EXPANSION OF CANCER STEM CELLS Breast cancer stem cells The cancer stem cell (CSC) theory postulates the existence of a sub-population of cancer cells with the ability to undergo self-renewal and also tumor differentiation[86]. The presence of these cells is a risk factor for carcinogenesis. CSC can recreate the bulk of the tumor, and are believed to be responsible for tumor initiation, cancer recurrence and metastatic progression[87]. CSC in breast cancer (BCSC) initiate and drive carcinogenesis and tumor differentiation[88]. BCSC can be identified by several molecular phenotypic markers. Networks of cytokines and growth factors, including leptin, have been implicated in BCSC interaction with the tumor micro-environment[89]. BCSC exhibit a high sensitized responses to leptin. It was reported that leptin mediates microenvironment effects on BCSC activity that establishes a self-reinforcing signaling circuit. Leptin upregulates several factors considered BCSC markers in several breast cancer cell lines like, including CD44, ALDH1[60], HER2[90], Oct-4 and Sox2[91]. Leptin is also involved in activation of transcriptional factors associated with BCSC, like STAT3[92] and NF-κB[93]. BCSC markers are shown in Table 1[60,90,91,94-105].

upregulates several factors considered BCSC markers in several breast cancer cell lines like, including CD44, ALDH1[60], HER2[90], Oct-4 and Sox2[91]. Leptin is also involved in activation of transcriptional factors associated with BCSC, like STAT3[92] and NF-κB[93]. BCSC markers are shown in Table 1[60,90,91,94-105]. Table 1 Breast cancer stem cells markers

upregulates several factors considered BCSC markers in several breast cancer cell lines like, including CD44, ALDH1[60], HER2[90], Oct-4 and Sox2[91]. Leptin is also involved in activation of transcriptional factors associated with BCSC, like STAT3[92] and NF-κB[93]. BCSC markers are shown in Table 1[60,90,91,94-105]. Table 1 Breast cancer stem cells markers Markers Localization Ref. Markers Localization Ref. CD44 Cell surface Guo et al[60], 2011 MET Cell surface Baccelli et al[100], 2013 CD24 Cell surface Kakarala et al[94], 2008 CD133 Cell surface Tume et al[101], 2016 Epcam Cell surface Chiotaki et al[95], 2015 CD338 Cell surface Leccia et al[102], 2014 CD49f Cell surface Chiotaki et al[95], 2015 ALDH1 Cytoplasm Guo et al[60], 2011 MUC1 Cell surface Nigam[96], 2013 Bmi I Cytoplasm Kim et al[103], 2015 CD29 Cell surface Yeo et al[97], 2016 GLI I Cytoplasm Fernandez-Zapico[104], 2013 CD61 Cell surface Yeo et al[97], 2016 Sox2 Cytoplasm Feldman et al[91], 2012 CD47 Cell surface Zhang et al[98], 2015 Oct-4 Cytoplasm Feldman et al[91], 2012 HER2 Cell surface Korkaya et al[90], 2008 NANOG Cytoplasm McClements et al[105], 2013 eHSP90 Cell surface Stivarou et al[99], 2016 PANCREATIC CANCER STEM CELLS Characterization of pancreatic cancer stem cells Pancreatic cancer stem cells (PCSC) are characterized by the expression of cell markers, including CD24+CD44+, CD133+, CD24+CD44+ and epithelial specific antigen (ESA+ or EpCAM+) and aldehyde dehydrogenase (ALDH+)[106-108]. PCSC represent a rare cell population of 0.5%-1% of total PC cells (Table 2). Remarkably, when isolated and inoculated into nude mice, PCSC generate tumors, whereas implantation of PC cells negative for these markers could not. Rasheed et al[109] showed that a subpopulation of PCSC, CD133+CXCR4+ was found in patients with PC metastatic disease. Additionally, PC ALDH+ cells showed enhanced clonogenic growth, migratory potential and affected negatively the overall survival of PC patients. In 2011, Li et al[106] described a new population of PCSC c-Met+ involved in PC growth and metastasis. Recent preclinical data suggest PC c-Met+ cells are involved in drug resistance. Indeed, the use of a c-Met inhibitor (Cabozantinib) in PC patients overcomes Gemcitabine resistance[110]. PCSC could also be identified by flow cytometry using Hoechst 33342 dye. PC side population that can exclude Hoechst 33342 dye correlated with chemoresistance and poor survival[111].

re involved in drug resistance. Indeed, the use of a c-Met inhibitor (Cabozantinib) in PC patients overcomes Gemcitabine resistance[110]. PCSC could also be identified by flow cytometry using Hoechst 33342 dye. PC side population that can exclude Hoechst 33342 dye correlated with chemoresistance and poor survival[111]. Wang et al[112] described a similar PC side population (Hoechst 33342 negative) showing high expression for CD133+, ABCG2+ and Notch1+, which were more chemoresistant compared to non-side population cells. A PCSC population marked by the expression of Doublecortin and Ca/Calmodulin- Dependent Kinase-Like 1 (Dclk1) was described by Bailey et al[113] in 2014. PCSC Dclk1+ were found in PanIN (pancreatic intraepithelial neoplasia) lesions, as well as in invasive stages of PC. These findings suggest that PCSC populations can be identified at the early stages of pancreatic tumorigenesis and may serve as a biomarker for early detection of this deadly disease. Table 2 Pancreatic cancer stem cells markers

Wang et al[112] described a similar PC side population (Hoechst 33342 negative) showing high expression for CD133+, ABCG2+ and Notch1+, which were more chemoresistant compared to non-side population cells. A PCSC population marked by the expression of Doublecortin and Ca/Calmodulin- Dependent Kinase-Like 1 (Dclk1) was described by Bailey et al[113] in 2014. PCSC Dclk1+ were found in PanIN (pancreatic intraepithelial neoplasia) lesions, as well as in invasive stages of PC. These findings suggest that PCSC populations can be identified at the early stages of pancreatic tumorigenesis and may serve as a biomarker for early detection of this deadly disease. Table 2 Pancreatic cancer stem cells markers Stem cell population Localization Ref. CD24+CD44+ Extracellular Li et al[106], 2007 CD24+CD44+ESA+ Extracellular Li et al[106], 2007 CD133+CXCR4+ Extracellular Hermann et al[107], 2007 CD133+CD44+ Extracellular Ji et al[123], 2011 C-Met Extracellular Li et al[106], 2007 DCLK1 Intracellular Bailey et al[113], 2014 ABCB1 Extracellular Van den broeck et al[111], 2013 Sox2 Intracellular Herreros-Villanueva et al[117], 2014 PCSC show self-renewal and multipotency, and can initiate and propagate the parental tumor while serial passage into immunocompromised mice[114]. CSC including PCSC have retained the expression of at least three of the transcription factors that are characteristic to embryonic stem cells (ESC) (Oct-4, Sox-2 and Nanog). Increased levels of Oct-4 and Nanog are correlated with early stages of carcinogenesis and worse prognosis. Oct-4 and Nanog play important roles in embryonic development, and also in maintaining the stemness of PCSC. In contrast, PCSC double knockdown of Oct-4 and Nanog show reduced proliferation, migration, invasion and tumorigenesis[115]. Additionally, Oct-4 contributes to metastasis and cancer multidrug resistance[116]. De novo Sox2 expression alone in PC is sufficient to promote self-renewal, differentiation and stemness. Although ESC and PCSC share the property of self-renewal, ESC favors differentiation, while PCSC act more toward proliferation and inhibition of apoptosis. Targeting PCSC may be a viable therapeutic strategy against PC. A better understanding of Oct-4, Sox-2 and Nanog regulation could facilitate the design of individualized therapies for PC patients[117].

rty of self-renewal, ESC favors differentiation, while PCSC act more toward proliferation and inhibition of apoptosis. Targeting PCSC may be a viable therapeutic strategy against PC. A better understanding of Oct-4, Sox-2 and Nanog regulation could facilitate the design of individualized therapies for PC patients[117]. Current studies demonstrate that PCSC determine tumor relapse and metastasis following chemotherapy[118]. From a clinical perspective, targeting PCSC populations would ensure tumor eradication. However, PCSC possess escape mechanisms shared with normal stem cells, such as over-expression of multi-drug transporters. These transporters increase the efflux of anticancer drugs, thereby reducing their accumulation inside the cancer cells[118]. ABCB1 protein was significantly augmented in CD44+ cells during acquisition of PC cells resistance to Gemcitabine. CD44 expression in PC was correlated with histologic grade and poor prognosis. These data indicate that cancer stem cells were expanded during the acquisition of Gemcitabine chemoresistance[119]. In line with these findings, the administration of anti-CD44 monoclonal antibody to a human PC xenograft mouse model increased Gemcitabine sensitivity[120]. Additionally, Metformin enhanced the capacity of Gemcitabine to inhibit the proliferation of PC cells by inhibiting the proliferation of CD133+ cells[121]. Side population PCSC identified by Van der Broeck in 2012[111] are resistant to Gemcitabine. Side population PC cells isolated from Panc-1 cell line have been found to express both ABCB1 and ABCG2, which contribute to chemoresistance[122]. Identification of enhanced stem cell populations within PC tumors might be used as biomarkers for personalized therapy.

der Broeck in 2012[111] are resistant to Gemcitabine. Side population PC cells isolated from Panc-1 cell line have been found to express both ABCB1 and ABCG2, which contribute to chemoresistance[122]. Identification of enhanced stem cell populations within PC tumors might be used as biomarkers for personalized therapy. Pancreatic cancer stem cell regulators Several factors could affect PCSC. Accumulated evidence suggested that microRNAs are involved in the regulation of PCSC. Specifically, miRNA34 affects the maintenance and survival of PCSC[123]. Obesity is associated with increased severity of acute pancreatitis[124] and decreased survival of PC patients. In obese mice, IL-6 contributes to prolonging inflammation and altering resolution from pancreatic damage, possibly contributing to a microenvironment favorable to tumorigenesis[125]. Cigarette smoking and nicotine, a major risk factor in PC, increase monocyte chemoattractant protein 1 (MCP-1) expression in PC cells. MCP-1 was found in 60% of invasive PC lesions, of whom 66% were smokers[126]. The concentration of six cytokines (IL-1β; IL-6, IL-8, VEGF, TGF, IL-10) were consistently reported to be increased in pancreatic ductal adenocarcinoma (PDAC) patients. These molecules were associated with the severity of PDAC (i.e., metastasis, tumor size, and advanced stage) that suggest these cytokines have prognostic biomarker for PC[127]. Additionally, IL-8/CXCR1 axis was associated with cancer stem cell properties in PC[128]. CXCR1 expression in PC patients correlates with lymph node metastasis and poor survival. MMP-13 has been shown to help mediate the effect of leptin on invasiveness and metastasis of PC. In addition, there was a positive correlation between the expression of PCSC markers CD133 and CD44, and CXCR1[129].

rties in PC[128]. CXCR1 expression in PC patients correlates with lymph node metastasis and poor survival. MMP-13 has been shown to help mediate the effect of leptin on invasiveness and metastasis of PC. In addition, there was a positive correlation between the expression of PCSC markers CD133 and CD44, and CXCR1[129]. P300 is a tumor suppressor gene. However, this factor is also upregulated in various cancer types and associated with worse prognosis. In PC, P300 is associated with chemoresistance from apoptosis upon Gemcitabine-induced DNA damage[130]. TGF-β negatively regulates ALDH1 expression (a PCSC marker) in a SMAD dependent manner in PC cells. This regulatory mechanism might be disrupted by SMAD4 mutations and deletions in PC cells[131]. The binding of stem cell factor (SCF, a protein involved in PC progression) to its receptor, c-kit, determines an increase in HIF-1α synthesis that affects cancerous transformation, chemoradiotherapy resistance, and tumor progression[132].

mechanism might be disrupted by SMAD4 mutations and deletions in PC cells[131]. The binding of stem cell factor (SCF, a protein involved in PC progression) to its receptor, c-kit, determines an increase in HIF-1α synthesis that affects cancerous transformation, chemoradiotherapy resistance, and tumor progression[132]. Additionally, high levels of leptin receptor, Ob-R, are associated with PC stage and lymph node metastasis and overall shorter survival. Ob-R and HIF-1α expression was highly associated in PC tissues. HIF-1α regulated the expression of Ob-R in PC[133]. Leptin binding to Ob-R was earlier found to induce HIF-1α in breast cancer cells. Leptin-induced HIF-1α was involved in the upregulation of VEGFR2 in these cells[55]. Therefore, it is possible that a leptin-induced HIF-1α feedback regulating Ob-R is present in PC. Moreover, robust expression of Ob-R is a characteristic of tumor initiating stem cells and pluripotent stem cells that was mediated directly by Oct-4 and Sox-2[91]. Furthermore, the expression of leptin in gastro-esophageal adenocarcinomas was associated with chemoresistance. The use of leptin receptor antagonist SHLA increased the sensitivity to Cisplatin in the resistant gastric cancer cell line, AGS Cis5, and the oesophageal adenocarcinoma cell line, OE33[134].

4 and Sox-2[91]. Furthermore, the expression of leptin in gastro-esophageal adenocarcinomas was associated with chemoresistance. The use of leptin receptor antagonist SHLA increased the sensitivity to Cisplatin in the resistant gastric cancer cell line, AGS Cis5, and the oesophageal adenocarcinoma cell line, OE33[134]. Chemoresistance and cancer stem cells In the absence of targeted therapeutic options, chemotherapy, along with surgery and radiotherapy are usually the last and only options for cancer treatment. Thus, resistance to chemotherapy is a vital area of research. Investigations on the mechanisms involved in chemoresistance are essential to overcome this issue. There are several mechanisms related to chemoresistance that have been identified in cancer cells, which include reduction or inhibition of drug-induced apoptosis, overexpression of detoxification and efflux proteins, increased expression of survival factor and pathways as nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) and PI-3K/Akt, hypoxia and hypoxia inducible factor HIF, and expansion of chemoresistant CSC among others[135-138].

drug-induced apoptosis, overexpression of detoxification and efflux proteins, increased expression of survival factor and pathways as nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) and PI-3K/Akt, hypoxia and hypoxia inducible factor HIF, and expansion of chemoresistant CSC among others[135-138]. Inhibition of apoptosis Numerous chemotherapies target the increased DNA synthesis that cancer cells undergo. Classes of chemotherapeutics such as platinum agents (Cisplatin), alkylating agents (Cytoxane) and anthracyclines (Adriamycin or Doxorubicin) inhibit DNA synthesis. A consequence of the action of these agents is increased apoptosis due to DNA damage. The p53 pathway plays an important role in cancer cell avoidance of apoptosis, with mutations in the p53 gene associated with increased drug resistance in cancer cell lines and poor survival in cancer patients[135,139]. In addition, cancer cells have been known to competitively inhibit Caspase 3, a central molecule in the apoptosis pathway. These cells show increased expression of B cell lymphoma 2 (BCL-2) and B cell lymphoma extra-large (BCL-xL) anti apoptotic proteins[140-143].

ll lines and poor survival in cancer patients[135,139]. In addition, cancer cells have been known to competitively inhibit Caspase 3, a central molecule in the apoptosis pathway. These cells show increased expression of B cell lymphoma 2 (BCL-2) and B cell lymphoma extra-large (BCL-xL) anti apoptotic proteins[140-143]. Detoxification and efflux proteins Aldehyde dehydrogenases (ALDH) are a class of enzymes that oxidise aldehydes. ALDH isoforms have been implicated in CSC and resistance to chemotherapeutics. ALDH1 is a marker of CSC and progenitor cells[144], whose expression correlated with poor response to Docetaxel therapy in advanced breast cancer[145]. Increased expression of ALDH1A1 and ALDH3A1 lead to greater inactivation of Cyclophosphamide in breast cancer[136]. ATP binding cassette (ABC) transporters are a family of transmembrane proteins involved in the efflux of drugs from cancer cells. ABC (ABCB1, ABCC1 and ABCG2) family of proteins are mainly found on CSC side-population (SP, Hoechst negative). ABCB1, also known as p-glycoprotein, CD243 or MDR1, is an efflux pump protein with broad substrate specificity. It is known to pump out chemotherapeutics such as Doxorubicin and Paclitaxel. ABCC1 is known to give cancer cell resistance to anthracyclines such as Doxorubicin. ABCG2, also called the breast cancer resistance protein or CDw338, allows cancer cell resistance to Mitoxantrone and Doxorubicin[146]. NFκB pathway

ATP binding cassette (ABC) transporters are a family of transmembrane proteins involved in the efflux of drugs from cancer cells. ABC (ABCB1, ABCC1 and ABCG2) family of proteins are mainly found on CSC side-population (SP, Hoechst negative). ABCB1, also known as p-glycoprotein, CD243 or MDR1, is an efflux pump protein with broad substrate specificity. It is known to pump out chemotherapeutics such as Doxorubicin and Paclitaxel. ABCC1 is known to give cancer cell resistance to anthracyclines such as Doxorubicin. ABCG2, also called the breast cancer resistance protein or CDw338, allows cancer cell resistance to Mitoxantrone and Doxorubicin[146]. NFκB pathway NFκB signaling pathway is a survival mechanism that controls DNA transcription of several genes. In non-malignant cells NFκB signaling plays a central role in immune response to infection. It is responsible for cellular responses to a wide range of stimuli which include reactive oxygen species, ionising radiation, bacterial lipopolysaccharide, IL-β and TNF-α. To drive oncogenesis, NFκB signaling cooperates or crosstalks with signaling pathways, oncogenic or cancer-related proteins such as STAT3, p53, ALDH1, glycogen synthase kinase (GSK-3β), PI-3K, MAPK, PKC, and others[147].

include reactive oxygen species, ionising radiation, bacterial lipopolysaccharide, IL-β and TNF-α. To drive oncogenesis, NFκB signaling cooperates or crosstalks with signaling pathways, oncogenic or cancer-related proteins such as STAT3, p53, ALDH1, glycogen synthase kinase (GSK-3β), PI-3K, MAPK, PKC, and others[147]. NFκB signaling is a critical mediator of chemoresistance in cancer. Glioblastoma multiforme’s resistance to Gemcitabine involves NFκB, ALDH and ROS actions[148]. Anti-ovarian cancer effects of MK5108 compound relied on the inhibition of the Aurora-A kinase and NFkB signaling, which induced polyploidy and cell cycle arrest[149]. In breast cancer, targeting NFκB signaling increased apoptosis and reduced proliferation in drug resistant breast cancer cell lines[150]. In mesothelioma, the STAT3-NFκB signaling crosstalk is essential in ALDH-mediated chemoresistance[151]. Abnormal activation of NFκB signaling is also implicated in cancer resistance to Paclitaxel therapy[152]. HIF and tumor hypoxia Hypoxia is a term which describes deficient oxygen supply to tissues due to poor vasculature, as it is in the case of obesity and cancer. Proliferation and expansion of adipose tissue induce tissue hypoxia and the expression of HIF. Hypoxia in cancer is associated with poor outcomes and chemoresistance[137,153]. In TNBC, chemotherapeutic treatment with Paclitaxel and Gemcitabine results in expression of HIF, and enrichment of CSC through IL-6 and IL-8 actions. Chemical inhibition of HIF results in the depletion of CSC and tumour abrogation in vitro and in vivo[154].

s associated with poor outcomes and chemoresistance[137,153]. In TNBC, chemotherapeutic treatment with Paclitaxel and Gemcitabine results in expression of HIF, and enrichment of CSC through IL-6 and IL-8 actions. Chemical inhibition of HIF results in the depletion of CSC and tumour abrogation in vitro and in vivo[154]. In addition, hypoxia promotes survival of TNBC MDA-MB231 from Paclitaxel-induced apoptosis via mTOR/JNK pathway[155]. CSC resistance to chemotherapy The presence of CSC within tumors make them resistant to chemotherapy. CSC are commonly more resistant to chemotherapeutics which target the bulk of the tumor that allow the proliferation of CSC[156]. The CSC stemness phenotype and chemoresistance involve TGF-β signaling, which plays a prominent role in stem biology, facilitating epithelial to mesenchymal transition in mammary cancer cells, which is a property of CSC[138]. TNBC cell lines treated with Paclitaxel showed an enrichment of cancer cells with stem like properties and increased TGF-β signaling both in vitro and in vivo. Chemical inhibition of TGF-β signaling abrogates tumor formation[157]. CSC show higher expression of ABC family of proteins that increase their capability to efflux chemotherapeutics from cells. CSC also show diminished apoptosis rate, and over activation of detoxification proteins and survival pathways as NFκB and PI-3K[158].

inhibition of TGF-β signaling abrogates tumor formation[157]. CSC show higher expression of ABC family of proteins that increase their capability to efflux chemotherapeutics from cells. CSC also show diminished apoptosis rate, and over activation of detoxification proteins and survival pathways as NFκB and PI-3K[158]. OBESITY, LEPTIN AND DRUG RESISTANCE Obesity and leptin signaling have been implicated in enhance capabilities of cancer cells to avoid apoptosis. Leptin expression was associated with higher expression of BCL-2 and BCL-xL expression in breast cancer cells[159]. Furthermore, leptin signaling has been reported to activate the PI-3K/Akt pathway that antagonizes apoptosis in various cancers such as colon cancer, liver cancers, endometrial cancers and lymphomas[44,160-163]. Additionally, obesity has been shown to influence breast cancer response to Doxorubicin therapy. Indeed, obese mice treated with Doxorubicin showed more proliferative tumors that also had more CSC as compared with non-obese mice[164]. Leptin increases the expression of ABC protein transporters in glioblastoma[165]. Our preliminary data further show that leptin increases the expression of ABCB1 in breast and pancreatic cancer cells.

treated with Doxorubicin showed more proliferative tumors that also had more CSC as compared with non-obese mice[164]. Leptin increases the expression of ABC protein transporters in glioblastoma[165]. Our preliminary data further show that leptin increases the expression of ABCB1 in breast and pancreatic cancer cells. Another mechanism involved in obesity-induced chemoresistant is NFκB signaling. It is known that NFκB is activated by leptin signaling and that can increase survival of cancer cells under chemotherapeutic treatment[55]. An additional link between obesity (via leptin signaling) and cancer chemoresistance is HIF, which correlates with activation of leptin signaling in several cancers including endometrial, pancreatic, breast and colon cancers[133,166-168]. A potential mechanism involved in obesity-mediated drug resistant is TGF-β signaling. Leptin and TGF-β are commonly co-expressed in breast cancer[34]. It is known that TGF-β signaling induces leptin expression. However, the connection between leptin and TGF-β signaling in breast cancer is still unclear[169].

,166-168]. A potential mechanism involved in obesity-mediated drug resistant is TGF-β signaling. Leptin and TGF-β are commonly co-expressed in breast cancer[34]. It is known that TGF-β signaling induces leptin expression. However, the connection between leptin and TGF-β signaling in breast cancer is still unclear[169]. Leptin increased proliferation and survival of breast cancer estrogen receptor positive cells, MCF-7 cells treated with Cisplatin. These data further assessed that leptin is a survival factor that induces drug resistance in breast cancer[170]. Moreover, leptin was found able to induce CSC expansion in breast[60] and pancreatic cancer[16]. Furthermore, our preliminary data suggest that leptin induces the expression of Oct-4 and Nanog in breast cancer cells. These factors are essential for the upregulation of Ob-R in cancer cells[91]. Thus, leptin can induce a feedback mechanism through Oct-4/Nanog to sustain Ob-R expression and its pro-oncogenic signals in breast cancer. LEPTIN ANTAGONISTS AND CANCER PROGRESSION Leptin signaling has numerous protumurogenic effects, including the increased chemoresistance found in several tumors. Therefore, leptin antagonism could be a new strategy to overcome drug resistance in cancer. Several molecules have been described as potential new agents to target leptin-induced cancer growth and drug resistance. Majority of the leptin antagonists reported are mutated or truncated versions of leptin molecule: Leptin muteins, Allo-aca and D-ser, LDFI, and leptin peptide receptor antagonists (LPrA).

in cancer. Several molecules have been described as potential new agents to target leptin-induced cancer growth and drug resistance. Majority of the leptin antagonists reported are mutated or truncated versions of leptin molecule: Leptin muteins, Allo-aca and D-ser, LDFI, and leptin peptide receptor antagonists (LPrA). SMLA and SHLA Leptin muteins or mutant proteins, were generated using random mutagenesis of the leptin sequence and screened for high affinity variants using a yeast surface display. This resulted in the creation and identification of high affinity muteins. Two mutein antagonists named superactive mouse leptin antagonist (SMLA) and superactive human leptin antagonist (SHLA) were made by the introduction of an Asp23 mutation. These antagonists showing 4 aminoacid residue mutations (D23L/L39A/D40A/F41A) were reported to have 60-fold increased affinity for Ob-R and 14 fold greater antagonistic activity as compared with the original leptin antagonist showing 3 mutations (L39A/D40A/F41A)[171]. These muteins were pegylated at the N terminus to increase bioavailability and stability. However, the pegylated muteins increased BW in mice. Pegylated SMLA induced higher BW gain as compared with the pegylated SHLA[171]. No effects of muteins on leptin-induced chemoresistance in cancer have been reported to date.

1]. These muteins were pegylated at the N terminus to increase bioavailability and stability. However, the pegylated muteins increased BW in mice. Pegylated SMLA induced higher BW gain as compared with the pegylated SHLA[171]. No effects of muteins on leptin-induced chemoresistance in cancer have been reported to date. Allo-aca and D-ser Allo-aca is a non-toxic, 9-residue peptide leptin antagonist based on the C terminal Ob-R binding leptin site III. Allo-aca was reported to increase survival of CD1 nude mouse hosting TNBC. The effective dose of the peptide was found after 9 to 13 d of treatment by injecting intraperiotoneally between 0.1 and 1 mg Allo-Aca/kg body weight (BW)/day. Allo-aca was nontoxic in C57Bl/6 and CD1 nude mice, but showed hepatotoxicity at 0.2 mg/kg BW/day in SCID mice[172]. Additionally, it induced weight gain of 6% to 10% of BW[172]. Treatment of TNBC MDAMB231 cell line with Allo-aca 50 pM inhibited leptin-induced proliferation in vitro[172]. D-ser, peptide inhibitor is an analogue of Allo-aca that at 1 nM concentration inhibited leptin-induced proliferation in Ob-R positive breast and colon cancer cells in vitro without exhibiting agonist activity[173]. However, no data on the effects of these antagonists on leptin-induced drug resistance and CSC are available.

, peptide inhibitor is an analogue of Allo-aca that at 1 nM concentration inhibited leptin-induced proliferation in Ob-R positive breast and colon cancer cells in vitro without exhibiting agonist activity[173]. However, no data on the effects of these antagonists on leptin-induced drug resistance and CSC are available. LDFI LDFI is a leptin peptide antagonist composed by amino acid 39 to 42 on the leptin binding site I (Leu-Asp-Phe-Ile). LDFI was reported to inhibit leptin-induced growth of breast cancer cells in vitro and in vivo[174]. This peptide antagonist inhibited proliferation, colony formation on soft agar and Boyden chamber transmigration of estrogen receptor positive as well as estrogen receptor negative breast cancer cells. LDFI effects correlated with reduced expression of key downstream leptin effectors such as JAK2, STAT3, AKT and MAPK. In vivo, the pegylated peptide (LDFI-PEG) was shown to inhibit the tumour growth in a murine mammary xenograft model. LDFI-PEG showed no toxicity or effects on BW of mice[174]. No reports on potential effects of LDFI on drug resistance in breast or other cancer types are available.

rs such as JAK2, STAT3, AKT and MAPK. In vivo, the pegylated peptide (LDFI-PEG) was shown to inhibit the tumour growth in a murine mammary xenograft model. LDFI-PEG showed no toxicity or effects on BW of mice[174]. No reports on potential effects of LDFI on drug resistance in breast or other cancer types are available. LPrAs LPrA1 and LPrA2 were earlier designed and tested in vitro and in vivo in mouse models[52,53,56,72,175,176]. LPrAs are composed by aminoacid sections of the binding site I (LPrA1) and III (LPrA2) of the leptin molecule[63]. LPrA2 was conjugate to polyethylene glycol 20 kDa (PEG-LPrA2) or to iron-oxide nanoparticles (IONP-LPrA2) to increase its bioavaibility and effectiveness to block leptin signaling in cancer cells. Unconjugated and conjugated LPrA2 effectively inhibited leptin-induced protumorigenic actions in breast and pancreatic cancer cells[52,53,56,72,175,176]. LPrA2 showed potent effects for the reduction of leptin-induced growth of tumors and expression of inflammatory (IL-1/IL-1R tI), proliferation (Ki67, PCNA), angiogenic factors (VEGF/VEGFR2) and Notch in tumors and endothelial cells[53,56,58,72]. The antagonist effects of LPrA2 on tumor growth and angiogenesis were more evident in obese than in lean mice[53,72]. However, unconjugated or conjugated LPrA2 showed no toxicity and did not affect energy balance (BW or food intake) or general health when it was applied (0.1 mM/i.v. per twice weekly) to many lean and obese mice for two months. Remarkably, LPrA2 negatively impact on leptin-induced expansion of CSC and Notch expression in breast and pancreatic cancer cells, derived tumorspheres and xenografts[16,74]. Moreover, LPrA2 significantly reduced the leptin-induced effects on drug resistance (Cisplatin, Sunitinib, Paclitaxel, Doxorubicin) in breast cancer cells[16,176].

negatively impact on leptin-induced expansion of CSC and Notch expression in breast and pancreatic cancer cells, derived tumorspheres and xenografts[16,74]. Moreover, LPrA2 significantly reduced the leptin-induced effects on drug resistance (Cisplatin, Sunitinib, Paclitaxel, Doxorubicin) in breast cancer cells[16,176]. CONCLUSION Combination of poor dietary habits and low physical activity, which are reinforced by accessibility of low-cost high caloric and fat foods have led to the obesity pandemic. Accumulated evidence supports a negative role of obesity on cancer risk, progression and management. Despite many efforts and social programs to tackle obesity, its effects on morbidity and mortality and its influences on cancer incidence and treatment are in crescendo[1-5]. It is known that obesity and leptin signaling not only affect cancer cells, but also tumor stroma. Moreover, leptin and paracrine factors secreted from cancer and stroma cells (adipocytes, fibroblasts, endothelial cells and inflammatory cells) could affect tumor progression, CSC and chemoresistance[16,176]. In this regards, the use of nontoxic leptin antagonists that do not affect energy balance could be a novel adjuvant therapy for cancer drugs. These compounds can increase chemotherapeutic effectiveness and allow reducing their dosage and undesired side effects in cancer patients.

ssion, CSC and chemoresistance[16,176]. In this regards, the use of nontoxic leptin antagonists that do not affect energy balance could be a novel adjuvant therapy for cancer drugs. These compounds can increase chemotherapeutic effectiveness and allow reducing their dosage and undesired side effects in cancer patients. Supported by Department of Defense (DOD), Congressionally Direct Medical Research Program (CDMRP), No. W81XWH- 13-1-0382; National Institute of Health (NIH)/National Cancer Institute (NCI), No. 1R41CA183399-01A; Pilot Project Award from MSM (Morehouse School of Medicine)/Tuskegee University/University of Alabama in Birmingham (UAB) Cancer Center partnership, No. 5U54CA118638; and the National Institute on Minority Health and Health Disparities (NIMHD) of NIH, No. 5S21MD00101. Conflict-of-interest statement: The authors declare no potential conflicts of interest. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): A Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: October 7, 2016 First decision: December 1, 2016 Article in press: February 28, 2017 P- Reviewer: Tu H, Vetvicka V, Zhou WQ S- Editor: Gong ZM L- Editor: A E- Editor: Lu YJ

Core tip: There are several reviews in the literature dedicated to breasts cancers. However, our manuscript is an updated review on the current knowledge and particularly on the molecular mechanisms involved in the relapse of patients on the current treatments. A summary of ongoing clinical trials gives a perspective for future therapeutic strategies. Our manuscript represents a working document for researchers/oncologists in the field of breast cancers. INTRODUCTION Breast cancer targeted therapies involve substances or drugs which block the growth of cancer by interfering with the function of specific molecules responsible for tumor cell proliferation and survival[1-21]. Breast cancer cells may overexpress specific receptors which, when activated can initiate downstream signaling resulting in the expression of genes for cancer cell proliferation, growth, survival, migration, angiogenesis and other vital cell cycle pathways[22,23]. There are various types of breast cancer, some have hormone receptors like estrogen or progesterone (some have both) and are called ER+ or PR+ breast cancer respectively.

INTRODUCTION Breast cancer targeted therapies involve substances or drugs which block the growth of cancer by interfering with the function of specific molecules responsible for tumor cell proliferation and survival[1-21]. Breast cancer cells may overexpress specific receptors which, when activated can initiate downstream signaling resulting in the expression of genes for cancer cell proliferation, growth, survival, migration, angiogenesis and other vital cell cycle pathways[22,23]. There are various types of breast cancer, some have hormone receptors like estrogen or progesterone (some have both) and are called ER+ or PR+ breast cancer respectively. The estrogen receptor ER is a major driver of the majority of breast cancers as it is expressed in 75% of breast cancers overall. It is more frequently related with postmenopausal women and there is a 99% survival rate at ten years. Hormone sensitive breast cancer has a strong correlation with lower tumor grade; lower levels of amplification of the human epidermal growth factor receptor 2 (HER2) oncogene and concomitant loss of p53 tumor suppressor gene; expression of progesterone receptor (PR), soft tissue and bone metastases and slower rates of disease recurrence. In cases of hormone positive breast cancer along with the expression of ER, multigene tests may be carried out to make treatment decisions particularly for adjuvant therapy and screen those patients who would benefit more from combination of endocrine plus chemotherapy[24-26].

es and slower rates of disease recurrence. In cases of hormone positive breast cancer along with the expression of ER, multigene tests may be carried out to make treatment decisions particularly for adjuvant therapy and screen those patients who would benefit more from combination of endocrine plus chemotherapy[24-26]. The most common receptors that are overexpressed in breast cancer cells are part of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases: EGFR and HER2 are overexpressed in approximately 40% and 25% of breast cancers respectively and are believed to be responsible for more aggressive tumor behavior and poor prognosis[27]. Triple negative breast cancer (TNBC) is defined by the lack of expression of both estrogen and progesterone as well as the HER2 protein and is often associated with an unfavorable prognosis as no treatment is yet available for this particular breast cancer subtype[28]. The rapid acquisition of resistance in breast cancer targeted therapies seems to limit the effectiveness of treatment and even though some of the genetic mutations and epigenetic changes in molecular pathways have been understood, it is sometimes necessary to combine several pathway blockades in order to achieve successful treatment results[29-35].

tance in breast cancer targeted therapies seems to limit the effectiveness of treatment and even though some of the genetic mutations and epigenetic changes in molecular pathways have been understood, it is sometimes necessary to combine several pathway blockades in order to achieve successful treatment results[29-35]. The identification of new target molecules in breast cancer and the use of combination therapies may have improved the understanding of compensatory pathways which lead to the emergence of resistance mechanisms, nevertheless, breast cancer subtypes like TNBCs seem to exploit alternative proliferative pathways which are not yet fully understood and need urgent attention and elucidation[11] (Figure 1). Figure 1 A schematic diagram of the most common resistance mechanisms to targeted therapies. (1) Alteration of the drug target (Treat.): This type of resistance involves mutations as well as amplifications of drug targets such as kinases; (2) Upstream and downstream pathway effect through the activation of receptor tyrosine kinase (RTK) (a) and/or the mutation/amplification of upstream (b) or downstream (c) components; (3) Bypass mechanisms occur as a result of a second receptor tyrosine kinase activation (a), through a mutation of a parallel kinase (b) or modulation of mRNA binding proteins (c). These alternative mechanisms of resistance especially through kinases activation result in the modification of gene expression via the phosphorylation or transcription factors (TF).

a second receptor tyrosine kinase activation (a), through a mutation of a parallel kinase (b) or modulation of mRNA binding proteins (c). These alternative mechanisms of resistance especially through kinases activation result in the modification of gene expression via the phosphorylation or transcription factors (TF). TARGETED THERAPIES IN BREAST CANCER Estrogen and estrogen receptors are key drivers in breast cancer progression. This is the reason why targeting estrogen has been used for many years to inhibit the estrogen signaling pathway in women with estrogen positive breast cancer. Selective estrogen receptor modulators or SERM have been used to suppress tumor growth in estrogen dependent breast cancers and tamoxifen was the first drug to be approved for estrogen positive metastatic breast cancer reducing recurrences by approximately 40%-50%[36]. Aromatase inhibitors (anastrozole, letrozole, exemestane) are also used as an alternative therapy to treat estrogen dependent breast cancers as they block the biosynthesis of androgens through inhibition of the aromatase enzyme resulting in reduction of estrogen levels in tumor cells[36]. Other therapies are available for other forms of breast cancer that are not hormone dependent. The HER2 protein represents the most common overexpressed receptor signature in breast cancer and is considered a relevant biomarker for treatment. The recombinant antibody trastuzumab ( Herceptin) targets HER2 and is the first drug that was approved by the FDA in 1998 for the treatment of HER2 positive breast cancers[37,38].

ed in 12.9% (4/31, all of which were managed by endoscopic methods)[63]. ESTD procedure involves creation of the submucosal tunnel, dissection of the submucosal tumor (SMT) and closure of mucosal entry with hemostatic clips[64]. Gong et al[64] evaluated the feasibility and safety of ESTD in upper gastrointestinal SMTs. Results showed that 58.3% (7/12) were GISTs, complete tumor resection was achieved in all patients, en bloc resection in 83.3% (10/12, other 2 lesions were resected in 2 pieces) and 2 patients had both pneumothorax and subcutaneous emphysema which were managed conservatively[64]. Disadvantages of endoscopic techniques include tumor recurrence and peritoneal seeding secondary to perforation. It is unclear whether there is remnant GIST tissue after dissection causing tumor recurrence, although the dissection site is usually ablated with electrical knife or snare. Perforation occurs due to pseudo capsule injury during difficult MP layer dissection which increases the chance of peritoneal seeding. Peritoneal seeding is associated with poor prognosis because of increased tumor recurrence.

Other therapies are available for other forms of breast cancer that are not hormone dependent. The HER2 protein represents the most common overexpressed receptor signature in breast cancer and is considered a relevant biomarker for treatment. The recombinant antibody trastuzumab ( Herceptin) targets HER2 and is the first drug that was approved by the FDA in 1998 for the treatment of HER2 positive breast cancers[37,38]. Other agents that followed such as pertuzumab and lapatinib have not shown immunity to the development of resistance mechanisms with significant side effects for the patients[7,39,40]. The conjugated monoclonal antibody TDM1 (trastuzumab emtansine) may be used in HER2 positive breast cancers as trastuzumab efficiently transports the DM1 drug, a microtubule inhibitor, directly into the breast cancer cells to inhibit growth. Triple negative cancers lacking hormone receptors and HER2 may respond to agents like PARP1 inhibitors and may have HER1 as a potential target. The monoclonal antibody cetuximab combined with cisplatin chemotherapy has shown promising results in a Phase II study, suggesting some subtypes of TNBC may be EGFR inhibition sensitive[41].

e cancers lacking hormone receptors and HER2 may respond to agents like PARP1 inhibitors and may have HER1 as a potential target. The monoclonal antibody cetuximab combined with cisplatin chemotherapy has shown promising results in a Phase II study, suggesting some subtypes of TNBC may be EGFR inhibition sensitive[41]. The conventional route to treat TNBC patients by taxol derivatives and anthracycline chemotherapy is still widely used until more “druggable” targets are identified[41]. Recent studies suggest that the microtubule-stabilizing agent ixabepilone in combination with capecitabine may be effective in TNBC that are resistant to anthracycline and taxane drugs and the PACS08 Phase III trial is evaluating this possible treatment strategy[28]. Targeted therapies have also been approved against the vascular endothelial growth factor (VEGF) and the drug bevacizumab has proven effective in the treatment of advanced metastatic breast cancer when used in association with paclitaxel or docetaxel[42,43]. Inhibitors of downstream pathways like PI3K/AKT/ mTOR and RAS/MEK/ERK are also available for therapeutic purpose as well as agents directed against other tyrosine kinases like SRC, insulin-like-growth-factor [IGF/IGF-receptor (IGFR)], poly-ADP ribose polymerase (PARP) Inhibitors and also matrix metalloproteases (MMPs) which are involved in cancer cell invasion and metastasis[8,29,31,44-48].

also available for therapeutic purpose as well as agents directed against other tyrosine kinases like SRC, insulin-like-growth-factor [IGF/IGF-receptor (IGFR)], poly-ADP ribose polymerase (PARP) Inhibitors and also matrix metalloproteases (MMPs) which are involved in cancer cell invasion and metastasis[8,29,31,44-48]. Compensatory survival pathways, increased phosphatydil-inositol-3-kinase (PI3K)[49-52] signaling, receptor tyrosine kinase signaling outside the ErbB/HER family and involvement of other HER receptors[53], may all play a key role in the development of alternative molecular pathways responsible for the development of therapeutic resistance in breast cancer cells. Indications of breast cancer targeted therapies Breast cancer targeted therapies are used to treat patients whose breast cancer cells overexpress certain characteristic proteins on their surface allowing an abnormal growth pattern. Antibodies are sometimes used as they work in a similar way as the human immune system.

Compensatory survival pathways, increased phosphatydil-inositol-3-kinase (PI3K)[49-52] signaling, receptor tyrosine kinase signaling outside the ErbB/HER family and involvement of other HER receptors[53], may all play a key role in the development of alternative molecular pathways responsible for the development of therapeutic resistance in breast cancer cells. Indications of breast cancer targeted therapies Breast cancer targeted therapies are used to treat patients whose breast cancer cells overexpress certain characteristic proteins on their surface allowing an abnormal growth pattern. Antibodies are sometimes used as they work in a similar way as the human immune system. The most efficient breast cancer targeted therapy today is the one targeting the HER2 protein overexpression on the surface of breast cancer cells. At present, there are seven widely used breast cancer targeted therapies which are effective in blocking several molecular pathways: Afinitor or everolimus, an m-TOR inhibitor, stops cancer cells from getting energy supplies[54-57]; Avastin or bevacizumab inhibits the growth of new blood vessels which supply oxygen and nutrients to cancer cells for growth and function[14,58]; Herceptin or trastuzumab blocks the ability of cancer cells to receive signals which tell them to grow[12,59]; Kadcyla or T-DM1 is a combination of Herceptin and emtansine[7,60]. In this case Herceptin is used as a transport method to deliver the emtansine chemotherapy to cancer cells; Perjita or pertuzumab works by stopping cancer cells from receiving growth signals[12,61]; Tykerb or lapatinib is a HER2 inhibitor that blocks signals of cell growth[4,42].

ation of Herceptin and emtansine[7,60]. In this case Herceptin is used as a transport method to deliver the emtansine chemotherapy to cancer cells; Perjita or pertuzumab works by stopping cancer cells from receiving growth signals[12,61]; Tykerb or lapatinib is a HER2 inhibitor that blocks signals of cell growth[4,42]. The HER2 protein The HER2 proto-oncogene is overexpressed in 10%-12% of over 2500 cases of human breast cancers and this is associated with malignant transformation and poorer overall survival rates particularly in breast tumors with lymph node metastasis[62]. The HER2 or neu oncogene (erbB2) is part of the EGFR family of tyrosine kinases and is located on chromosome 17 (17q12). It represents the most common overexpressed receptor in breast cancers and is considered a relevant therapeutic target[59,63-69].

The HER2 protein The HER2 proto-oncogene is overexpressed in 10%-12% of over 2500 cases of human breast cancers and this is associated with malignant transformation and poorer overall survival rates particularly in breast tumors with lymph node metastasis[62]. The HER2 or neu oncogene (erbB2) is part of the EGFR family of tyrosine kinases and is located on chromosome 17 (17q12). It represents the most common overexpressed receptor in breast cancers and is considered a relevant therapeutic target[59,63-69]. The EGFR family is composed of four receptors: EGFR/HER1, ErbB2/HER2, ErbB3/HER3 and ErbB4/HER4. These receptors share common domains: an extracellular region characterized by leucine-rich repeats; cysteine rich repeats in the intracellular domain; a single transmembrane spanning region; a short juxtamembrane region; a kinase region and a cytoplasmic tail with various tyrosine phosphorylation sites[5,10]. Binding of ligands to the extracellular domain of EGFR, HER3 and HER4 allows for the formation of kinase active homo- and hetero-dimers to which HER2 is recruited as a preferential partner. Heterodimer formation between HER2/HER3 is the most common occurrence in these receptors’ preferences. HER3 is often responsible for the activation of the PI3K/AKT signaling pathway via six docking sites for the p85 adaptor subunit of PI3K. The HER3/PI3K axis plays a key role in the survival of HER2-dependent cells as the loss of HER3 inhibits the survival of HER2-overexpressing breast cancer cells[70,71].

preferences. HER3 is often responsible for the activation of the PI3K/AKT signaling pathway via six docking sites for the p85 adaptor subunit of PI3K. The HER3/PI3K axis plays a key role in the survival of HER2-dependent cells as the loss of HER3 inhibits the survival of HER2-overexpressing breast cancer cells[70,71]. Trastuzumab resistance mechanisms The first recombinant antibody approved by the FDA to target HER2-positive breast cancers was trastuzumab or Herceptin followed by other agents like pertuzumab and lapatinib. Trastuzumab binds to the juxtamembrane region of the HER2 receptor tyrosine kinase resulting in the uncoupling of the HER2/HER3 heterodimers and consequent inhibition of downstream signaling and cytotoxicity.

Trastuzumab resistance mechanisms The first recombinant antibody approved by the FDA to target HER2-positive breast cancers was trastuzumab or Herceptin followed by other agents like pertuzumab and lapatinib. Trastuzumab binds to the juxtamembrane region of the HER2 receptor tyrosine kinase resulting in the uncoupling of the HER2/HER3 heterodimers and consequent inhibition of downstream signaling and cytotoxicity. Resistance mechanisms to trastuzumab develop often as a result of HER2 gene amplification and RNA/protein overexpression. HER2 overexpressing tumor cells continue to depend on the HER2 oncogene even after bypassing trastuzumab action possibly due to signaling from receptor tyrosine kinases (RTK) outside the ErbB family, increased PI3K signaling and the presence of alternative forms of HER2 which are not detected by trastuzumab. Also, the modulation of Cdk inhibitor p27 by IGF-1 may be a key player in resistance to trastuzumab as overexpressed IGF-1 is responsible for the activation of the PI3K downstream signaling pathway and further effects on Akt[72,73]. One of the key players in trastuzumab-resistance in HER2 positive breast cancer was the inhibition of expression of miR-375, a tumor suppressor gene which targets IGF1R[74]. Also, molecular pathway crosstalk may have resulted in increased cell survival and division by interference with HER2 accessibility, independent downstream signaling activation as well as HER2 mutations, particularly the expression of p95HER2, an active truncated form of HER2. Blocking IGFR1 completely resulted in restored sensitivity of HER2 positive cancer cells to trastuzumab in vitro. The loss of miR-375 with consequent epigenetic changes such as DNA methylation and histone deacetylation may drive the upregulation of IGFR1 and hence the development of trastuzumab-resistant cancer cells; in this case, targeting miR-375 may prove to be worthy of further investigation as a potential therapeutic target to restore trastuzumab sensitivity in HER2 positive breast cancer cells[74].

on and histone deacetylation may drive the upregulation of IGFR1 and hence the development of trastuzumab-resistant cancer cells; in this case, targeting miR-375 may prove to be worthy of further investigation as a potential therapeutic target to restore trastuzumab sensitivity in HER2 positive breast cancer cells[74]. The new antibody-drug conjugate trastuzumab-DM1 (TDM1) which has been recently developed for the treatment of HER2 positive cancer has proved to be effective in inhibiting trastuzumab sensitive and resistant HER2 positive breast cancer cell lines in vitro. TDM1 drives both apoptosis and mitotic catastrophe in the trastuzumab resistant breast cancer cell line Jimt-1, acting as a potent inhibitor of microtubule assembly. These cells are characterized for having several co-existing trastuzumab resistance mechanisms like a mutation in the PIK3CA gene, low PTEN expression, overexpression of NRG1 and a moderate expression of the HER2 receptor. Interestingly, in the T-DM1 treated Jimt-1 cell line model, an accumulation of HER2 was observed in organelles which resembled enlarged lysosomes, suggesting sequestration of the protein in these intracellular granules[75]. The integrin αvβ6, involved in promoting migration, invasion and cancer cell survival, seems to play a significant role in the development of trastuzumab resistance mechanisms. Targeting αvβ6 with the 264RAD antibody in HER2 positive breast cancer cell lines expressing both HER2 and the integrin seems to slow down the growth of trastuzumab resistant tumors[62].

gration, invasion and cancer cell survival, seems to play a significant role in the development of trastuzumab resistance mechanisms. Targeting αvβ6 with the 264RAD antibody in HER2 positive breast cancer cell lines expressing both HER2 and the integrin seems to slow down the growth of trastuzumab resistant tumors[62]. Resistance of breast cancer cells to trastuzumab mediated cytotoxicity has been implicated in the secretion of soluble factors by adipocytes and preadipocytes in adipose tissue proximal to breast cancer cells. The development of resistance mechanisms in this case occurs by inhibition of trastuzumab-mediated tumor lysis by natural killer cells in vitro and by adipose tissue in vivo. A reduced antitumor effect was observed in mice which had tumors in close proximity to a lipoma, while in another group of mice which had tumors located distant to the lipoma, the trastuzumab anti-tumor effects were enhanced. The inhibition of antitumor activity was enhanced when the adipocytes were in hypoxic conditions, these factors might suggest a link between patient obesity and development of trastuzumab resistance mechanisms[76].

of mice which had tumors located distant to the lipoma, the trastuzumab anti-tumor effects were enhanced. The inhibition of antitumor activity was enhanced when the adipocytes were in hypoxic conditions, these factors might suggest a link between patient obesity and development of trastuzumab resistance mechanisms[76]. The dual targeting of HER family receptors with antibody therapy may prove to be a strategy to overcome acquired resistance mechanisms by cancer cells to cetuximab. When both HER3 and EGFR were neutralized by cetuximab and the anti HER3 monoclonal antibody U3-1287, cetuximab sensitive tumor cells showed a significant decrease in proliferation possibly due to inhibition of both MAPK and AKT pathways and a diminished signaling from all three HER family receptors[77]. The efficacy of trastuzumab in inhibiting proliferation of breast cancer cells might be dependent on the presence of endogenous HER-receptor activating ligands EGF and heregulin-β1; the receptor density of HER-family members and growth ligands are key players in the development of resistance mechanisms to trastuzumab therapy, which interfers with cell cycle kinetics by inducing a G1 accumulation in HER-2 positive breast adenocarcinomas[78].

ous HER-receptor activating ligands EGF and heregulin-β1; the receptor density of HER-family members and growth ligands are key players in the development of resistance mechanisms to trastuzumab therapy, which interfers with cell cycle kinetics by inducing a G1 accumulation in HER-2 positive breast adenocarcinomas[78]. An unexpected mechanism of resistance is associated with down-stream mutations especially those targeting the mRNA binding protein tristetraprolin (TTP). ttp gene germinal mutation generates a form of TTP mRNA which is inefficiently translated in protein. The lack of TTP and the general increase of the TTP competitor the ELAV-like protein 1 (HuR) results in the increase of the half-life of mRNAs encoding oncogenes, inflammatory and angiogenic factors. The mutation of TTP is predictive of trastuzumab resistance[79]. Hence TTP is considered as a tumor suppressor for breast cancers[80-82]. TTP and HuR are phosphorylated by the same kinases and phosphorylation has antagonistic effects on both proteins (inactivation/degradation for TTP and activation/stabilization for HuR). Hence, activation of intracellular signaling pathways results in a general increase of proteins associated with oncogenic properties[83] (Figure 1). The main drawback in trastuzumab therapy is represented by the emergence of serious cardiac side effects resulting from administration of this monoclonal antibody. Analysis of HER2 specific mutation may predict cardiac toxic effect[84].

An unexpected mechanism of resistance is associated with down-stream mutations especially those targeting the mRNA binding protein tristetraprolin (TTP). ttp gene germinal mutation generates a form of TTP mRNA which is inefficiently translated in protein. The lack of TTP and the general increase of the TTP competitor the ELAV-like protein 1 (HuR) results in the increase of the half-life of mRNAs encoding oncogenes, inflammatory and angiogenic factors. The mutation of TTP is predictive of trastuzumab resistance[79]. Hence TTP is considered as a tumor suppressor for breast cancers[80-82]. TTP and HuR are phosphorylated by the same kinases and phosphorylation has antagonistic effects on both proteins (inactivation/degradation for TTP and activation/stabilization for HuR). Hence, activation of intracellular signaling pathways results in a general increase of proteins associated with oncogenic properties[83] (Figure 1). The main drawback in trastuzumab therapy is represented by the emergence of serious cardiac side effects resulting from administration of this monoclonal antibody. Analysis of HER2 specific mutation may predict cardiac toxic effect[84]. HER-2 is expressed in the adult human myocardium and trastuzumab therapy unfortunately carries the risk of inducing cardiac dysfunction and congestive heart failure. When adjacent chemotherapy is applied in addition to trastuzumab, one has to take into consideration anthracyclin-associated cardiotoxicity following the inhibition of the HER-2/erbB2 receptor to ensure safety for patients. Some of the cardiotoxicity side effects of trastuzumab may be reversible over time and in some cases, administering the monoclonal antibody after chemotherapy or radiotherapy may decrease the risk of potential cardiac side effects. Trastuzumab therapy seems to represent clear overall benefits for patients in the long run, therefore, should be still considered as an appropriate standard choice of treatment as a HER-2/erbB2 inhibitor as long as care is taken to minimize its side effects[85].

may decrease the risk of potential cardiac side effects. Trastuzumab therapy seems to represent clear overall benefits for patients in the long run, therefore, should be still considered as an appropriate standard choice of treatment as a HER-2/erbB2 inhibitor as long as care is taken to minimize its side effects[85]. Endocrine therapy resistance mechanisms Resistance to hormone therapy is a major challenge within hormone sensitive breast cancers even though ER and PR targeted therapy has proven to be very effective, improving the quality of life of hormone sensitive breast cancer patients. The major pathways responsible for endocrine resistance mechanisms might be several: The HER tyrosine kinase receptor family; receptors for insulin/IGF1, FGF and VEGF, Src, AKT, stress related kinases, might each play a pivotal role in contributing to endocrine therapy resistance when their cognate ligands are amplified or overexpressed. Cross-talk between the estrogen receptor (ER) and growth factor receptor signaling with hyperactivation of the PI3K pathway have also been associated the development of endocrine resistance[86]. Nuclear receptors and the androgen receptors may also act as alternative growth stimulators by post translational modification, enabling the bypass of ER inhibition. Co-targeting the EGFR and HER2 pathway simultaneously seems to be the most promising way forward in circumventing endocrine resistance as these two seem to be the most important factors responsible in this particular resistance scenario[26].

tors by post translational modification, enabling the bypass of ER inhibition. Co-targeting the EGFR and HER2 pathway simultaneously seems to be the most promising way forward in circumventing endocrine resistance as these two seem to be the most important factors responsible in this particular resistance scenario[26]. The mTOR pathway The mTOR pathway seems to be a master regulator of cell physiology and may be a key player in the targeted therapy of cancer[87]. When the natural product rapamycin was discovered in the early 1970’s as an antifungal agent, it emerged in later studies that the molecule could halt growth in many types of eukaryotic cells and have a powerful immunosuppressive function. In 1999 the FDA approved sirolimus as a drug used against rejection of transplanted organs particularly the kidneys. Rapamycin binds to another molecule, FKBP12 and once this complex is formed it associates with a protein called mTOR[88]; a serine/threonine kinase, resembling the kinase domain of PI3 kinase and its related enzymes. The circuitry of the mTOR pathway is of interest as it represents a key element of the mammalian cell cycle integrating incoming signals and vital mechanisms such as glucose import and protein synthesis, as well as phosphorylating two kinases involved in translation: S6 kinase (S6KI) and 4E-BP1[89,90]. The activation of S6KI is followed by the activation of the small 40-S ribosomal subunit which can initiate protein synthesis after associating to the large ribosomal subunit. mTOR is also a key upstream regulator which controls the AKT signaling pathway for the regulation of apoptosis and proliferation; inhibiting the mTOR complex results in a shutdown of the AKT signaling stream resulting in an hyperactivated PI3K/loss of PTEN expression[91,92].

associating to the large ribosomal subunit. mTOR is also a key upstream regulator which controls the AKT signaling pathway for the regulation of apoptosis and proliferation; inhibiting the mTOR complex results in a shutdown of the AKT signaling stream resulting in an hyperactivated PI3K/loss of PTEN expression[91,92]. The PI3K/AKT/mTOR pathway is overactivated in 70% of breast cancers and the protein kinases found along these pathways may be potential drug targets for breast cancer therapy. Due to the large scale involvement of this pathway the cell cycle regulation, selectively silencing of the PI3K/AKT/mTOR pathway represents an attractive approach for patients who might have shown resistance mechanisms to previous types of therapy. The combination of mTOR inhibitors with other targeted therapies might be a winning formula to circumvent resistance mechanisms of breast cancer patients. Inhibition of the mTOR pathway by the drug everolimus in combination with HER-2 or estrogen receptor inhibitors may be a promising future strategy to apply, in order to reinstate sensitivity of breast cancer cells to traditional therapies and overcome resistance mechanisms which seem to emerge when the mTOR pathway is functioning in hyperactive mode[93]. Molecular alterations like mutations in EGFR, BRAF, AKT, or PI3K are associated with activation of downstream signaling pathways resulting in unrestricted proliferation in cancer cells.

tional therapies and overcome resistance mechanisms which seem to emerge when the mTOR pathway is functioning in hyperactive mode[93]. Molecular alterations like mutations in EGFR, BRAF, AKT, or PI3K are associated with activation of downstream signaling pathways resulting in unrestricted proliferation in cancer cells. Glaysher et al[94] have shown that targeting a breast epithelial cell line after having knocked-in mutations and using EGFR and mTOR inhibitors, there was an increased sensitivity to therapeutic drugs. As development of resistance in breast cancer cells may be a result of the activation of the PI3K/AKT/mTOR pathway, Glaysher et al[94] studied the effects of inhibiting both mTOR and EGFR by combined drug action of ZSTK474/sirolimus and erlotinib/gefitinib, observing a more effective signaling blockade, as opposed to use of single agents on the parental cell line and irrespective of the knocked-in mutations in EGFR,KRAS,PI3K,BRAF or AKT[94]. Receptor tyrosine kinase inhibitors resistance mechanisms Lapatinib is a dual EGFR/HER2 tyrosine kinase inhibitor which acts as an ATP competitor. It is used as a first line monotherapy in patients with HER2 positive metastatic breast cancer in addition to conventional chemotherapy like paclitaxel.

Glaysher et al[94] have shown that targeting a breast epithelial cell line after having knocked-in mutations and using EGFR and mTOR inhibitors, there was an increased sensitivity to therapeutic drugs. As development of resistance in breast cancer cells may be a result of the activation of the PI3K/AKT/mTOR pathway, Glaysher et al[94] studied the effects of inhibiting both mTOR and EGFR by combined drug action of ZSTK474/sirolimus and erlotinib/gefitinib, observing a more effective signaling blockade, as opposed to use of single agents on the parental cell line and irrespective of the knocked-in mutations in EGFR,KRAS,PI3K,BRAF or AKT[94]. Receptor tyrosine kinase inhibitors resistance mechanisms Lapatinib is a dual EGFR/HER2 tyrosine kinase inhibitor which acts as an ATP competitor. It is used as a first line monotherapy in patients with HER2 positive metastatic breast cancer in addition to conventional chemotherapy like paclitaxel. Unfortunately, the activation of compensatory pathways after onset of therapy with lapatinib seems to be responsible for the emergence of resistance mechanisms, particularly when inhibition of AKT phosphorylation leads to increased estrogen receptor-α transcription and estrogen receptor signaling. This mechanism of resistance can be circumvented by administering an ER-down-regulator fulvestrant, which can prevent the proliferation of lapatinib resistant cells. In addition, mutations in the HER2 protein, particularly a YVMA insertion at G776 in exon 20, seems to be responsible for mechanisms of de novo resistance to lapatinib as well as trastuzumab[73].

mvented by administering an ER-down-regulator fulvestrant, which can prevent the proliferation of lapatinib resistant cells. In addition, mutations in the HER2 protein, particularly a YVMA insertion at G776 in exon 20, seems to be responsible for mechanisms of de novo resistance to lapatinib as well as trastuzumab[73]. The inhibitory effects of lapatinib may be bypassed as downstream signaling is amplified and upregulation of activated HER3 becomes responsible for compromising the inhibitory effects of tyrosine kinases. Activation of the PI3K/AKT pathway results from HER3 upregulation with a subsequent nuclear increase in FoxO3A family of transcription factors responsible for control of cell cycle, neoplastic transformation and epithelial-to-mesenchymal transition[30]. Targeting erb-B3 (HER3) with an antibody has proven to be quite effective in both preclinical and clinical studies although tumor cells eventually develop resistance as the antibody is only active in inhibiting signaling without altering the actual expression of the erb-B3 receptors. New strategies which aim at reducing erb-B3 levels are being investigated such as the HDAC inhibitor entinostat and the antisense oligonucleotide EZN-3920[95].

tumor cells eventually develop resistance as the antibody is only active in inhibiting signaling without altering the actual expression of the erb-B3 receptors. New strategies which aim at reducing erb-B3 levels are being investigated such as the HDAC inhibitor entinostat and the antisense oligonucleotide EZN-3920[95]. The hepatocyte growth factor receptor HGFR/c-Met tyrosine kinase responsible for cell proliferation, protection from apoptosis and cell invasion, seems to be implicated in the emergence of resistance to targeted therapies particularly lapatinib and trastuzumab and recent preclinical studies suggested that inhibition of c-MET in gastric cancer cell lines circumvented resistance mechanisms as well as restored growth inhibition[96]. The overexpression of the receptor tyrosine kinase AXL is associated with poor prognosis and a more aggressive phenotype in ovarian, breast colon, esophageal, thyroid and lung cancers and may be implicated in the emergence of lapatinib acquired resistance in in vitro models of preclinical breast cancer studies.

The hepatocyte growth factor receptor HGFR/c-Met tyrosine kinase responsible for cell proliferation, protection from apoptosis and cell invasion, seems to be implicated in the emergence of resistance to targeted therapies particularly lapatinib and trastuzumab and recent preclinical studies suggested that inhibition of c-MET in gastric cancer cell lines circumvented resistance mechanisms as well as restored growth inhibition[96]. The overexpression of the receptor tyrosine kinase AXL is associated with poor prognosis and a more aggressive phenotype in ovarian, breast colon, esophageal, thyroid and lung cancers and may be implicated in the emergence of lapatinib acquired resistance in in vitro models of preclinical breast cancer studies. Lapatinib resistance has been also associated with SRC tyrosine kinase activity; overexpression of SRC in breast cancer cell lines seems to result in an increased interaction with EGFR rather than HER2. According to Formisano et al[97], when EGFR was inhibited with the monoclonal antibody cetuximab and SRC was inhibited by the small molecule saracatinib, lapatinib resistant breast cancer cells would not survive and sensitivity was restored. The combined treatment of lapatinib with cetuximab both in vitro and in vivo resulted in the reduction of EGFR/HER2 signaling and proved to be effective[97]. As observed by Wilson et al[98], autocrine tumor cell production might be responsible for increased levels of receptor tyrosine kinase-ligand levels and in breast cancer cell lines the HER3 ligand neuregulin-1 seems to induce complete rescue from lapatinib.

Lapatinib resistance has been also associated with SRC tyrosine kinase activity; overexpression of SRC in breast cancer cell lines seems to result in an increased interaction with EGFR rather than HER2. According to Formisano et al[97], when EGFR was inhibited with the monoclonal antibody cetuximab and SRC was inhibited by the small molecule saracatinib, lapatinib resistant breast cancer cells would not survive and sensitivity was restored. The combined treatment of lapatinib with cetuximab both in vitro and in vivo resulted in the reduction of EGFR/HER2 signaling and proved to be effective[97]. As observed by Wilson et al[98], autocrine tumor cell production might be responsible for increased levels of receptor tyrosine kinase-ligand levels and in breast cancer cell lines the HER3 ligand neuregulin-1 seems to induce complete rescue from lapatinib. An additional mechanism of resistance to lapatinib may occur as a result of crosstalk between the estrogen receptor and the HER2 pathway. Lapatinib induced upregulation of ER by inhibition of the PI3K/AKT signaling pathway results in overexpression of the anti-apoptotic protein Bcl-2 leading to the emergence of lapatinib resistance and cell death escape[99]. The VEGF The VEGF and its cell surface receptors represent the main modulators in the emergence of tumor angiogenesis. Avastin or bevacizumab, a humanized anti-VEGF antibody, has played a key role in anti-angiogenic therapy for cancer treatment in concomitance with small molecule VEGF receptor kinase inhibitors[43].

An additional mechanism of resistance to lapatinib may occur as a result of crosstalk between the estrogen receptor and the HER2 pathway. Lapatinib induced upregulation of ER by inhibition of the PI3K/AKT signaling pathway results in overexpression of the anti-apoptotic protein Bcl-2 leading to the emergence of lapatinib resistance and cell death escape[99]. The VEGF The VEGF and its cell surface receptors represent the main modulators in the emergence of tumor angiogenesis. Avastin or bevacizumab, a humanized anti-VEGF antibody, has played a key role in anti-angiogenic therapy for cancer treatment in concomitance with small molecule VEGF receptor kinase inhibitors[43]. The VEGF ligand presents itself as an antiparallel homodimeric structure in which each monomer is made mostly of β strands stabilized by a disulfide knot and two symmetrically disposed intermolecular disulfide bridges that are responsible for linking the monomers together. On the extracellular domain of each of the three VEGF receptors (VEGF-1, VEGF-2, VEGF-3) there are seven immunoglobulin-like structures (Ig domain)[100,101].

f β strands stabilized by a disulfide knot and two symmetrically disposed intermolecular disulfide bridges that are responsible for linking the monomers together. On the extracellular domain of each of the three VEGF receptors (VEGF-1, VEGF-2, VEGF-3) there are seven immunoglobulin-like structures (Ig domain)[100,101]. All four members of the VEGF family and the placenta growth factor bind to three endothelial cell tyrosine kinase receptors which have each different functions. VEGFR1 is responsible for promoting differentiation and vascular maintenance, VEGFR2 induction of endothelial cell proliferation and vascular permeability, VEGFR3 stimulation of lynphangiogenesis. Isoform specific receptors neuropilin-1 and neuropilin-2 may bind to class 3 semaphorins involved in axonal growth and also to some isoforms of VEGF1 as co-receptors which results in additional VEGF binding to VEGFR2[102]. Several other pathways are implicated by the function of VEGF as proteolytic and heparin activation further modulates receptor sites resulting in various cellular effects like the increase of vascular permeability, endothelial cell proliferation, survival and tubular formation. The VEGFR are usually endothelial in origin but in some instances they may be located in the stroma as macrophages and tumor cells themselves. Under abnormally low oxygen conditions (hypoxia), the hypoxia-inducible factor (HIF) plays a central role in transcription of genes like VEGF.

n, survival and tubular formation. The VEGFR are usually endothelial in origin but in some instances they may be located in the stroma as macrophages and tumor cells themselves. Under abnormally low oxygen conditions (hypoxia), the hypoxia-inducible factor (HIF) plays a central role in transcription of genes like VEGF. In normoxic conditions, the alpha-subunit of the HIF heterodimer (alpha, beta) is degraded by ubiquitylation as HIF-alpha binds to the von Hippel-Lindau tumor suppressor protein (p-VHL) forming the E3 ubiquitin ligase complex, a recognition component leading to proteasome-dependent degradation. In hypoxic conditions, as the HIF-alpha subunit is stabilized by heterodimerization with HIF-beta and hypoxia response elements (HRE), regulatory elements of HIF target genes are activated including VEGF, genes controlling cell proliferation and cell metabolism[103,104]. VEGF is one of the genes that is upregulated in hypoxia microenvironments eliciting a particular vascular phenotype; the high expression of VEGF is a common prognostic factor in human breast cancer malignancies representing an important therapeutic target. Other family members though play a role in angiogenesis even when VEGF is not expressed, in addition to the function of these homologues, the switching of angiogenic pathways may represent an area for further investigation to be possibly circumvented by multiple pathway inhibition[105].

an important therapeutic target. Other family members though play a role in angiogenesis even when VEGF is not expressed, in addition to the function of these homologues, the switching of angiogenic pathways may represent an area for further investigation to be possibly circumvented by multiple pathway inhibition[105]. Emerging patient data suggests that the combination of the anti-angiogenic drug bevacizumab with chemotherapy agents such as paclitaxel has proven to be a very dangerous therapeutic choice in terms of fatal side effects including hemorrhage, neutropenia, perforations of the gastrointestinal tract, blockage of arteries and stroke[106]. VEGF resistance mechanisms Several mechanisms are implicated in the emergence of resistance mechanisms to anti-angiogenic therapy (Figure 2). The most prominent one relates to the promiscuity of cancer cells to produce many types of alternative angiogenic signals which limit drug efficacy. The rescue of tumor vascularization may occur as escape mechanisms are induced by anti-angiogenic therapy and hypoxia of tumor tissue.

anti-angiogenic therapy (Figure 2). The most prominent one relates to the promiscuity of cancer cells to produce many types of alternative angiogenic signals which limit drug efficacy. The rescue of tumor vascularization may occur as escape mechanisms are induced by anti-angiogenic therapy and hypoxia of tumor tissue. Figure 2 Resistance mechanisms to anti-angiogenic therapy. During the initial development, tumor cells that are in the core of the tumor, become hypoxic and secrete pro-angiogenic factors (a); Proangiogenic factors are also produced by immune cells (b) and bone marrow cell participate in tumor vascularization (c); The amplification of cancer cell genome stimulates high gene expression levels, consequently, requiring an increased anti-angiogenic drug concentration (d); Tumors have evolved to switch from various modes of vascularization, in order to ensure a sufficient supply of nutrients, such as sprouting angiogenesis, vasculogenesis, vessel co-option as well as vascular mimicry (e); Various pro-angiogenic factors that are redundant of VEGF are secreted by tumor and stromal cells in malignant cancers (f); In response to the treatments, blood vessels regress (g) and tumor cell produced alternative proangiogenic prolymphangiogenic factors with the development of a lymphatic network (h); Tumor cells also express immune checkpoints proteins resulting in immune tolerance (i).

tumor and stromal cells in malignant cancers (f); In response to the treatments, blood vessels regress (g) and tumor cell produced alternative proangiogenic prolymphangiogenic factors with the development of a lymphatic network (h); Tumor cells also express immune checkpoints proteins resulting in immune tolerance (i). Cancer cells may amplify angiogenic genes which in return do not respond to low doses of anti-angiogenic drugs; they may switch from vessel sprouting to vessel co-option, vasculogenesis or vascular mimicry in order to ensure tumor nutrients. The recruitment of bone-marrow derived cells by cancer cells may result in the secretion of pro-angiogenic factors like angiopoietin, fibroblast growth factor or ephrins. The VEGF receptors may induce the release of a cytokine cascade which results in an inflamed microenvironment allowing for the emergence of tumor extravasation and metastasis.

rrow derived cells by cancer cells may result in the secretion of pro-angiogenic factors like angiopoietin, fibroblast growth factor or ephrins. The VEGF receptors may induce the release of a cytokine cascade which results in an inflamed microenvironment allowing for the emergence of tumor extravasation and metastasis. Some of the alternative targets to overcome drug resistance to anti-angiogenesis therapies might be to target the placental growth factor and Bv8 (Bombina variegata) to reduce tumor inflammation, reduce leakiness of vessels, moderate hypoxia and reduce angiogenesis; the Notch pathway by anti-delta like ligands 4 (DII4) and secretase inhibitors to reduce excessive sprouting and reduce leaky dysfunctional vessels. Vessel normalization may be achieved by PHD2 inhibition improving vessel function and reducing metastasis and hypoxia. Lymphangiogenesis may be targeted by inhibiting neuropilin-2 (Npn2) and by targeting neuropilin-1, tumor growth and angiogenesis can be significantly reduced[107]. Several alternative pathways may take over as resistance develops to anti-angiogenic therapy through intrinsic tumor resistance or acquired resistance: angiogenic redundancy involves the production of redundant pro-angiogenic factors like the fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), placenta growth factor (PlGF), tumor necrosis factor-α (TNF-α). As these pro-angiogenic factors allow for the growth of tumor vasculature despite the VEGF pathway being inhibited it would be appropriate to target several of them synergistically.

fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), placenta growth factor (PlGF), tumor necrosis factor-α (TNF-α). As these pro-angiogenic factors allow for the growth of tumor vasculature despite the VEGF pathway being inhibited it would be appropriate to target several of them synergistically. The increase of tumor hypoxia as a result of anti-angiogenic therapy is often implicated in angiogenic redundancy: The overexpression of the hypoxia-induced factor-1 (HIF-1) is correlated with chemotherapy resistance and selection of aggressive cancer cells as it is directly involved in the induction of transcription of genes involved in angiogenesis. The important role of activating the membrane tyrosine kinase receptor c-MET by the hepatocyte growth factor during angiogenesis, allows for downstream activation of SRC, AKT, MEK, STAT3 with an increased expression of VEGF and its receptor by endothelial cells. The HGF/c-MET collaboration is often associated with invasive cancer phenotypes and increased metastasis. In these cases, the selection of more invasive tumor cells may occur as hypoxic environments pressure cancer cells to move rapidly toward normoxic locations. The recruitment of bone marrow derived pro-angiogenic cells and inflammatory cell invasion may contribute to adaptive mechanisms of resistance as low oxygen concentrations induce these cells to release large amounts of pro-angiogenic factors. As alterations in endothelial cells and pericytes may be responsible for crosstalk between angiogenic pathways resulting in the emergence of anti-angiogenesis therapy resistance, inhibiting the VEGF pathway and the platelet derived growth factor receptor with a tyrosine kinase inhibitor simultaneously might be a promising strategy to enhance treatment efficacy. The process of vessel co-option may result in cancer cells displaying a normal looking vasculature which is less sensitive to anti-angiogenic therapy and early stage tumors may escape inhibition as they grow in an angiogenesis independent fashion[108].

eously might be a promising strategy to enhance treatment efficacy. The process of vessel co-option may result in cancer cells displaying a normal looking vasculature which is less sensitive to anti-angiogenic therapy and early stage tumors may escape inhibition as they grow in an angiogenesis independent fashion[108]. The future of anti-angiogenic therapy seems to depend on how different tumors become vascularized and by what alternative pathways these manage to escape therapeutic effects. Elucidation of the complexity of the biology of angiogenesis, coupled with the function of key biomarkers, may prove to be a promising way forward to enhance the function of anti-angiogenic therapy to achieve vascular normalization and increase the effects of complementary chemotherapy. TNBC and PARP inhibitors TNBC represent 10%-20% of invasive breast cancers in the general population and have been associated with the African-American ethnic group where a clear prevalence of the disease affects up to 28% of all patients within that group[109].

The future of anti-angiogenic therapy seems to depend on how different tumors become vascularized and by what alternative pathways these manage to escape therapeutic effects. Elucidation of the complexity of the biology of angiogenesis, coupled with the function of key biomarkers, may prove to be a promising way forward to enhance the function of anti-angiogenic therapy to achieve vascular normalization and increase the effects of complementary chemotherapy. TNBC and PARP inhibitors TNBC represent 10%-20% of invasive breast cancers in the general population and have been associated with the African-American ethnic group where a clear prevalence of the disease affects up to 28% of all patients within that group[109]. About 80% of breast tumors which lack the overexpression of the HER-2/erbB2 protein, the estrogen receptor (ER) and the progesterone receptor (PgR) fall under the category of TNBC. They may be characterized by elevated levels of PARP enzymes and often originate from basal-like cell types. TNBC represent the most aggressive phenotype of the disease with no specific targeted therapies available for treatment. Twelve percent of TNBC are characterized by a claudin-low subtype; these can be identified by DNA microarray expression profiling, a method slowly emerging in clinical practice for the detection of this rare form of breast cancer. These tumors seem to respond to molecules which target DNA repair systems to induce synthetic lethality if used in combination with other drugs. PARP inhibitors are an example of therapeutic choice when one of the genes in a synthetic lethal pair, with one gene already defective, is targeted resulting in cell death. PARP iso-enzymes include a group of 18 molecules which are central to base-excision repair pathways of single strand DNA breaks. An example is the BRCA1-2 mutation in breast cancer, this scenario allows for PARP inhibitors to target and block the only functioning DNA repair system, hence, the selective killing of tumor cells while sparing healthy ones and limiting toxicity for the patient[110]. Nuclear basic fibroblast growth factor (bFGF) is a protein found in a subset of TNBC which contributes to the emergence of resistance following chemotherapy[111]. In vitro studies have shown that a residual TNBC subpopulation remains after short-term chemotherapy and this resumes proliferation over time. When bFGF was knocked down in these residual cancer cells using short hairpin RNA, the number of residual TNBC cells decreased. This phenomenon is linked to a down-regulation of DNA-dependent protein kinase (DNA-PK) responsible for accelerated DNA repair. This study might suggest that expression of bFGF in TNBC cells could be a prognostic predictor of incomplete chemotherapy response and future tumor recurrence in TNBC patients[111].

decreased. This phenomenon is linked to a down-regulation of DNA-dependent protein kinase (DNA-PK) responsible for accelerated DNA repair. This study might suggest that expression of bFGF in TNBC cells could be a prognostic predictor of incomplete chemotherapy response and future tumor recurrence in TNBC patients[111]. The main challenge of circumventing treatment induced resistance mechanisms and the emergence of alternative escape pathways, significantly lowers the overall survival rate of breast cancer patients belonging to this particular subtype as they often exhibit an incomplete pathological response[93]. Sunitinib seems to suppress angiogenesis, tumor proliferation, migration and growth of basal like breast cancer cells; xenograft models indicate that tumor volumes decrease under sunitinib action but due to its effects on the Notch-1 protein expression and hypoxia through HIF-1, there was an increase in proliferation of breast cancer stem cells. The use of a γ-secretase inhibitor in addition to sunitinib may represent a promising treatment option for TNBC while simultaneously targeting cancer stem cells and angiogenesis[112]. Sunitinib may prove to be an effective treatment choice for patients with TNBC as this breast cancer subtype may express increased levels of VEGF. High levels of VEGF may act as a potential prognostic factor in TNBC as the vascular pathway is a key component when targeting this particularly rare subtype of breast cancer[113].

ib may prove to be an effective treatment choice for patients with TNBC as this breast cancer subtype may express increased levels of VEGF. High levels of VEGF may act as a potential prognostic factor in TNBC as the vascular pathway is a key component when targeting this particularly rare subtype of breast cancer[113]. As targeted therapies have not yet been discovered for TNBC, the conventional route is to treat patients with chemotherapy particularly anthracycline and taxane. The multitude of pathways which drive proliferation of this particular breast cancer subtype need to be further investigated in order to isolate potential therapeutic targets. Patients with BRCA1 and BRCA2 gene mutations which are present in 20% of TNBC, may be sensitive to the function of PARP inhibitors in addition to chemotherapy[6]. In a Phase II clinical trial carried out to evaluate the combined administration of the PARP1 inhibitor iniparib with cisplatin and gemcitabine on patients with TNBC, iniparib seemed to show significant anticancer activity enhancing the antiproliferative and cytotoxic effects of cisplatin and gemcitabine[114]. Combination therapy of cisplatin, gemcitabine and iniparib is currently under Phase III clinical trial to see if this association could represent the new standard of care for the treatment of TNBC (ClinicalTrials.gov No.NCT00938652).

hancing the antiproliferative and cytotoxic effects of cisplatin and gemcitabine[114]. Combination therapy of cisplatin, gemcitabine and iniparib is currently under Phase III clinical trial to see if this association could represent the new standard of care for the treatment of TNBC (ClinicalTrials.gov No.NCT00938652). Immunotherapy for breast cancer Breast cancer has been considered non-immunogenic for quite a long time and only recently, data has suggested that TNBC and HER2 positive types are characterized by an immune infiltrate, which might prove to be a promising target to complement the function of other synergistic drugs. Solid tumors like melanoma and lung cancer have already responded to immunotherapeutic agents like ipilimumab and sipuleucel-T has proven a successful vaccine against castration-resistant prostate cancers. Ongoing studies are also evaluating to what extent immune response is correlated to prognosis in breast cancer (Table 1). Table 1 Recapitulative breast cancer targeted therapy scheme cited in this article

Immunotherapy for breast cancer Breast cancer has been considered non-immunogenic for quite a long time and only recently, data has suggested that TNBC and HER2 positive types are characterized by an immune infiltrate, which might prove to be a promising target to complement the function of other synergistic drugs. Solid tumors like melanoma and lung cancer have already responded to immunotherapeutic agents like ipilimumab and sipuleucel-T has proven a successful vaccine against castration-resistant prostate cancers. Ongoing studies are also evaluating to what extent immune response is correlated to prognosis in breast cancer (Table 1). Table 1 Recapitulative breast cancer targeted therapy scheme cited in this article Target pathway Current therapy Combination therapy HER2 (HER2-positive breast cancer) Trastuzumab/herceptin Pertuzumab lapatinib Combination trastuzumab/lapatinib (EPHOS-B trial) trastuzumab/264RAD m-TOR pathway Everolimus Possible combination everolimus/HER2 inhibitor Angiogenesis (VEGF) Bevacizumab paclitaxel Docetaxel Targeting the placental growth factor and Bv8/Targeting the Notch pathway by anti-delta like ligands 4 and secretase inhibitors inhibiting simultaneously the VEGF pathway and the platelet derived growth factor receptor with a TK inhibitor DNA repair mechanisms (TNBC) Notch-1 protein over-expression/breast cancer stem cells proliferation (TNBC) Parp inhibitors/anthracyclins and taxanes Possible combination cisplatin/gemcitabine/iniparib Possible combination of g-secretase inhibitor in addition to sunitinib Immune system response Cell cycle checkpoints Immunotherapeutic agents Antibodies against PD-1 T-cell inhibitory molecule or its ligand PD-L1 Nelipepimut-S(human leukocyte antigen)/GM-CSF Pembrolizumab in TNBC/PD-L1 positive (KEYNOTE-086 trial) HER2: Human epidermal growth factor receptor 2; DII4: Delta like ligands 4; TNBC: Triple negative breast cancer; GM-CSF: Granulocyte-macrophage colony stimulating factor; VEGF: Vascular endothelial growth factor.

D-L1 Nelipepimut-S(human leukocyte antigen)/GM-CSF Pembrolizumab in TNBC/PD-L1 positive (KEYNOTE-086 trial) HER2: Human epidermal growth factor receptor 2; DII4: Delta like ligands 4; TNBC: Triple negative breast cancer; GM-CSF: Granulocyte-macrophage colony stimulating factor; VEGF: Vascular endothelial growth factor. The aim of immunotherapy is that of activating the human immune response to recognize tumors as a foreign entity and eventually kill the tumor cells. The tumor microenvironment (TME) including T-regulatory cells (T-Reg) involves a complex structure of intercellular communication which represent a very promising area of research aiming at the isolation of key immunogenic targets which may enhance the function of existing therapies[115].

entually kill the tumor cells. The tumor microenvironment (TME) including T-regulatory cells (T-Reg) involves a complex structure of intercellular communication which represent a very promising area of research aiming at the isolation of key immunogenic targets which may enhance the function of existing therapies[115]. The immune-checkpoint receptor PD-1 is expressed on tumor-infiltrating lymphocytes (TILs) with the role of inhibiting the activity of effector T-cells, preventing autoimmunity and inflammatory response; it is often upregulated on tumor cell surface in many types of solid tumors. The PD-1 ligand PDL1 engages with T-cells resulting in upregulation of the receptor followed by an immunosuppressive signal, which inhibits kinases involved in the activation of the immune response[116]. Clinical blockade of the PD-1/PDL1 axis should enhance antibody function in cancer patients underlining the importance of further investigation in this particular area of breast cancer research (Table 2). Pro-inflammatory cytokines and the overexpression of PDL1 inhibitory ligand may play a key role in the development of cancer immune resistance mechanisms, resulting in a state of exhausted or tolerant immune T-cell response hence the importance of studying the possible role of PDL1 expression as a resistance biomarker. Overall, main role of PD-1 blockade results in the reversal of chronic antigen response which is often found in cancer and viral infection scenarios[117]. The anti PD-1 antibody nivolumab has shown successful activity in melanoma and lung cancer patients targeting these immunoregulatory proteins and enhancing tumor response. There are several other ligands being investigated at present which might be potential targets like: CD80, CD86, PDL2, ICOS-L, B7-H3, B7-H4 and B7-H6 and future directions in cancer immunotherapy research point towards the effects of combined checkpoint blockade to maximize clinical response[118].

ncing tumor response. There are several other ligands being investigated at present which might be potential targets like: CD80, CD86, PDL2, ICOS-L, B7-H3, B7-H4 and B7-H6 and future directions in cancer immunotherapy research point towards the effects of combined checkpoint blockade to maximize clinical response[118]. Table 2 Some of the current clinical trials in breast cancer targeted immunotherapy (http://www.cancerresearch.org./cancer-immunotherapy/impacting-all-cancers/breast-cancer) Title of clinical trial Type of breast cancer Phase III clinical trial: NEUVAX: nelipepimut-S or E75NCT01479244 HER1+ HER2+ Phase II clinical trial: NEUVAX NCTO1570036 Node positive or TNBC Phase I clinical trial: Pembrolizumab PD1 antibody + dendritic cell vaccine NCTO2479230 Metastatic breast cancer Phase II trial: Pembrolizumab PD1 antibody + HDAC inhibitor and anti-estrogen therapy NCT02395627 Breast cancer Phase II first line neo adjuvant trial: Atezolizumab + chemotherapy NCTO2530489 TNBC Phase I clinical trial: Atezolizumab and HER2 inhibitors NCTO2605915 HER2+ Phase I/II clinical trial: PDR001(PD1 antibody) Advanced breast cancer, TNBC Phase I/II clinical trial: MEDI6469 anti OX40 antibody NCTO1642290 Stage 4 breast cancer (patients with prior failure of hormone or chemotherapy) Pilot study of QBX258 targeting IL-4 and IL-13 NTCO2494206 Advanced TNBC whose cancer cells make a protein called glycoprotein NMB to which CDX-011 binds IL: Interleukin; HER2: Human epidermal growth factor receptor 2; TNBC: Triple negative breast cancer.

cancer (patients with prior failure of hormone or chemotherapy) Pilot study of QBX258 targeting IL-4 and IL-13 NTCO2494206 Advanced TNBC whose cancer cells make a protein called glycoprotein NMB to which CDX-011 binds IL: Interleukin; HER2: Human epidermal growth factor receptor 2; TNBC: Triple negative breast cancer. Future direction: Breast cancer combination therapy Over the last few years, new agents have been introduced in breast cancer targeted therapy resulting in overall improved treatments and greater patient overall survival rates. Some of the most widely used combination therapies involve the use of agents which target the PI3K/AKT/mTOR pathways such as everolimus combined to exemestane. The everolimus-FKBP12 complex that forms when the m-TOR inhibitor binds with high affinity to the intracellular receptor FKBP12, is very effective in inhibiting down strem signaling in cancer cells. The BOLERO study has demonstrated the efficacy of the m-TOR inhibitor everolimus used in combination with exemestane (endocrine therapy) to restore hormonal sensitivity in breast cancer patients[6]. Palbocib has been combined with letrozole in treating women with ER positive (estrogen positive), HER2 negative, advanced breast cancers as a first line endocrine therapy in metastatic cases. Trastuzumab and lapatinib have been used successfully in combination to treat metastatic breast cancers that overexpress HER2[6]. Trastuzumab and pertuzumab have been used in combination for the treatment of HER2 positive metastatic breast cancers and have shown a statistically significant increase in overall survival of patients[6]. The trastuzumab/lapatinib/hormonal therapy combination has proven to be effective in cases of hormonal receptor positive and overexpressed HER2 protein breast cancers like the luminal B/HER2 enriched type. Iniparib, a PARP1 inhibitor, in combination with gemcitabine and carboplatin chemotherapy have been evaluated in a Phase I clinical trial for the treatment of metastatic TNBCs and a clinical benefit of 56% was observed in the combined therapy arm, compared to the gemcitabine/carboplatin arm which had a 34% clinical benefit[114].

a PARP1 inhibitor, in combination with gemcitabine and carboplatin chemotherapy have been evaluated in a Phase I clinical trial for the treatment of metastatic TNBCs and a clinical benefit of 56% was observed in the combined therapy arm, compared to the gemcitabine/carboplatin arm which had a 34% clinical benefit[114]. A promising area of clinical research for breast cancer targeted therapy involves the use of immune checkpoints inhibitors or immune checkpoint stimulatory molecules. In order to unleash anti-cancer immune responses, inhibitory molecules are blocked or stimulatory molecules are activated to allow the immune system to attack directly cancer cells as foreign invaders. An example would be the anti PD1 antibody pembrolizumab (Keytruda), anti CTLA antibodies, the anti PD-L1 antibody atezolizumab, anti CD73 antibodies or anti OX40 antibodies being tested currently in Phase I/II clinical trials (Table 2).

ted to allow the immune system to attack directly cancer cells as foreign invaders. An example would be the anti PD1 antibody pembrolizumab (Keytruda), anti CTLA antibodies, the anti PD-L1 antibody atezolizumab, anti CD73 antibodies or anti OX40 antibodies being tested currently in Phase I/II clinical trials (Table 2). As the importance of the TME is being discovered with its potential contribution to cancer therapy, novel agents are being developed to target the non-malignant tumor stroma like trabectedin which inhibits macrophage differentiation; other drugs target the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) pathway such as mapatumumab and dulanermin; immunomodulators used alone or in combination to cytotoxic agents should be also investigated as a strategy to decrease the immunosuppression caused by T-effector cell upregulation in the quest to increase the innate immune response against cancer cells, keeping the right balance as immune over-stimulation could be potentially harmful to patients[86].

combination to cytotoxic agents should be also investigated as a strategy to decrease the immunosuppression caused by T-effector cell upregulation in the quest to increase the innate immune response against cancer cells, keeping the right balance as immune over-stimulation could be potentially harmful to patients[86]. The main future challenge for breast cancer combination therapy is to design a winning formula that is simultaneously effective against the many subtypes of breast cancers like luminal A, luminal B, basal-like and overexpressing HER2. This approach would represent a hopeful avenue to explore in the quest to inhibit the multitude of pathways being exploited by the various breast cancer subtypes. The phenotype of each breast cancer subtype should be thoroughly investigated as well, to allow researchers to gather a general picture describing in detail the different mechanisms of action for cell survival. Only then, more precise targets can be identified allowing for the discovery of more inclusive breast cancer combination therapies. A more precise and personalized characterization of each cancer as well as the identification of factors involved in resistance for each patient may provide useful improvements in current therapeutic approaches.

targets can be identified allowing for the discovery of more inclusive breast cancer combination therapies. A more precise and personalized characterization of each cancer as well as the identification of factors involved in resistance for each patient may provide useful improvements in current therapeutic approaches. CONCLUSION Decoding of the human genome has allowed for the isolation of key gene signatures for many types of known cancers; unfortunately, targeted therapies to inhibit the function of these genes have proven quite elusive as the quest to circumvent the emergence of resistance mechanisms continues. Breast cancer subtypes, particularly TNBCs, still represent a major challenge; future studies should revolve around the discovery of new prognostic biomarkers as no targets for these rare types of breast cancer have yet been identified.

lusive as the quest to circumvent the emergence of resistance mechanisms continues. Breast cancer subtypes, particularly TNBCs, still represent a major challenge; future studies should revolve around the discovery of new prognostic biomarkers as no targets for these rare types of breast cancer have yet been identified. The EPHOS-B trial carried out by researchers in The Institute of Cancer Research, London, the University of Manchester and University Hospital of South Manchester NHS Foundation Trust investigating the response of HER2 positive breast cancer to dual lapatinib and trastuzumab therapy shortly after diagnosis and surgery to remove the tumors, has released very promising data in which of 257 women who were administered the two drugs synergistically 11 d before surgery, 17% had only minimal residual disease with invasive tumor smaller than 5 mm in size, 11% had a pathological complete response with no biological invasive tumor present in the breast and 3% had a complete response. This dramatic response after only 11 d suggests that combination anti-HER2 targeted therapy prior surgery may reduce the number of breast cancer patients requiring chemotherapy in the future and significantly eliminate long term chemotherapy associated side effects[4,119].

in the breast and 3% had a complete response. This dramatic response after only 11 d suggests that combination anti-HER2 targeted therapy prior surgery may reduce the number of breast cancer patients requiring chemotherapy in the future and significantly eliminate long term chemotherapy associated side effects[4,119]. Resistance mechanisms in breast cancer targeted therapies represent the main challenge to current research; the combination of different molecules used to target different levels of signaling pathways by synergistically blocking cancer cell escape routes and minimizing the emergence of survival mechanisms, could prove to be a promising way forward, keeping in mind that specific molecular profiling particularly for metastatic relapses should be carried out to elucidate further resistance phenotypes and allow for the design of specific new targets. Several clinical trials are underway to try to improve survival of the worse cases (Table 3). Table 3 Some of the current clinical trials in breast cancer targeted therapy (http://www.breastcancertrials.org)

Resistance mechanisms in breast cancer targeted therapies represent the main challenge to current research; the combination of different molecules used to target different levels of signaling pathways by synergistically blocking cancer cell escape routes and minimizing the emergence of survival mechanisms, could prove to be a promising way forward, keeping in mind that specific molecular profiling particularly for metastatic relapses should be carried out to elucidate further resistance phenotypes and allow for the design of specific new targets. Several clinical trials are underway to try to improve survival of the worse cases (Table 3). Table 3 Some of the current clinical trials in breast cancer targeted therapy (http://www.breastcancertrials.org) Title of clinical trial Type of cancer Randomized open label Phase II trial: Kadcyla, tykerb and abraxane vs herceptin, tykerb and taxol before Surgery for HER2+ tumors NCT02073487 HER2+ Phase III randomised, placebo controlled clinical trial: Chemotherapy and a PARP-inhibitor for BRCA1/2+, HER2- advanced breast cancer NCTO2163694 HER2-, BRCA1/2+ metastatic or locally advanced unresectable breast cancer Phase II, multicenter, randomized clinical trial: Alisertib with taxol for advanced ER+/HER2- or TNBC NCTO2187991 ER+/HER2- TNBC Phase II Clinical trial: Gemzar, herceptin and perjeta for HER2+ metastatic breast cancer NCTO2252887 HER2+ metastatic breast cancer Phase I clinical trial: CD-839 for advanced breast tumors NCTO2071862 Advanced breast cancer and solid tumors Phase I clinical trial: Saracatinib and anastrozole for ER-positive disease NCTO1216176 ER+ Randomised Phase III clinical trial: Hormone therapy with or without ibrance for HR+, HER2- stage II-III breast cancer NCTO2513394 HR+, HER2- Phase II clinical trial: CDK-inhibitor for previously treated metastatic disease NCTO1037790 Previously treated metastatic breast cancer Phase I clinical trial: GS-5745 in metastatic HER2- breast cancer and other solid tumors NCTO1813282 Metastatic HER2- breast cancer not responding to other treatments Supported by The French Association for Cancer Research (ARC); the Fondation de France; the French National Institute for Cancer Research (INCA); the Fondation Estée Lauder (Pink Ribbon Award); Roche France and “Cordon de Vie” Monaco.

tumors NCTO1813282 Metastatic HER2- breast cancer not responding to other treatments Supported by The French Association for Cancer Research (ARC); the Fondation de France; the French National Institute for Cancer Research (INCA); the Fondation Estée Lauder (Pink Ribbon Award); Roche France and “Cordon de Vie” Monaco. Conflict-of-interest statement: There is no conflict of interest to declare. Manuscript source: Unsolicited manuscript Specialty type: Oncology Country of origin: France Peer-review report classification Grade A (Excellent): A Grade B (Very good): B Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0 Peer-review started: July 21, 2016 First decision: September 5, 2016 Article in press: October 18, 2016 P- Reviewer: Song J, Shao R, Wei JF, Wang L S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: Gastrointestinal stromal tumors (GISTs) are most common mesenchymal tumors in the gastrointestinal tract. The management of GISTs is revolutionized with the advent of tyrosine kinase inhibitors (TKIs) and newer advanced endoscopic techniques. Accurate identification and differentiation of GISTs from other submucosal tumors is achieved with the help of endoscopic ultrasound. The management of small to medium GISTs are feasible by newer advanced endoscopic and/or laparoscopic techniques. Team approach involving endoscopist, pathologist, radiologist, medical oncologist and surgeon is key in optimal management of GISTs. This article focuses on role of TKIs and endoscopist perspective in the management of GISTs.

mall to medium GISTs are feasible by newer advanced endoscopic and/or laparoscopic techniques. Team approach involving endoscopist, pathologist, radiologist, medical oncologist and surgeon is key in optimal management of GISTs. This article focuses on role of TKIs and endoscopist perspective in the management of GISTs. INTRODUCTION Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal (sub epithelial) tumor, and are frequently found in stomach and small intestine[1]. GISTS are hypothesized to originate from interstitial cells of cajal (ICC) which coordinate gut motility[2]. GISTs are rarely found in the peritoneum, mesentery and omentum[3]. GISTs have varied malignant potential, with about 40% of GISTs that are localized at initial diagnosis give rise to metastasis[4], and about 10%-20% of GISTs present with distant metastasis[5,6]. In Europe, the annual incidence of GISTs is about 10 cases per million[7]. In the United States, the annual incidence of GIST ranges from 4000 to 6000 new cases per year (7-20 cases per million population per year)[8]. The mean age at diagnosis is 63 years[9]; men and women are equally affected. The majority of GISTs are sporadic and may be associated with mutations like NF1, C-kit, platelet derived growth factor receptor-alpha (PDGFRA), succinate dehydrogenase (SDH) and deletions in chromosome 1 involving SDH c[10].

on per year)[8]. The mean age at diagnosis is 63 years[9]; men and women are equally affected. The majority of GISTs are sporadic and may be associated with mutations like NF1, C-kit, platelet derived growth factor receptor-alpha (PDGFRA), succinate dehydrogenase (SDH) and deletions in chromosome 1 involving SDH c[10]. PATHOGENESIS OF GIST Overall, GISTs are defined by the presence of KIT gene or PDGFRA mutation. Majority (80%) of GISTs have KIT gene mutations and biologic response of KIT receptor is produced without a bound ligand[11]. KIT receptor tyrosine kinase activity in normal cells is regulated by binding of endogenous KIT ligand or stem cell factor (SCF)[12]. In the majority of cases, spontaneous receptor dimerization and activation occurs when exon 11 is affected by KIT gene mutation. However, in few cases, a different mechanism results in uncontrolled KIT signaling if mutation occurs in Exon 9, 13 or 17. In cases with NF1, uncontrolled KIT activation may be present even in the absence of KIT gene mutation (wild type)[13]. A subset of GISTs which are negative for KIT gene mutations are positive for receptor tyrosine kinase PDGFRA mutations. GISTs expressing PDGFRA or KIT gene mutations have similar biologic consequences[14]. About 10% of adult GISTs have neither KIT gene nor PDGFRA mutation[15]. SDH-ubiquinone complex 2 is composed of subunits A, B, C and D which is part of Krebs cycle and respiratory chain[16]. In mutant SDH, dysfunction of electron transport chain in mitochondria leads to defective oxidative phosphorylation, which ultimately leads to abnormal stabilization of hypoxia inducible factors (HIF)[17]. Carney-Stratakis syndrome is caused by germline mutation in SDH subunits B, C or D which leads to GIST and paraganglioma[18].

DH, dysfunction of electron transport chain in mitochondria leads to defective oxidative phosphorylation, which ultimately leads to abnormal stabilization of hypoxia inducible factors (HIF)[17]. Carney-Stratakis syndrome is caused by germline mutation in SDH subunits B, C or D which leads to GIST and paraganglioma[18]. Histologically GISTs are subdivided in to spindle cell (60%-70%), epithelioid (30%-40%) or both (10%). GISTs with spindle cells are compact, highly cellular, arranged in fascicular or whorled pattern with minimal amount of stroma and contain eosinophilic, basophilic or amphophilic cytoplasm. Epithelioid tumors have abundant cytoplasm which is amphophilic to clear and cellular borders are clearly defined[19]. Antibodies to CD34 and CD117 appear in most GISTs[20]. CD34 is a transmembrane glycoprotein present on vascular endothelium and human hematopoietic progenitor cells[21]. CD34 is expressed in a wide variety of tumors and it is detected in about 50%-80% of GISTs[2,11,20]. CD 117 is expressed in 80%-100% of GISTs and it is not expressed in smooth muscle or neural tumors which helps in distinguishing GISTs from other gastrointestinal mesenchymal tumors[20] (Figure 1). Figure 1 Pathogenesis. GIST: Gastrointestinal stromal tumor; PDGFRA: Platelet derived growth factor receptor-alpha; HIF: Hypoxia inducible factors.

Histologically GISTs are subdivided in to spindle cell (60%-70%), epithelioid (30%-40%) or both (10%). GISTs with spindle cells are compact, highly cellular, arranged in fascicular or whorled pattern with minimal amount of stroma and contain eosinophilic, basophilic or amphophilic cytoplasm. Epithelioid tumors have abundant cytoplasm which is amphophilic to clear and cellular borders are clearly defined[19]. Antibodies to CD34 and CD117 appear in most GISTs[20]. CD34 is a transmembrane glycoprotein present on vascular endothelium and human hematopoietic progenitor cells[21]. CD34 is expressed in a wide variety of tumors and it is detected in about 50%-80% of GISTs[2,11,20]. CD 117 is expressed in 80%-100% of GISTs and it is not expressed in smooth muscle or neural tumors which helps in distinguishing GISTs from other gastrointestinal mesenchymal tumors[20] (Figure 1). Figure 1 Pathogenesis. GIST: Gastrointestinal stromal tumor; PDGFRA: Platelet derived growth factor receptor-alpha; HIF: Hypoxia inducible factors. CLINICAL PRESENTATION AND DIAGNOSTIC TOOLS Clinical manifestations of GISTs are highly variable and it depends on tumor size and location. GISTs are usually asymptomatic and found incidentally by imaging or endoscopy[22]. Symptoms include melena, hematemesis, abdominal pain, discomfort, fullness, early satiety and palpable mass. GISTs in proximal stomach can cause dysphagia and tumors in pylorus can present as gastric outlet obstruction[23]. Rectal GISTs can present with hematochezia[24]. Rarely, they can present as intraperitoneal rupture of large tumor causing hemoperitoneum[25]. GISTs can occur as part of a syndrome; Carneys triad (gastric GIST, pulmonary chondroma, paraganglioma)[26], or neurofibromatosis type 1 (mostly spindle cell GIST)[27]. Overall, about 50% of GISTs have local or distant metastasis at the time of presentation[28], with the liver being the most frequent site of metastasis. Other common sites of metastasis include the bone, peritoneum, retroperitoneum, lung, pleura, and subcutaneous (scar) tissue[29].

ype 1 (mostly spindle cell GIST)[27]. Overall, about 50% of GISTs have local or distant metastasis at the time of presentation[28], with the liver being the most frequent site of metastasis. Other common sites of metastasis include the bone, peritoneum, retroperitoneum, lung, pleura, and subcutaneous (scar) tissue[29]. Computed tomography (CT) is the primary modality of choice for diagnosing GISTs[30,31]. CT tumor characteristics such as size greater than 10 cm, calcifications, irregular margins, heterogeneous, lobulated, regional lymphadenopathy, ulceration, extraluminal and mesenteric fat infiltration are more likely to be associated with metastasis[29]. CT enterography uses large volumes of oral contrast and it is superior to conventional CT. It has advantage of displaying the entire thickness of the small bowel, better visualization of deep ileal loops without superimposition and evaluation of surrounding mesentery[32]. MRI is more accurate than CT for delineating rectal GISTs and in detecting liver metastasis, hemorrhage and necrosis[33].

conventional CT. It has advantage of displaying the entire thickness of the small bowel, better visualization of deep ileal loops without superimposition and evaluation of surrounding mesentery[32]. MRI is more accurate than CT for delineating rectal GISTs and in detecting liver metastasis, hemorrhage and necrosis[33]. Esophagogastroduodenoscopy (EGD) shows most sub epithelial lesions as a bulge with a smooth, intact, normal appearing mucosa in the gastrointestinal tract. Hwang et al[34] did a prospective study and patients were referred for endoscopic ultrasound (EUS) to evaluate sub epithelial masses diagnosed previously by EGD, sigmoidoscopy or colonoscopy. The size of the mass during endoscopic exam was measured by open biopsy forceps for size reference. Results showed endoscopy was 98% sensitive and 64% specific in identifying intramural lesions. Intramural size measurement of endoscopy correlated with EUS (r = 0.88, P < 0.001) but, for extramural lesions, it was suboptimal (r = 0.56)[34]. Overall, the study concluded endoscopy had a high sensitivity but low specificity in identifying the location of sub epithelial lesions and histologic confirmation by EUS-fine-needle aspiration (FNA) should be obtained for masses originating from 3rd (submucosa) and 4th layer (muscularis propria)[34].

l (r = 0.56)[34]. Overall, the study concluded endoscopy had a high sensitivity but low specificity in identifying the location of sub epithelial lesions and histologic confirmation by EUS-fine-needle aspiration (FNA) should be obtained for masses originating from 3rd (submucosa) and 4th layer (muscularis propria)[34]. Endosonographically GISTs appear as oval or hypoechoic mass arising from the muscularis propria. EUS features suggestive of malignancy include enlarged lymph nodes, size greater than 4 cm, irregular borders and cystic spaces with in the mass[35]. EUS has 92% sensitivity and 100% specificity in differentiating submucosal tumor from extrinsic compression[36]. Chen et al[37], retrospectively evaluated EUS characteristics to predict the malignant potential of GISTs. EUS features of GISTs were compared to National Institutes of Health (NIH) criteria for classification of malignant potential and were divided in to very low/low risk, intermediate/high risk. Results showed that GISTs at high risk for malignancy were associated with EUS characteristics like lesion size (P < 0.0001), cystic change (P = 0.015) and surface ulceration (P = 0.036)[37]. EUS-FNA cannot accurately differentiate benign from malignant GIST due to lack of mitotic activity on smears. The definitive method for assessment of GIST malignant potential requires surgical resection.

EUS characteristics like lesion size (P < 0.0001), cystic change (P = 0.015) and surface ulceration (P = 0.036)[37]. EUS-FNA cannot accurately differentiate benign from malignant GIST due to lack of mitotic activity on smears. The definitive method for assessment of GIST malignant potential requires surgical resection. Dewitt et al[38] evaluated the diagnostic yield and complications of EUS-Trucut biopsy (EUS-TCB) for gastrointestinal mesenchymal tumor (GIMT). EUS-FNA was performed in 33/38 (87%), and was diagnostic on final cytology in 25/33 (76%) and by FNA-immunochemistry (FNA-IC) in 12/24 (50%). EUS-TCB obtained visible tissue specimen in 37/38 (97%), and diagnostic in the final TCB histology in 30/38 (79%) and TCB-IC in 30/31 (97%)[38]. Overall, the authors concluded that EUS-TCB should be considered as an alternative to EUS-FNA when technically feasible[38]. Na et al[39] evaluated the yield and utility of 19-gauge (G) TCB vs 22-G FNA for diagnosing gastric sub epithelial tumors (SETs). The diagnostic yield of TCB vs FNA were 77.8% vs 38.7% (P < 0.0001). The Accuracy of TCB vs FNA for diagnosing GISTs was 90.9% vs 68.8%; and for non-GIST SETs was 81.1% vs 14.3% respectively. There were 9 technical failures with TCB likely due to stiffness, poor maneuverability of the needle and location of the tumor[39]. The most common procedure associated adverse events were pain, hemorrhage (requiring endoscopic hemostasis) and fever[39]. Procedure related events in TCB vs FNA were [3/90 (3.3%) vs 5/62 (8.1%); P = 0.27] respectively[39].

h TCB likely due to stiffness, poor maneuverability of the needle and location of the tumor[39]. The most common procedure associated adverse events were pain, hemorrhage (requiring endoscopic hemostasis) and fever[39]. Procedure related events in TCB vs FNA were [3/90 (3.3%) vs 5/62 (8.1%); P = 0.27] respectively[39]. Positron emission tomography (PET)-CT using 18F-fluorodeoxy glucose (FDG) detects cancer based on changes in tissue metabolism[40,41]. PET-CT is used for initial staging and to monitor disease progression. A baseline 18FDG-PET should be obtained before treatment so that the results can be used to compare with future studies[42]. Liver metastasis from GIST often appear as isodense lesions on CT, but may be detected by PET. Hence PET compliments CT in resolving ambiguity of liver lesions in patients with GISTs[42].

ession. A baseline 18FDG-PET should be obtained before treatment so that the results can be used to compare with future studies[42]. Liver metastasis from GIST often appear as isodense lesions on CT, but may be detected by PET. Hence PET compliments CT in resolving ambiguity of liver lesions in patients with GISTs[42]. Gayed et al[43] showed that the sensitivity and positive predictive value of 18F-FDG PET were 86% and 98% respectively and it is superior to CT in predicting early response to therapy in recurrent or metastatic GISTs[43]. Yoshikawa et al[40] evaluated the efficacy of PET-CT to predict the malignant potential of GIST. Standardized uptake value maximum (SUVmax) and GIST parameters (Ki-67 labeling index and mitotic index) were compared. SUVmax and Ki67 labeling index were significantly elevated in high risk group when compared to low/intermediate risk group[40]. Tumor response to treatment with imatinib mesylate may be detected by a decrease in CT attenuation units (Hounsfield units, HU)[44]. However, there may be delay in measurement of cellular and macroscopic changes after treatment with imatinib by CT. In contrast, PET using 18F-FDG can detect early effects induced by imatinib and decrease in FDG uptake after the initiation of imatinib treatment indicates good prognosis[45].

(Hounsfield units, HU)[44]. However, there may be delay in measurement of cellular and macroscopic changes after treatment with imatinib by CT. In contrast, PET using 18F-FDG can detect early effects induced by imatinib and decrease in FDG uptake after the initiation of imatinib treatment indicates good prognosis[45]. The “Response Evaluation Criteria in Solid Tumors” (RECIST) classification was previously used, however, due to limitations in assessing malignant response to immunotherapy such as imatinib, RECIST has been replaced by the Choi criteria[46]. Limitations of RECIST were primarily because the response to therapy can occur not only in tumor size but also in structure like decreased tumor density and enhancement of intratumoral nodules[31,47]. The Choi criteria of contrast-enhanced CT is based on decrease in tumor size by 10% in any dimension or decrease in structure by 15%, and was found to be more predictive of time to tumor progression (TTP) than RECIST[48].

but also in structure like decreased tumor density and enhancement of intratumoral nodules[31,47]. The Choi criteria of contrast-enhanced CT is based on decrease in tumor size by 10% in any dimension or decrease in structure by 15%, and was found to be more predictive of time to tumor progression (TTP) than RECIST[48]. PROGNOSIS AND RISK STRATIFICATION Mitotic index, tumor size, location (gastric vs non-gastric) and tumor rupture are independent risk factors for GIST metastases[4]. Joensuu et al[49] analyzed the association between KIT and PDGFRA mutation and RFS in GIST patients treated with surgery alone. The authors concluded that tumor mutation status should not be interpreted in isolation from other risk factors[49]. The American College of Surgeons Oncology trial (ACOSOG) Z90001 study found that tumor size, location and mitotic rate were important in RFS but not tumor mutation status[50]. Gold et al[51] developed a nomogram by calculating concordance probabilities and by comparing three commonly employed staging systems NIH-Miettinen[52], NIH-Fletcher[53] and Armed Forces Institute of Pathology (AFIP)-Miettinen[54]. The investigators concluded that the nomogram can accurately predict RFS after the resection of localized, primary GIST[51].

by calculating concordance probabilities and by comparing three commonly employed staging systems NIH-Miettinen[52], NIH-Fletcher[53] and Armed Forces Institute of Pathology (AFIP)-Miettinen[54]. The investigators concluded that the nomogram can accurately predict RFS after the resection of localized, primary GIST[51]. MANAGEMENT OF GIST Surgery is the treatment of choice for primary and localized GISTs[55]. The goal of surgery is complete tumor resection (negative microscopic and macroscopic margins) with functional preservation (often accomplished by wedge resection), while avoiding tumor rupture and injury to the pseudo capsule[55]. McCarter et al[56] analyzed factors associated with R0 (grossly and histologically negative margin), R1 (grossly negative but histologically positive margins), R2 resection (grossly positive margins) and assessed the risk of recurrence with and without imatinib[56]. Factors associated with R1 resection included tumor size (> or = 10 cm), tumor rupture and location[56]. The authors concluded there was no significant difference in recurrence free survival (RFS) in patients who underwent R1 vs R0 resection of GIST with or without adjuvant imatinib[56]. Although the management of R1 resection after complete resection is not clear, options include careful observation (watchful waiting), re-excision and adjuvant imatinib treatment.

ant difference in recurrence free survival (RFS) in patients who underwent R1 vs R0 resection of GIST with or without adjuvant imatinib[56]. Although the management of R1 resection after complete resection is not clear, options include careful observation (watchful waiting), re-excision and adjuvant imatinib treatment. Laparoscopic wedge resection (LWR) is recommended for gastric GIST smaller than 5 cm. To prevent tumor seeding in laparoscopy, plastic bag is recommended to collect the tumor sample and direct handling of tumor with forceps is contraindicated. Wedge resection of gastric GIST is considered standard treatment[57] and lymphadenectomy is not indicated as nodal metastasis is rare[28]. LWR has the advantage of early resumption of diet, early return of bowel function, shorter hospital stay and decreased duration of parenteral or epidural analgesia[58]. Lee et al[59] study concluded that LWR can be safely performed and have better outcome in terms of recovery after surgery regardless of tumor size and location. Kim et al[60] study concluded that LWR is safe and feasible for small to medium sized gastroduodenal tumors irrespective of location in cardia or pylorus. However, they recommended careful consideration of direction of stapling for exogastric resection of submucosal tumors located in antrum, lesser curvature and pylorus to prevent gastric outlet obstruction.

s safe and feasible for small to medium sized gastroduodenal tumors irrespective of location in cardia or pylorus. However, they recommended careful consideration of direction of stapling for exogastric resection of submucosal tumors located in antrum, lesser curvature and pylorus to prevent gastric outlet obstruction. Endoscopic enucleation and other related procedures are more feasible for GISTs less than 5 cm[61]. Complete resection of GIST is indicated with endoscopic enucleation in the presence of a pseudo capsule. According to location in the gastric wall, GISTs are classified in to several types such as type 1 [very narrow connection with muscularis propria (MP) layer which protrudes in to the lumen], type 2 (wide based connection with MP layer and protrudes in the luminal side at obtuse angle), type 3 (located in the middle of gastric wall) and type 4 (protrudes into the serosal surface of gastric wall)[61]. This classification is very important when considering endoscopic enucleation. Endoscopic enucleation is best suitable for type 1 because of narrow connection to the MP layer and can be attempted for type 2. Type 3 and type 4 cannot be completely resected by endoscopic enucleation and hence endoscopic full-thickness resection (EFTR), laparoscopic and endoscopic cooperative surgery (LECS), laparoscopic-assisted endoscopic full-thickness resection (LAEFR) and non-exposed wall-inversion surgery (NEWS) should be considered[61]. Endoscopic enucleation includes various techniques like endoscopic submucosal dissection (ESD)[62], endoscopic muscularis dissection (EMD)[63] and endoscopic submucosal tunnel dissection (ESTD)[64]. Bialek et al[62] evaluated the efficacy, safety and outcomes of ESD for gastric sub epithelial tumors. Results showed 47% (17/37) sub epithelial tumors were GISTs, overall rate of R0 resection was 81.1% (30/37), and perforation rate was 5.4%[62]. Liu et al[63] evaluated the feasibility and safety of EMD. Results showed that 51.6% (16/31) were GISTs, 96.8% (30/31) were completely resected, perforation occurred in 12.9% (4/31, all of which were managed by endoscopic methods)[63]. ESTD procedure involves creation of the submucosal tunnel, dissection of the submucosal tumor (SMT) and closure of mucosal entry with hemostatic clips[64]. Gong et al[64] evaluated the feasibility and safety of ESTD in upper gastrointestinal SMTs.

r recurrence, although the dissection site is usually ablated with electrical knife or snare. Perforation occurs due to pseudo capsule injury during difficult MP layer dissection which increases the chance of peritoneal seeding. Peritoneal seeding is associated with poor prognosis because of increased tumor recurrence. EFTR without laparoscopic assistance procedure involves introducing a single-chamber gastroscope into the stomach with a transparent cap attached to its tip. Dots are marked around the lesion and submucosal injection is done using normal saline with 1% indigo carmine and epinephrine (1:100000). Hook knife and IT knife are used to incise superficial layers overlying the SMT and snare is used to remove the mucosal and submucosal layers of gastric wall. Hook knife and IT knife are used to make circumferential dissection around the border of SMT. To visualize the SMT clearly, submucosal injection can be done again in the lower border of the tumor as needed. After the MP layer is reached and root of the tumor is exposed, gastric fluid is extracted as much as possible. Active perforation is made with the help of hook knife. After the tumor is completely exposed, SMT is removed en bloc with the snare. Dual channel gastroscope can be used for tumors with a broad basement which has the advantage of passing two snares through the accessory channels in to the gastric cavity. Tumor body is grasped with one snare and the other snare is used to en bloc enucleate the tumor along with the attached serosal layer. Titanium clips are used to close the defect in gastric wall. Paracentesis can be performed if there are signs of pneumoperitoneum during the procedure. Feng et al[65] evaluated the efficacy and safety of EFTR in 48 patients with gastric SMTs. Results showed that 43/48 had GIST, no post-EFTR complication such as bleeding or peritonitis, 5 had moderate postoperative abdominal distension because of air filtration (3 had abdominal paracentesis and the other 2 were managed conservatively)[65]. Zhou et al[66] evaluated the efficacy, feasibility and safety of EFTR for gastric SMTs originating from MP layer. Results showed that 16/26 were GISTs, en bloc resection rate was 100% and no major complications[66]. In general, there is a risk of peritoneal seeding with EFTR because it involves creating an active large perforation and hence gentle handling of GIST is necessary to maintain an intact pseudo capsule to prevent peritoneal seeding.

d that 16/26 were GISTs, en bloc resection rate was 100% and no major complications[66]. In general, there is a risk of peritoneal seeding with EFTR because it involves creating an active large perforation and hence gentle handling of GIST is necessary to maintain an intact pseudo capsule to prevent peritoneal seeding. LECS has advantage over LWR especially for gastric SMTs located near esophagogastric junction or pyloric region because SMTs can be located accurately using endoscope and the resection of healthy stomach can be minimized[67]. The best indication for LECS is for gastric GISTs originating from MP layer which are intraluminal[61]. First, Argon plasma coagulation (APC) can be used to mark the periphery of the tumor[67]. A small incision is made on the marked area using standard needle knife after injecting 10% glycerin into submucosal layer. Using the IT knife, three-fourth of the marked area is cut circumferentially. Next, laparoscopic dissection of seromuscular layer is performed by making an artificial perforation and seromuscular dissection is carried out with ultrasonically activated device[67]. The incision is closed with the help of laparoscopic stapling device[67]. Hiki et al[67] analyzed seven patients who underwent LECS for gastric GISTs. Results showed that 6/7 were GISTs, no postoperative complications like bleeding, stenosis or anastomotic leakage, and successful tumor resection was done irrespective of tumor location (esophagogastric junction or pyloric ring). Tsujimoto et al[68] evaluated the feasibility and surgical outcomes of LECS for gastric SMTs. The authors found 16/20 were GISTs, no postoperative complications like bleeding, stenosis or anastomotic leakage, and there was no recurrence of tumor[68].

pective of tumor location (esophagogastric junction or pyloric ring). Tsujimoto et al[68] evaluated the feasibility and surgical outcomes of LECS for gastric SMTs. The authors found 16/20 were GISTs, no postoperative complications like bleeding, stenosis or anastomotic leakage, and there was no recurrence of tumor[68]. NEWS is a new technique developed to prevent peritoneal seeding from large active perforation and minimize resected tissue volume of stomach[69]. Mitsui et al[69] evaluated the efficacy and safety of NEWS in 6 patients with suspected gastric GIST. Results showed that 5/6 were GIST, en bloc resection was achieved in all GISTs, perforation occurred in 2/6 cases (1 case had muscle injury leading to perforation during mucosal cutting by endoscopic knife and the other case had laparoscopic mucosal injury leading to perforation during seromuscular cutting), and no postoperative complications[69]. Future studies with large cohort are needed to validate the safety of NEWS before it is standardized for GISTs treatment.

perforation during mucosal cutting by endoscopic knife and the other case had laparoscopic mucosal injury leading to perforation during seromuscular cutting), and no postoperative complications[69]. Future studies with large cohort are needed to validate the safety of NEWS before it is standardized for GISTs treatment. IMATINIB AS ADJUVANT THERAPY Tumor size, location, mitotic index and tumor rupture are the most important independent prognostic indicators to determine RFS[4]. Multiple stratification schema like National Institutes of Health (NIH) consensus criteria, Armed Forces Institute of Pathology (AFIP) criteria and the modified NIH consensus criteria were developed to predict risk of recurrence[4,70-72]. The most commonly used stratification method is AFIP criteria[73]. AFIP groups 3a and above are considered high risk for recurrence. This corresponds to 5-year recurrence rate of 30% based on nomogram evaluation[73]. DeMatteo et al[74] evaluated the overall survival (OS) in 106 patients who had undergone complete gross tumor removal but were considered high risk for recurrence. It was a phase II Z9000 trial lead by ACOSOG and all patients were treated with imatinib 400 mg per day for 1 year[74]. Results showed that OS for 1, 3 and 5-year was 99%, 97% and 83% respectively after a mean follow up of 7.7 years[74]. RFS rate for 1, 3 and 5-year was 96%, 60% and 40% respectively[74]. In the subsequent trial, patients were randomly assigned to receive imatinib 400 mg per day or placebo for one year[75]. RFS at the end of 1 year for imatinib vs placebo was 98% vs 83% respectively and OS for imatinib vs placebo was 99.2% vs 99.7% respectively[75]. Li et al[76] evaluated RFS in Chinese patients after complete tumor resection of GISTs. All patients in treatment group (56/105) were treated with imatinib 400 mg once a day for 3 years and 49/105 were not treated (control group)[76]. RFS for imatinib vs control group at the end of 1year, 2 year and 3 years were 100% vs 90%, 96% vs 57% and 89% vs 48% respectively[76]. All GISTs with size ≥ 3 cm, small bowel site and high mitotic index were shown to benefit from adjuvant imatinib treatment[50,75]. Joensuu et al[77] evaluated the RFS and OS in KIT-positive GISTs treated with imatinib for 3 year vs 1 year who had undergone complete tumor resection but considered high risk for recurrence.

l GISTs with size ≥ 3 cm, small bowel site and high mitotic index were shown to benefit from adjuvant imatinib treatment[50,75]. Joensuu et al[77] evaluated the RFS and OS in KIT-positive GISTs treated with imatinib for 3 year vs 1 year who had undergone complete tumor resection but considered high risk for recurrence. Results showed that RFS for patients treated with imatinib for 3 year vs 1 year were 65.6% vs 47.9% respectively and OS for 3 year vs 1 year were 92% vs 81.7% respectively[77]. Kang et al[78] evaluated the efficacy of adjuvant imatinib for 2 years in high risk GISTs with KIT exon 11 mutation after complete resection at four South Korean centers. The results showed median RFS was 58.9 mo compared to 22.7 mo in pre-imatinib era[78]. They also concluded that imatinib is effective in GIST recurrence even after completion of adjuvant imatinib therapy[78].

for 2 years in high risk GISTs with KIT exon 11 mutation after complete resection at four South Korean centers. The results showed median RFS was 58.9 mo compared to 22.7 mo in pre-imatinib era[78]. They also concluded that imatinib is effective in GIST recurrence even after completion of adjuvant imatinib therapy[78]. NEOADJUVANT OR PREOPERATIVE IMATINIB THERAPY National comprehensive cancer network (NCCN) guidelines recommend neoadjuvant imatinib therapy to reduce tumor size before surgery and minimize morbidity in patients with primary GISTs considered unresectable or resectable with high risk morbidity[73]. Eisenberg et al[79] evaluated the safety and efficacy of neoadjuvant imatinib (600 mg/d) in patients with KIT positive primary GIST (≥ 5 cm, 32 patients) or with operable metastatic/recurrent GIST (≥ 2 cm, 20 patients). It was a prospective nonrandomized trial and imatinib was continued postoperatively for 2 years[79]. In primary GIST group, preoperative response was partial in 2 patients (7%), stable in 25 (83%) and unknown in 3 (10%); in metastatic or recurrent group, partial in 1 (4.5%), stable in 20 (91%), and progression in 1 (4.5%)[79]. Only 7 (13%) patients did not have any surgery (5 inoperable or unresectable, 1 patient refusal and 1 physician refusal)[79]. The estimated 2-year rate of TTP, PFS, OS in primary vs metastatic/recurrent GIST was 13.9% vs 13.6%, 82.7% vs 77.3% and 93.3% vs 90.9% respectively[79].

(91%), and progression in 1 (4.5%)[79]. Only 7 (13%) patients did not have any surgery (5 inoperable or unresectable, 1 patient refusal and 1 physician refusal)[79]. The estimated 2-year rate of TTP, PFS, OS in primary vs metastatic/recurrent GIST was 13.9% vs 13.6%, 82.7% vs 77.3% and 93.3% vs 90.9% respectively[79]. Fiore et al[80] prospectively evaluated the PFS in locally advanced or unresectable primary GISTs treated with preoperative imatinib. All patients who were considered high risk or needed extensive surgery (3 considered unresectable underwent complete resection, 7 who were initially considered to undergo extensive surgery were conservatively operated, 4 who were considered high perioperative risk underwent safe surgery) improved after preoperative imatinib therapy. PFS after 3 years was 77% from the time of initial imatinib treatment[80].

sectable underwent complete resection, 7 who were initially considered to undergo extensive surgery were conservatively operated, 4 who were considered high perioperative risk underwent safe surgery) improved after preoperative imatinib therapy. PFS after 3 years was 77% from the time of initial imatinib treatment[80]. IMATINIB IN METASTATIC GIST The outcome of advanced GISTs treated with imatinib is not clear. Demetri et al[81] evaluated the efficacy of imatinib on antitumor response, safety and tolerability in advanced GISTs. Results showed that 79 patients (53.7%) had partial response, 41 patients (27.9%) had stable disease and in 7 patients (4.8%) response could not be evaluated[81]. Adverse effects related to imatinib therapy were diarrhea, edema (periorbital and leg), fatigue and gastrointestinal bleeding[81]. Overall, the therapy was well tolerated. Blanke et al[82] conducted a multicenter randomized phase II trial and they evaluated the efficacy and long-term safety of imatinib (group A 400 vs group B 600 mg) in advanced GISTs positive for CD117 antigen. In group A (400 mg, 73 patients), the authors observed GISTs with complete response 0 (0%), partial response 50 (68.5%), stable 10 (13.7%), progressive 11 (15.1%) and unknown 2 (2.7%)[82]. In group B (600 mg, 74 patients), the authors reported GISTS with complete response 2 (2.7%), partial 48 (64.9%), stable 13 (17.6%), progressive 6 (8.1%) and unknown 5 (6.8%)[82]. Overall, imatinib was well tolerated[82]. In the subsequent phase III trial, Blanke et al[83] evaluated PFS or OS with standard imatinib dose (400 mg) vs higher dose (400 mg twice daily) in patients with incurable GISTs. After a median follow up of 4.5 years, median PFS for standard vs high dose imatinib was 18 mo vs 20 mo, median OS for standard vs high dose imatinib was 55 mo vs 51 mo respectively[83]. Treatment response in standard vs high dose imatinib were divided in to complete response (5% vs 3%), partial (40% vs 42%), stable (25% vs 22%), progressive disease (12% vs 10%) and inadequate assessment (10% vs 15%) respectively[83]. This study concluded that 400 mg twice daily imatinib was more toxic than 400 mg dose in treatment of incurable GISTs[83]. Debiec-Rychter et al[84] evaluated the efficacy of standard dose imatinib (400 mg) vs higher dose (400 mg two times daily) in advanced GIST based on mutational status (KIT or PDGFRA). There was a 61% relative risk reduction of PFS in GISTs expressing exon 9 mutation treated with high dose imatinib[84].

rable GISTs[83]. Debiec-Rychter et al[84] evaluated the efficacy of standard dose imatinib (400 mg) vs higher dose (400 mg two times daily) in advanced GIST based on mutational status (KIT or PDGFRA). There was a 61% relative risk reduction of PFS in GISTs expressing exon 9 mutation treated with high dose imatinib[84]. Overall, this study concluded that tumor genotype determines PFS and OS in advanced GISTs and also GISTs with KIT exon 9 benefited from 400 mg two times daily imatinib[84]. Heinrich et al[85] showed that presence of KIT exon-11 mutation (71.7%) had better treatment outcome with imatinib when compared to KIT exon-9 (44.4%) and wild-type mutation (44.6%) in advanced GISTs. The authors also showed that there was an improved response rate (complete/partial response) in patients with KIT exon-9 mutation treated with imatinib 800 mg vs 400 mg (67% vs 17%, P = 0.02)[85]. GIST meta-analysis group (MetaGIST) evaluated PFS and OS with imatinib (400 mg vs 800 mg) in advanced GISTs[86]. The results showed that there was a small but significant PFS (P = 0.04) advantage in high dose (400 mg twice daily) group and no difference in OS between both (400 and 800 mg) groups[86].

7%, P = 0.02)[85]. GIST meta-analysis group (MetaGIST) evaluated PFS and OS with imatinib (400 mg vs 800 mg) in advanced GISTs[86]. The results showed that there was a small but significant PFS (P = 0.04) advantage in high dose (400 mg twice daily) group and no difference in OS between both (400 and 800 mg) groups[86]. SUNITINIB AFTER TREATMENT FAILURE WITH IMATINIB IN ADVANCED GIST Demetri et al[87] evaluated patients treated with sunitinib in advanced GISTS who were intolerant or resistant to previous imatinib treatment. They concluded that median TTP with sunitinib vs placebo was 27.3 wk vs 6.4 wk respectively[87]. Overall, sunitinib was well tolerated and side effects like nausea, fatigue, skin discoloration and diarrhea were common[87]. REGORAFENIB AFTER TREATMENT FAILURE WITH IMATINIB AND SUNITINIB IN ADVANCED GIST Demetri et al[88] evaluated the efficacy and safety of regorafenib after failure of treatment with imatinib and sunitinib. Results showed that the median PFS in regorafenib vs placebo group were 4.8 mo vs 0.9 mo respectively[88]. There was no statistical significance in terms of OS between regorafenib and placebo group[88]. Drug related adverse events occurred in 130/132 (98.5%) in regorafenib group and 45/66 (68.2%) in placebo group[88]. The most common adverse effects of regorafenib include hypertension (31/132, 23.5%), hand foot skin reaction (26/132, 19.7%) and diarrhea (7/132, 5.3%)[88]. Overall, this study concluded that regorafenib significantly improved PFS in patients with advanced GISTs who failed treatment with imatinib and sunitinib[88].

8]. The most common adverse effects of regorafenib include hypertension (31/132, 23.5%), hand foot skin reaction (26/132, 19.7%) and diarrhea (7/132, 5.3%)[88]. Overall, this study concluded that regorafenib significantly improved PFS in patients with advanced GISTs who failed treatment with imatinib and sunitinib[88]. FOLLOW-UP AFTER TREATMENT The goal of follow-up after surgery is early detection and treatment of relapse. CT abdomen and pelvis is used for follow-up. Metastasis of GISTs outside the abdomen is infrequent. MRI or PET-CT can be used as an alternative for follow-up. Annual CT abdomen and pelvis for 5 years is recommended for low risk GISTs after surgery[89]. During adjuvant treatment with imatinib for high risk GISTs, CT abdomen and pelvis is recommended every 6 mo[89]. After adjuvant therapy is stopped, CT is repeated every 3-4 mo for first 2 years and there after every 6-12 mo for 10 years[89].

n and pelvis for 5 years is recommended for low risk GISTs after surgery[89]. During adjuvant treatment with imatinib for high risk GISTs, CT abdomen and pelvis is recommended every 6 mo[89]. After adjuvant therapy is stopped, CT is repeated every 3-4 mo for first 2 years and there after every 6-12 mo for 10 years[89]. CONCLUSION With increasing availability of EUS and improved knowledge of the pathogenesis of GISTs, accurate identification and differentiation of GISTs from other submucosal tumors are achieved. Although surgery is preferred, newer endoscopic techniques can be attempted by experienced endoscopists with the assistance of surgeons in suitable candidates. Neoadjuvant imatinib therapy is recommended for primary GISTs considered unresectable or resectable with high morbidity to reduce the tumor size before surgery and minimize morbidity. Adjuvant therapy with imatinib in intermediate and high risk GISTs improves OS and RFS. Sunitinib and regorafenib can be used in advanced GISTs after treatment failure with imatinib. Multidisciplinary approach involving endoscopist, pathologist, radiologist, medical oncologist and surgeon is required for optimal management of GIST. Conflict-of-interest statement: None of the authors have any potential conflicts (financial, professional, or personal) that are relevant to the manuscript. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0

Conflict-of-interest statement: None of the authors have any potential conflicts (financial, professional, or personal) that are relevant to the manuscript. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: October 15, 2016 First decision: December 15, 2016 Article in press: February 20, 2017 P- Reviewer: Deng MM, Gu MJ, Lee SW S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: This paper is a review that outlines the most recent updates on the immunotherapy treatment of sarcomas. After a brief review of the concept of immunotherapies and the different treatment modalities, we discuss the available data, the limitations and future perspectives of each treatment option. INTRODUCTION Sarcomas are malignant tumors that derive from embryonic mesodermic tissues including fat, muscles, bones, nerves and blood vessels[1]. Epidemiologic studies report its predominance in the pediatric populations and its rare occurrence in adults[2]. Sarcomas are characterized by a wide diversity of subtypes with various cytogenetic profiles conferring treatment resistances. These findings combined with an advanced stage at diagnosis substantially increase the years of life lost[3]. The standard treatment modalities combining chemotherapy, radiotherapy, and surgery have failed to improve overall survival (OS)[4]. Despite the major breakthroughs in the treatment armamentarium, the recent data reports a relative 5-year survival rate limited to 66% for bone and soft tissue sarcomas, 53.9% for osteosarcomas, 75.2% for chondrosarcomas, and 50.6% for Ewing’s sarcomas[5]. Interestingly, Coley described in 1891 a complete regression of sarcomas secondary to severe episodes of erysipelas but failed to regenerate these results in other patients[6]. The Food and Drug Administration thereafter banned the use of toxin therapy without a new drug-approval process. Fortunately, Coley’s paper has encouraged scientists to analyze the role of the immune system in carcinogenesis[7].

severe episodes of erysipelas but failed to regenerate these results in other patients[6]. The Food and Drug Administration thereafter banned the use of toxin therapy without a new drug-approval process. Fortunately, Coley’s paper has encouraged scientists to analyze the role of the immune system in carcinogenesis[7]. After more than a century since Coley’s research efforts that marked the history of immunotherapy, we present a review on this elegant treatment modality in the management of sarcomas including adoptive cell therapies (ACT), monoclonal antibodies, vaccines, and immune checkpoint inhibitors (ICI).

severe episodes of erysipelas but failed to regenerate these results in other patients[6]. The Food and Drug Administration thereafter banned the use of toxin therapy without a new drug-approval process. Fortunately, Coley’s paper has encouraged scientists to analyze the role of the immune system in carcinogenesis[7]. After more than a century since Coley’s research efforts that marked the history of immunotherapy, we present a review on this elegant treatment modality in the management of sarcomas including adoptive cell therapies (ACT), monoclonal antibodies, vaccines, and immune checkpoint inhibitors (ICI). APPROVED THERAPIES IN SARCOMAS FROM CHEMOTHERAPY TO TARGETED THERAPIES Specialized centers in the management of sarcomas have demonstrated a better OS and low recurrence rate[8]. Yet, all patients are managed uniformly according to their prognosis dictated by the stage of the disease, which is determined by the grade, depth and size of the tumor[9]. For patients with localized disease, a complete resection with wide 2-3 cm margins followed by adjuvant radiation therapy is the mainstay treatment for a curative approach. However, survival is not only determined by local control since most patients die from systemic disease. The choice of the chemotherapy regimen depends on the tumor chemosensitivity which varies with the tumor subtype and grade, the patient’s performance status, and the timing of metastatic disease[10]. Unfortunately, the benefits of adjuvant chemotherapy are limited to rhabdomyosarcomas, osteosacromas and Ewing’s sarcomas. Moreover, Trabectidine is showing promising results encountered in the adjuvant and neoadjuvant settings of patients with myxoid liposarcomas[11]. The role of adjuvant and neoadjuvant chemotherapy in the management of soft tissue sarcomas is yet to be clearly established. The actual recommendations by NCCN and ESMO are to address this issue on a case by case basis according to the patient’s performance status, comorbid factors, disease location, tumor size, and histologic subtype. In case of advanced and recurrent sarcomas, induction regimens include Cyclophosphamide and Ifosphamide, Vincristine, Doxorubicin, Dactinomycin, and Etoposide[12]. For patients with unresectable or metastatic disease, the management plan is limited to a palliative approach with Trabectedin or Ifosfamide and Doxorubicin based chemotherapy[13,14].

urrent sarcomas, induction regimens include Cyclophosphamide and Ifosphamide, Vincristine, Doxorubicin, Dactinomycin, and Etoposide[12]. For patients with unresectable or metastatic disease, the management plan is limited to a palliative approach with Trabectedin or Ifosfamide and Doxorubicin based chemotherapy[13,14]. The rationale of using targeted therapies in sarcomas goes back to 1984 when sarcomagenesis was correlated to recurrent translocations[15]. Genetic profiling thus defined two groups of sarcomas. The first group is characterized by a simple karyotype associated with specific tumor genetic alterations that include chromosomal translocations, oncogenetic mutations, and recurrent gene amplifications. The second group is characterized by a complex karyotype associated with nonspecific and nonrecurring genetic alterations[16]. Subsequent to these advances, Pazopanib, a multitargeted tyrosine kinase inhibitor against VEGFR1-3, PDFGRA-B, and KIT was approved for pretreated metastatic nonlipomatous sarcomas based on the phase III PALETTE study[17]. Clinical and preclinical mechanistic studies are being conducted to validate a possible therapeutic role of the various targeted therapies available. Among these novel targeted therapies, we report the trials of Cediranib and Sunitinib in alveolar soft part sarcoma, Tivantinib and Cabozantinib in clear cell sarcoma, Imatinib in dermatofibrosarcoma protuberans, Cabozantinib in endometrial stromal tumors, and Everolimus in perivascular epitheloid cell tumor[18].

able. Among these novel targeted therapies, we report the trials of Cediranib and Sunitinib in alveolar soft part sarcoma, Tivantinib and Cabozantinib in clear cell sarcoma, Imatinib in dermatofibrosarcoma protuberans, Cabozantinib in endometrial stromal tumors, and Everolimus in perivascular epitheloid cell tumor[18]. ADVANCES IN IMMUNO-ONCOLOGY In fact, the previous cancer treatment approaches addressed distinctive and complementary hallmarks of carcinogenesis that included sustained proliferative signaling, evasion of growth suppressors, resistance of cell death, enabling of replicative immortality, induction of angiogenesis and activation of invasions and metastasis[19]. The well-known conventional cytotoxic drugs and targeted therapies have reached a plateau in effect that required a re-assessment of the six hallmarks of carcinogenesis. Recent conceptual progress has added two new hallmarks, namely reprogramming of energy metabolism and signaling interactions of the tumor microenvironment[20].

known conventional cytotoxic drugs and targeted therapies have reached a plateau in effect that required a re-assessment of the six hallmarks of carcinogenesis. Recent conceptual progress has added two new hallmarks, namely reprogramming of energy metabolism and signaling interactions of the tumor microenvironment[20]. The later resides in the concept of the cancer-immunity cycle and is actually a turning point in the history of cancer therapy[21]. This cycle is the result of a counterbalance between immune-stimulatory and inhibitory factors. It occurs physiologically and starts with the release of cancer cell antigens and ends with the apoptosis of cancer cells via the activated effectors of the immune system[22]. Subsequently, cancer immunoediting may proceed with any of the three following phases[23]. The elimination phase describes an activation of the innate and adaptive immune effectors in response to cytokine secretion. The equilibrium phase occurs in the setting of a balance between tumor immune destruction and proliferation. The immunologic phase takes place when the tumor cells are capable of evading the immune system[23].

n phase describes an activation of the innate and adaptive immune effectors in response to cytokine secretion. The equilibrium phase occurs in the setting of a balance between tumor immune destruction and proliferation. The immunologic phase takes place when the tumor cells are capable of evading the immune system[23]. Recent advances recommend addressing only one step of the immune cycle to avoid potential unwanted activation of autoimmunity mechanism and normal cells damage. Therefore, immunotherapy aims at initiating or maintaining the cancer-immunity cycle by acting on its rate limiting step. Consequently, ICI often address the immunostar function of the tumor microenvironment[24]. The PD-1/PD-L1 axis is a potential therapeutic target in view of the confirmed expression of PD-L1 in various sarcomas[25]. Inhibition of this axis enables the immune system to quickly adapt to cancer resistances thus allowing durable responses with ICI[26]. IMMUNOTHERAPEUTIC MODALITIES EVALUATED IN SARCOMAS Sarcomas mainly occur either secondary to the activation of oncogenes via translocations and inversions, or secondary to the natural expression of germ cell peptides[27,28]. The issuing peptides generate an immune cascade directed against the aberrant cells[29]. Consequently, multiple rationales to immunotherapy including ACT, therapeutic vaccines, and ICI have been assessed in the treatment of sarcomas (Table 1). Table 1 Summary of the phase I/II trials of immunotherapies in sarcoma

IMMUNOTHERAPEUTIC MODALITIES EVALUATED IN SARCOMAS Sarcomas mainly occur either secondary to the activation of oncogenes via translocations and inversions, or secondary to the natural expression of germ cell peptides[27,28]. The issuing peptides generate an immune cascade directed against the aberrant cells[29]. Consequently, multiple rationales to immunotherapy including ACT, therapeutic vaccines, and ICI have been assessed in the treatment of sarcomas (Table 1). Table 1 Summary of the phase I/II trials of immunotherapies in sarcoma Treatment modality Ref. Agent Phase/Patients Indication RR Survival Adoptive cell therapy Robbins et al[31], 2011 Adoptively transferred autologous T cells transduced with a T-cell receptor directed against NY-ESO-1 I/6 Metastatic synovial cell sarcoma expressing NY-ESO-1 RR: 4/6 N/A Vaccines Mahvi et al[34], 2002 GM-CSF treated tumor cells I/16 Melanoma and sarcomas RR: 1/16 N/A Dillman et al[35], 2004 Autologous tumor cell line-derived vaccines I, II/23 Recurrent or metastatic sarcoma No objective response assessed 10 patients lived more than 1 year Kawaguchi et al[36], 2005 Vaccination By SYT-SSX junction peptide I/6 Disseminated synovial sarcoma RR: 0/6 N/A Kawaguchi et al[38], 2012 SYT-SSX breakpoint peptide vaccines I, II/21 Metastatic synovial sarcoma RR: 1/21 SD: 6/21 N/A Takahashi et al[39], 2013 Personalized peptide vaccination II/20 Refractory bone and soft tissue sarcoma SD in all patients Median OS: 9.6 mo Finkelstein et al[40], 2012 Combination of external beam radiotherapy with intratumoral injection of dendritic cells I, II/17 Neoadjuvant treatment in high-risk soft tissue sarcoma RR: 9/17 One-year PFS: 70.6% Ghisoli et al[41], 2015 FANG autologous immunotherapy I/12 Advanced and metastatic Ewing's sarcoma RR: 1/12 One-year OS: 75% Checkpoint inhibitors Makki et al[44], 2013 Ipilimumab II/6 Advanced synovial sarcoma RR: 0/6 (closed prematurely) Median OS: 8.75 mo GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor; N/A: Not available; OS: Overall survival; PFS: Progression free survival; RR: Response rate.

s sarcoma RR: 1/12 One-year OS: 75% Checkpoint inhibitors Makki et al[44], 2013 Ipilimumab II/6 Advanced synovial sarcoma RR: 0/6 (closed prematurely) Median OS: 8.75 mo GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor; N/A: Not available; OS: Overall survival; PFS: Progression free survival; RR: Response rate. Adoptive cell therapy in sarcomas Adoptive cell therapy is a new therapeutic strategy based on the modulation, manipulation and selection of autologous T-cells in vitro to overcome the tolerance of the immune system to the tumor cells. Those T-cells may be harvested from tumor infiltrating lymphocytes (TIL) and re-transfused into the same patient after ensuring their expansion. Lymphocyte T-cells may also be harvested from peripheral blood, and those that recognize tumor antigens are selectively expanded. Alternatively, lymphocyte T-cells may be genetically engineered either by modifying a T-cell receptor for cancer antigen (transgenic TCR) or by adding a chimeric antigen receptor (CAR) that recognizes a specific cancer antigen[30,31]. Apart from T-cells, NK ACT has also been proven efficacious with several advantages over the classical T-cell ACT in the absence of MHC/HLA restriction, namely their NKG2D-dependent cytotoxicity against autologous tumor cells[32,33].

ng a chimeric antigen receptor (CAR) that recognizes a specific cancer antigen[30,31]. Apart from T-cells, NK ACT has also been proven efficacious with several advantages over the classical T-cell ACT in the absence of MHC/HLA restriction, namely their NKG2D-dependent cytotoxicity against autologous tumor cells[32,33]. To our knowledge, the use of TIL has never been reported in the treatment of sarcomas whilst the use of NK ACT has been limited to case reports[33]. On the other hand, tumor antigens such as GD2 (93% of sarcomas) and NY-ESO-1 (80% to 100% of different subtype of sarcomas) were found to represent interesting targets for adoptive cells therapies. Moreover, other cancer testis antigens such as LAGE, MAGE-A3 and PRAME were frequently expressed in sarcomas and would be potential immunotherapeutic targets. In this setting, a phase I study evaluated the ability of adoptively transferred autologous T-cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic synovial cell sarcoma expressing NY-ESO-1. The results showed an objective clinical response in 4 out of 6 patients[31]. Two ongoing trials are evaluating genetically engineered NY-ESO-1 T-cells for children and adults in metastatic synovial sarcoma (NCT01343043). Another phase I trial is testing the role of CAR T-cell therapy targeting the GD2 protein in children and young adults with sarcomas and rhabdomyosarcomas (NCT00743496).

To our knowledge, the use of TIL has never been reported in the treatment of sarcomas whilst the use of NK ACT has been limited to case reports[33]. On the other hand, tumor antigens such as GD2 (93% of sarcomas) and NY-ESO-1 (80% to 100% of different subtype of sarcomas) were found to represent interesting targets for adoptive cells therapies. Moreover, other cancer testis antigens such as LAGE, MAGE-A3 and PRAME were frequently expressed in sarcomas and would be potential immunotherapeutic targets. In this setting, a phase I study evaluated the ability of adoptively transferred autologous T-cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic synovial cell sarcoma expressing NY-ESO-1. The results showed an objective clinical response in 4 out of 6 patients[31]. Two ongoing trials are evaluating genetically engineered NY-ESO-1 T-cells for children and adults in metastatic synovial sarcoma (NCT01343043). Another phase I trial is testing the role of CAR T-cell therapy targeting the GD2 protein in children and young adults with sarcomas and rhabdomyosarcomas (NCT00743496). Therapeutic vaccines in sarcomas The therapeutic effects of cancer vaccines rely on the activation of dendritic cells upon the presence of an immunogenic predetermined antigen. However, most of the initial studies of vaccines in sarcomas did not determine specific antigens and used inefficaciously the entirety of the tumor cells[34,35]. Later studies used SYT-SSX, a fusion derived peptide present in 90% of synovial sarcoma, and also failed to demonstrate an objective response[36-38]. Takahashi et al[39] personalized the peptide vaccination patients with refractory sarcoma and administered multiple tumor antigens chosen according to preexisting peptide-specific IgG titers. The median OS was 9.6 mo with disease stabilization occurring in 30% of patients but no objective responses were seen. Another vaccination modality used in situ vaccination through combining preoperative gamma radiation (50 Gy) with intratumoral dendritic cells injection. The studied population was limited to high risk, localized, and resected extremity soft tissue sarcoma and resulted in 71% progression free survival at one year[40].

Another vaccination modality used in situ vaccination through combining preoperative gamma radiation (50 Gy) with intratumoral dendritic cells injection. The studied population was limited to high risk, localized, and resected extremity soft tissue sarcoma and resulted in 71% progression free survival at one year[40]. Major efforts in this field are being conducted namely in children with Ewing sarcomas. Recent data demonstrated a 75% OS at one year with FANG immunotherapy in adolescent patients with Ewing’s sarcoma. The treatment was well tolerated with a favorable OS[41]. A seemingly interesting phase I trial designed for the treatment of pediatric patients with relapsed high-risk Ewing sarcoma, osteogenic sarcoma, rhabdomyosarcoma, synovial sarcoma, and neuroblastoma is using a combination of Decitabine demethylating agent and a cancer vaccine composed of dendritic cells pulsed with overlapping peptides of NY-ESO-1, MAGE-A1, and MAGE-A3 (NCT0 1241162). Another dendritic cell vaccine is also being assessed in combination with Gemcitabine in a phase I trial for adults and children with soft tissue and bone sarcomas (NCT01803152).

ing agent and a cancer vaccine composed of dendritic cells pulsed with overlapping peptides of NY-ESO-1, MAGE-A1, and MAGE-A3 (NCT0 1241162). Another dendritic cell vaccine is also being assessed in combination with Gemcitabine in a phase I trial for adults and children with soft tissue and bone sarcomas (NCT01803152). Immune checkpoint inhibitors in sarcomas The concept of ICI relies on deactivating the suppressed activity of the immune system. ICI remove the brakes (PD-1 and CTLA4) thus enhancing the immune function of already sensitized T-cells. Effectively, PD-1 and CTLA4 inhibitors are showing interesting results with acceptable response rates in different cancers, including those considered for a long time as non-immunogenic[42]. Unlike CTLA4 inhibitors, the response to PD1 and PDL-1 inhibitors has been correlated with the expression of PD-1 and PDL-1 on tumor cells and to the mutational load of the tumors[42]. Moreover, PD-1 and PDL-1 expression seems to vary between sarcoma subtypes, a finding that may direct immunotherapy management in patients with sarcomas[43].

s, the response to PD1 and PDL-1 inhibitors has been correlated with the expression of PD-1 and PDL-1 on tumor cells and to the mutational load of the tumors[42]. Moreover, PD-1 and PDL-1 expression seems to vary between sarcoma subtypes, a finding that may direct immunotherapy management in patients with sarcomas[43]. Unfortunately, the efficacy of ICI in sarcomas has been evaluated in only one study so far. It is a phase II study that administered Ipilimumab (3 mg/kg intravenously every 3 wk for 3 cycles), a CTLA-4 inhibitor, to six patients with synovial sarcoma. The median OS was 8.75 mo ranging between 0.8 and 19.7 mo. The study was closed prematurely when none of the patients had an objective tumor response. All patients expressed NY-ESO-1 but its titers did not change after treatment administration[44]. PD-1 and PDL-1 inhibitors present a different mechanism of action compared to anti-CTLA4 agents and consequently may present better response rates[43]. Many ongoing phase I trials are assessing the role of anti-PD1 agents in sarcomas as single agent or in combination with Ipilimumab and Dasatinib (NCT0 1643278).

n[44]. PD-1 and PDL-1 inhibitors present a different mechanism of action compared to anti-CTLA4 agents and consequently may present better response rates[43]. Many ongoing phase I trials are assessing the role of anti-PD1 agents in sarcomas as single agent or in combination with Ipilimumab and Dasatinib (NCT0 1643278). PERSPECTIVE The proof of the immunotherapy concept in sarcomas has been undoubtedly validated with the benefits encountered upon the use of liposomal muramyl-tripeptide-phosphatidyl-ethanolamine, an immunoactivator agent derived from BCG. However, its role remains controversial in view of the discordant results between the preliminary data and final results in both the adjuvant and metastatic setting. Even though the actual trend is moving towards immunotherapy as an essential tool in the treatment of cancer, the recent ASCO 2016 meeting was unfortunately disappointing in this regard. Five studies have been presented, of which one trial of chemotherapy (Busulphan and Melphalan), three trials of tyrosine kinase inhibitors, monotherapy (Anlotinib and Regorafenib) or in combination with chemotherapy (Gemcitabine plus Pazopanib), and one study reporting the evident detrimental impact of disease progression and altered quality of life on the long-term care and survival of patients with sarcomas. The ongoing trials including the promising results of immunotherapies are awaited. The available results reported a failure of Pembrolizumab in multiple soft tissue sarcomas (NCT02301039) and Nivolumab in metastatic uterine leiomyosarcoma (NCT0 2428192) despite the promising findings encountered with Nivolumab in retrospective experiences[45]. In fact, the biological preclinical rationale is not fully elucidated in view of the absence of any correlation between PD-L1 expression and OS[46]. Thus, the actual state of knowledge does not predict the patient profile that might benefit from immunotherapy.

ncountered with Nivolumab in retrospective experiences[45]. In fact, the biological preclinical rationale is not fully elucidated in view of the absence of any correlation between PD-L1 expression and OS[46]. Thus, the actual state of knowledge does not predict the patient profile that might benefit from immunotherapy. CONCLUSION The cornerstone treatment for sarcomas consists of complete surgical resection, chemotherapy, and radiotherapy. Unfortunately, these treatment options fall short from achieving an optimal clinical outcome. Immunotherapy is therefore expected to further improve the survival of patients with sarcomas. Until recently, the field of immunotherapy has not yet matured enough to present robust effects. The better understanding of onco-immunotherapy principles is essential to adjust the design of clinical trials and the selection of inclusion criteria. The published data shows that ACT is yet to be more elucidated and evaluated, vaccine therapy requires tailoring and personalization, and ICI, preferably PD-1 and PDL-1 inhibitors, necessitate better patient selection. Such results would allow more understanding of the antitumor immunity mechanisms and improvement of the treatment arsenal against sarcomas.

et to be more elucidated and evaluated, vaccine therapy requires tailoring and personalization, and ICI, preferably PD-1 and PDL-1 inhibitors, necessitate better patient selection. Such results would allow more understanding of the antitumor immunity mechanisms and improvement of the treatment arsenal against sarcomas. Conflict-of-interest statement: The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Lebanon Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: September 23, 2016 First decision: October 20, 2016 Article in press: January 18, 2017 P- Reviewer: Leithner A, Mehdi I, Rapidis AD S- Editor: Kong JX L- Editor: A E- Editor: Lu YJ

Core tip: The purpose of this study was to stratify the malignancy risks in thyroid nodules in a tertiary care referral center using the Bethesda system. The study found that there were 124 total cases of malignancy on resection, giving an overall surgical yield malignancy of 33.6%. Majority of the thyroid cancer nodules in Bethesda VI category followed by Bethesda IV. Almost 40% of the cancer nodules in 31-45 age group in both sex. Papillary Thyroid Carcinoma was the most common form of thyroid cancer among the study population followed by follicular thyroid carcinoma, medullary carcinoma and anaplastic carcinoma. INTRODUCTION According to epidemiological and clinical studies thyroid nodules are commonly encountered in clinical exams, palpable in 5% of the population on thyroid examination and detectable in nearly 60% of those subjected to thyroid ultrasound. While the majority of the nodules are benign (non-cancerous), they are normally the first indicators of thyroid cancer; therefore, further investigations are required to identify the cancerous nodule[1,2].

5% of the population on thyroid examination and detectable in nearly 60% of those subjected to thyroid ultrasound. While the majority of the nodules are benign (non-cancerous), they are normally the first indicators of thyroid cancer; therefore, further investigations are required to identify the cancerous nodule[1,2]. The last decades have revealed a constant and remarkable rise in the occurrence of thyroid cancer across the world, including Saudi Arabia[3-5]. The Saudi Cancer Registry (SCR) report has registered 890 thyroid cancer cases, in nearly 8.1% of all the newly diagnosed cases in 2012. However, studies revealed variations in the incidence of thyroid cancer globally. Thyroid cancer is the 5th most common cancer among females in the United States, whereas in Saudi Arabia it is the 2nd commonest identified cancer in females, and 8th among males[6]. However, compared with the developed countries, research regarding the malignancy risks in thyroid nodules is still insufficient due to lack of appropriate studies being conducted in these specified areas.

ited States, whereas in Saudi Arabia it is the 2nd commonest identified cancer in females, and 8th among males[6]. However, compared with the developed countries, research regarding the malignancy risks in thyroid nodules is still insufficient due to lack of appropriate studies being conducted in these specified areas. One of the most widely used diagnostic tools is fine-needle aspiration (FNA) cytology with ultrasound imaging to determine the necessity for the surgical excision of a thyroid nodule. Today, molecular genetic biomarker analyses are employed to increase the diagnostic accuracy of the FNA biopsies, and can at times drastically change clinical decision procedures as they become more commonly available and better assessed. FNA cytology (FNAC) continues to remain the initial investigation mode for malignancy in patients with thyroid nodules and the selection of patients for thyroid surgery[7]. This minimally invasive and useful method is highly effective in identifying a large percentage of thyroid nodules as benign and eliminating unnecessary surgery for patients with benign disease[8]. However, because a standardized reporting system is still unavailable, pathologists have been employing varying terminologies and diagnostic criteria, thus causing misunderstanding among the referring clinicians while interpreting cytopathology reports, resulting in non-definitive clinical management[9-11]. In 2007, the National Cancer Institute (NCI) established guidelines employing a standardized nomenclature to interpret thyroid FNAs called the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) which is now accepted as the proposed diagnostic categories for thyroid cancer[12]. This study attempts to stratify the malignancy risks in thyroid nodules in a tertiary care referral center in Saudi Arabia utilizing the Bethesda system.

hyroid FNAs called the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) which is now accepted as the proposed diagnostic categories for thyroid cancer[12]. This study attempts to stratify the malignancy risks in thyroid nodules in a tertiary care referral center in Saudi Arabia utilizing the Bethesda system. MATERIALS AND METHODS Study design and setting From January, 2012 to December, 2014 (36 mo), a retrospective analysis was performed among 1188 patients (15-90 years old) who had 1433 thyroid nodules and FNA at Prince Sultan Military Medical City (PSMMC), a 1200 bedded tertiary care center, Riyadh, Saudi Arabia. The PSMMC caters to the patients referred from different regions of Saudi Arabia and considered a worthy representative of Saudi Arabia in general. The study protocol was approved by the Research and Ethics Committee of PSMMC, Riyadh, Saudi Arabia. Data collection All thyroid cytopathological slides and ultra sound reports were reviewed and classified according to the BSRTC system. Age, gender, cytological features and histological types of the study population were collected from patients’ medical chart and cyto-pathology reports.

MATERIALS AND METHODS Study design and setting From January, 2012 to December, 2014 (36 mo), a retrospective analysis was performed among 1188 patients (15-90 years old) who had 1433 thyroid nodules and FNA at Prince Sultan Military Medical City (PSMMC), a 1200 bedded tertiary care center, Riyadh, Saudi Arabia. The PSMMC caters to the patients referred from different regions of Saudi Arabia and considered a worthy representative of Saudi Arabia in general. The study protocol was approved by the Research and Ethics Committee of PSMMC, Riyadh, Saudi Arabia. Data collection All thyroid cytopathological slides and ultra sound reports were reviewed and classified according to the BSRTC system. Age, gender, cytological features and histological types of the study population were collected from patients’ medical chart and cyto-pathology reports. Bethesda system Currently, the Bethesda system of reporting thyroid cytology (TBSRTC) is used for reporting FNAC specimens of thyroid. According to Cibas[13], this system was innovated in 2007 and consists of six categories: (1) Unsatisfactory (UNS) or nondiagnostic (ND); (2) Benign and nonneoplastic; (3) Atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS); (4) Follicular neoplasm or suspicious for follicular neoplasm (FNS/SFN); (5) Suspicious for, but not diagnostic of, malignancy; and (6) Malignant (Table 1). Table 1 The Bethesda system

Bethesda system Currently, the Bethesda system of reporting thyroid cytology (TBSRTC) is used for reporting FNAC specimens of thyroid. According to Cibas[13], this system was innovated in 2007 and consists of six categories: (1) Unsatisfactory (UNS) or nondiagnostic (ND); (2) Benign and nonneoplastic; (3) Atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS); (4) Follicular neoplasm or suspicious for follicular neoplasm (FNS/SFN); (5) Suspicious for, but not diagnostic of, malignancy; and (6) Malignant (Table 1). Table 1 The Bethesda system Diagnostic category Cytological diagnosis Risk of malignancy, % Usual management I Nondiagnostic or unsatisfactory 1-4 Repeat FNA with ultrasound guidance II Benign 0-3 Clinical follow-up III AUS/FLUS 5-15 Repeat FNA IV FNS/SFN 15-30 Surgical lobectomy V Suspicious for malignancy 60-75 Near-total thyroidectomy or surgical VI Malignant 97-99 Near-total thyroidectomy FNA: Fine-needle aspiration; AUS/FLUS: Atypia of undetermined significance or follicular lesion of undetermined significance; FNS/SFN: Follicular neoplasm or suspicious for follicular neoplasm. All FNAs were performed by one of five interventional radiologists under ultrasound (US) guidance, performing 3-5 passes by using 25 gauge needles. On-site FNAs stained with the Diff-Quik stain and adequacy assessment was performed for all samples. All slides interpreted by among of five accredited cyto-pathologists.

Diagnostic category Cytological diagnosis Risk of malignancy, % Usual management I Nondiagnostic or unsatisfactory 1-4 Repeat FNA with ultrasound guidance II Benign 0-3 Clinical follow-up III AUS/FLUS 5-15 Repeat FNA IV FNS/SFN 15-30 Surgical lobectomy V Suspicious for malignancy 60-75 Near-total thyroidectomy or surgical VI Malignant 97-99 Near-total thyroidectomy FNA: Fine-needle aspiration; AUS/FLUS: Atypia of undetermined significance or follicular lesion of undetermined significance; FNS/SFN: Follicular neoplasm or suspicious for follicular neoplasm. All FNAs were performed by one of five interventional radiologists under ultrasound (US) guidance, performing 3-5 passes by using 25 gauge needles. On-site FNAs stained with the Diff-Quik stain and adequacy assessment was performed for all samples. All slides interpreted by among of five accredited cyto-pathologists. Histological diagnoses The histological diagnoses of thyroid nodules were classified into two types: Benign and nonneoplastic and malignant. For papillary thyroid carcinoma (PTC), subtype variants were documented such as the follicular variant, classical variant, conventional variant and tall cell variant. Also were follicular thyroid carcinoma (FTC) subdivided to minimally invasive follicular thyroid carcinoma (MIFTC) and Widely Invasive follicular thyroid carcinoma (WIFTC).

id carcinoma (PTC), subtype variants were documented such as the follicular variant, classical variant, conventional variant and tall cell variant. Also were follicular thyroid carcinoma (FTC) subdivided to minimally invasive follicular thyroid carcinoma (MIFTC) and Widely Invasive follicular thyroid carcinoma (WIFTC). Statistical analysis All statistical calculations were performed using IBM SPSS Statistics (IBM SPSS Statistics for Windows, Version 22, SPSS Inc. an IBM Company) program and Microsoft Excel 2010 (Microsoft Corporation, Seattle, WA, United States). The descriptive analysis of the epidemiological data presented as frequencies, percentages and mean ± standard deviation (SD). χ2 test was performed to find out the variables associated with cancer among the surgical patients. RESULTS A total of 1188 patients (range 15-90 years) included in this study. The mean age of the study population was 46.3 ± 15.1 (SD), median 45 years, and mode 49 years. Of the 1188 (212 male; 976 female) patients, 245 patients had two thyroid nodules, which resulted in a total of 1433 FNA cases (nodules). Among the study population, a total of 311 patients underwent surgery and 877 patients did not undergo surgery. Of the 311 patients who underwent surgery, 58 patients had two thyroid nodules, which resulted in a total of 369 cases (245 benign and 124 malignant) (Figure 1). Among patients who underwent surgery, no statistically significant differences were observed on the presence of cancer among both gender (P = 0.463), and different age groups (P = 0.928).

went surgery, 58 patients had two thyroid nodules, which resulted in a total of 369 cases (245 benign and 124 malignant) (Figure 1). Among patients who underwent surgery, no statistically significant differences were observed on the presence of cancer among both gender (P = 0.463), and different age groups (P = 0.928). Figure 1 Flowchart of thyroid nodules description among 1188 patients and the risk of malignancy among 311 surgically excised nodules during January, 2012 to December, 2014. FNA: Fine needle aspiration. As shown in Table 2, the distribution of all cases in the six Bethesda diagnostic categories were as follows: 46 cases (3.2%) of category I, 1080 cases (75.3%) of category II, 131 cases (9.1%) of category III, 71 cases (5%) of category IV, 32 cases (2.2%) of category V and 73 cases (5.1%) of category VI. Table 2 Age and sex distribution of thyroid lesion (based on fine-needle aspiration cytology according to Bethesda system) Age (yr) Total number of patients Gender All FNAs (n = 1433) n, % F/M Bethesda I Bethesda II Bethesda III Bethesda IV Bethesda V Bethesda VI Total 15-30 176 (14.8) 159/17 9 (4.5) 149 (74.9) 17 (8.5) 12 (6) 4 (2) 8 (4) 199 31-45 420 (35.4) 362/58 12 (2.4) 375 (74.7) 41 (8.2) 28 (5.6) 14 (2.8) 32 (6.4) 502 46-60 374 (31.5) 301/73 15 (3.3) 347 (75.1) 40 (8.8) 22 (4.8) 9 (2) 23 (5) 456 61-75 175 (14.7) 126/49 10 (4.5) 162 (72.3) 33 (14.7) 7 (3.1) 4 (1.8) 8 (3.6) 224 > 75 43 (3.6) 28/15 0 47 (90.4) 0 2 (3.8) 1 (1.9) 2 (3.8) 52 Total 1188 976/212 46 (3.2) 1080 (75.3) 131 (9.1) 71 (5) 32 (2.2) 73 (5.1) 1433 FNA: Fine-needle aspiration; F: Female; M: Male.

3) 347 (75.1) 40 (8.8) 22 (4.8) 9 (2) 23 (5) 456 61-75 175 (14.7) 126/49 10 (4.5) 162 (72.3) 33 (14.7) 7 (3.1) 4 (1.8) 8 (3.6) 224 > 75 43 (3.6) 28/15 0 47 (90.4) 0 2 (3.8) 1 (1.9) 2 (3.8) 52 Total 1188 976/212 46 (3.2) 1080 (75.3) 131 (9.1) 71 (5) 32 (2.2) 73 (5.1) 1433 FNA: Fine-needle aspiration; F: Female; M: Male. The distributions of follow-up diagnoses for each initial Bethesda diagnostic classification are shown in Table 3. There were 124 total cases of malignancy on resection, giving an overall surgical yield of malignancy of 33.6%. Eight of (2.2%) 369 thyroid nodules were diagnosed as ND, 181 (49.1%) diagnosed as benign, 42 (11.4%) diagnosed as AUS/FLUS, 53 (14.4%) as FNS/SFN. Category V (SM) diagnoses (26 cases) reminded benign in 8 cases, but histologically confirmed as carcinoma in 18 case (69.2%). Finally, category VI diagnoses (59 cases) reminded benign in 2 cases, but histologically confirmed as carcinoma in 57 cases (96.7%). Table 3 Cyto-Histopathological correlation of thyroid lesion

The distributions of follow-up diagnoses for each initial Bethesda diagnostic classification are shown in Table 3. There were 124 total cases of malignancy on resection, giving an overall surgical yield of malignancy of 33.6%. Eight of (2.2%) 369 thyroid nodules were diagnosed as ND, 181 (49.1%) diagnosed as benign, 42 (11.4%) diagnosed as AUS/FLUS, 53 (14.4%) as FNS/SFN. Category V (SM) diagnoses (26 cases) reminded benign in 8 cases, but histologically confirmed as carcinoma in 18 case (69.2%). Finally, category VI diagnoses (59 cases) reminded benign in 2 cases, but histologically confirmed as carcinoma in 57 cases (96.7%). Table 3 Cyto-Histopathological correlation of thyroid lesion Cytopathology Histopathological diagnosis Total Benign Malignant, n (%) Bethesda I 6 2 (25) 8 Bethesda II 165 16 (8.9) 181 Bethesda III 36 6 (14.3) 42 Bethesda IV 28 25 (47.2) 53 Bethesda V 8 18 (69.3) 26 Bethesda VI 2 57 (96.7) 59 Total 245 124 (33.6) 369 Table 4 shows the comparison rates of malignancy on surgical resection for FNA diagnostic categories and malignancy risk of the present findings and previously published data. Table 5 shows the age and sex distribution of thyroid cancer. Majority of the thyroid cancer nodules (n = 57, 46%) in Bethesda VI category followed by Bethesda IV (n = 25, 20.2%) and Bethesda V (n = 18, 14.5%). Among the Bethesda IV category 17 (68%) were PTC and 8 (32%) were follicular carcinoma. Almost 40% of the cancer nodules in 31-45 age groups in both sex.

f thyroid cancer. Majority of the thyroid cancer nodules (n = 57, 46%) in Bethesda VI category followed by Bethesda IV (n = 25, 20.2%) and Bethesda V (n = 18, 14.5%). Among the Bethesda IV category 17 (68%) were PTC and 8 (32%) were follicular carcinoma. Almost 40% of the cancer nodules in 31-45 age groups in both sex. Table 4 Comparison rates of malignancy (%) on surgical resection for fine-needle aspiration diagnostic categories and malignancy risk of recent studies Published year Comparison of diagnostic categories I (ND) II (Benign) III (AUS/FLUS) IV (FN/SFN) V (SM) VI (malignant) Recent studies Park et al[22] 2014 13.3 40.6 9.1 0.4 19.3 17.6 Mondal et al[10] 2013 1.2 87.5 1 4.2 1.4 4.7 Mufti et al[29] 2012 11.6 77.6 0.8 4 2.4 3.6 Wu et al[30] 2012 20.1 39 27.2 8.4 2.6 2.7 Bongiovanni et al[31] 2012 2 54.7 6.3 25.3 6.3 5.4 Present study 3.2 75.3 9.1 5 2.2 5.1 Comparison of malignancy risk Haugen et al[32] (meta-analysis) 2016 9-32 1-10 6-48 14-34 53-97 94-100 Pantola et al[33] 2016 2016 0 0 8.3 10 100 100 Park et al[22] 2014 35.3 5.6 69 50 38.7 98.9 Mondal et al[10] 2013 0 4.5 20 30.6 75 97.8 Mufti et al[29] 2012 20 3.1 50 20 80 100 Wu et al[30] 2012 12 8 27 33 68 100 Present study 25 8.9 14.3 47.2 69.3 96.7 ND: Nondiagnostic; AUS/FLUS: Atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN: Follicular neoplasm/suspicious for follicular neoplasm; SM: Suspicious for malignancy. Table 5 Age and sex distribution of thyroid cancer

Published year Comparison of diagnostic categories I (ND) II (Benign) III (AUS/FLUS) IV (FN/SFN) V (SM) VI (malignant) Recent studies Park et al[22] 2014 13.3 40.6 9.1 0.4 19.3 17.6 Mondal et al[10] 2013 1.2 87.5 1 4.2 1.4 4.7 Mufti et al[29] 2012 11.6 77.6 0.8 4 2.4 3.6 Wu et al[30] 2012 20.1 39 27.2 8.4 2.6 2.7 Bongiovanni et al[31] 2012 2 54.7 6.3 25.3 6.3 5.4 Present study 3.2 75.3 9.1 5 2.2 5.1 Comparison of malignancy risk Haugen et al[32] (meta-analysis) 2016 9-32 1-10 6-48 14-34 53-97 94-100 Pantola et al[33] 2016 2016 0 0 8.3 10 100 100 Park et al[22] 2014 35.3 5.6 69 50 38.7 98.9 Mondal et al[10] 2013 0 4.5 20 30.6 75 97.8 Mufti et al[29] 2012 20 3.1 50 20 80 100 Wu et al[30] 2012 12 8 27 33 68 100 Present study 25 8.9 14.3 47.2 69.3 96.7 ND: Nondiagnostic; AUS/FLUS: Atypia of undetermined significance/follicular lesion of undetermined significance; FN/SFN: Follicular neoplasm/suspicious for follicular neoplasm; SM: Suspicious for malignancy. Table 5 Age and sex distribution of thyroid cancer Age (yr) Total number of nodules Gender All FNAs (n = 124) n, % F/M Bethesda I Bethesda II Bethesda III Bethesda IV Bethesda V Bethesda VI 15-30 18 (14.5) 3/15 0 3 1 3 4 7 31-45 49 (39.5) 39/10 1 5 2 9 7 25 46-60 43 (34.7) 35/8 1 7 3 9 5 18 61-75 12 (9.7) 8/4 0 1 0 3 2 6 > 75 2 (1.6) 2/0 0 0 0 1 0 1 Total 124 87/37 2 (1.6) 16 (12.9) 6 (4.8) 25 (20.2) 18 (14.5) 57 (46) FNA: Fine-needle aspiration; F: Female; M: Male.

III Bethesda IV Bethesda V Bethesda VI 15-30 18 (14.5) 3/15 0 3 1 3 4 7 31-45 49 (39.5) 39/10 1 5 2 9 7 25 46-60 43 (34.7) 35/8 1 7 3 9 5 18 61-75 12 (9.7) 8/4 0 1 0 3 2 6 > 75 2 (1.6) 2/0 0 0 0 1 0 1 Total 124 87/37 2 (1.6) 16 (12.9) 6 (4.8) 25 (20.2) 18 (14.5) 57 (46) FNA: Fine-needle aspiration; F: Female; M: Male. Type and variants of thyroid cancer among histopathological diagnosis are shown in Table 6. Papillary carcinoma was the most common form of thyroid cancer among the study population (111, 89.6%). Among PTC (n = 111), four histologic variants exist, with classic variant PTC accounting for 51.4% of PTC followed by follicular-variant PTC (30.6%). Furthermore, 8.9% of malignancies were FTC (including 0.8% of the highest risk widely invasive phenotype), 0.8% of medullary thyroid carcinoma (MTC) and 0.8% of anaplastic thyroid carcinoma (ATC). Among the Bethesda IV category 17 (68%) thyroid nodules were PTC and 8 (32%) were FTC. Table 6 Type and variants of thyroid cancer among histopathological diagnosis

Type and variants of thyroid cancer among histopathological diagnosis are shown in Table 6. Papillary carcinoma was the most common form of thyroid cancer among the study population (111, 89.6%). Among PTC (n = 111), four histologic variants exist, with classic variant PTC accounting for 51.4% of PTC followed by follicular-variant PTC (30.6%). Furthermore, 8.9% of malignancies were FTC (including 0.8% of the highest risk widely invasive phenotype), 0.8% of medullary thyroid carcinoma (MTC) and 0.8% of anaplastic thyroid carcinoma (ATC). Among the Bethesda IV category 17 (68%) thyroid nodules were PTC and 8 (32%) were FTC. Table 6 Type and variants of thyroid cancer among histopathological diagnosis Type of cancer Total = 124 (n, %) BETHESDA (n, %) I II III IV (n = 25) V VI PTC Classic variant 57 1 5 1 3 8 39 Follicular variant 34 1 8 2 11 6 6 Conventional 19 0 2 2 3 3 9 Tall-cell variant 1 0 0 0 0 0 1 Total PTC 111 (89.6) 2 15 5 17 (68) 17 55 FTC MIFTC 10 0 1 1 7 1 0 WIFTC 1 0 0 0 1 0 0 Total FTC 11 (8.9) 0 1 1 8 (32) 1 0 MTC 1 (0.8) 0 0 0 0 0 1 ATC 1 (0.8) 0 0 0 0 0 1 PTC: Papillary thyroid carcinoma; MTC: Medullary thyroid carcinoma; ATC: Anaplastic thyroid carcinoma; FTC: Follicular thyroid carcinoma; MIFTC: Minimally invasive follicular thyroid carcinoma; WIFTC: Widely invasive follicular thyroid carcinoma.

11 (8.9) 0 1 1 8 (32) 1 0 MTC 1 (0.8) 0 0 0 0 0 1 ATC 1 (0.8) 0 0 0 0 0 1 PTC: Papillary thyroid carcinoma; MTC: Medullary thyroid carcinoma; ATC: Anaplastic thyroid carcinoma; FTC: Follicular thyroid carcinoma; MIFTC: Minimally invasive follicular thyroid carcinoma; WIFTC: Widely invasive follicular thyroid carcinoma. DISCUSSION Over the last few decades thyroid cancer has been on the rise considerably, globally, while mortality has steadily dropped, including in Saudi Arabia[14]. This reduction in the mortality resulting from thyroid cancer reflects the variations in the exposure to risk factors and alters the diagnosis and treatment of the disease, while the rise in the incidence is probably due to the improvement in the identification of this neoplasm[14]. However, in comparison with the developed countries, research on the incidence, prevalence and type of thyroid cancer in Saudi Arabia is still inadequate due to the lack of suitable studies being done on this specific aspect. Therefore, the objective of the current study is to stratify the risk of malignancy in the thyroid nodules based on the Bethesda system, which enhances the interpretation of the FNAC reports and enables a more accurate study and diagnosis of such thyroid nodules[13,15]. In this study, the distribution of age and gender among the patients is almost similar to those recorded in identical studies[1,2,16]. Besides, the female/male ratio reported in this study for thyroid cancer (4.7:1) concurs with the concept that thyroid cancer occurs more commonly among women. In the present study we found that the overall malignant rate was 33.6% which exactly matches the percentage (33.8%) of 25445 thyroid FNAs used in the meta-analysis done by Bongiovanni et al[17], as well as Jo et al[18] who reported 30.9%. However, this high malignancy rate is not unusual if it is understood that the FNAC is consistently being performed today for most patients with thyroid nodules. This has resulted in a drop in the number of unwarranted surgeries and thereby to an increase in the percentage for reported malignancies[1]. It is noteworthy that the number of FNA cases in this study steadily rose from 2012 (n = 357) to 2014 (n = 449).

tly being performed today for most patients with thyroid nodules. This has resulted in a drop in the number of unwarranted surgeries and thereby to an increase in the percentage for reported malignancies[1]. It is noteworthy that the number of FNA cases in this study steadily rose from 2012 (n = 357) to 2014 (n = 449). From various studies it was evident that the percentage of cases that were subjected to surgery differed widely among different institutions, reporting a range from 11.8%[19] to 45.1%[20] with an average rate of 25%[17]; the current study identified 26.2% of the study population who had surgical outcome.

tly being performed today for most patients with thyroid nodules. This has resulted in a drop in the number of unwarranted surgeries and thereby to an increase in the percentage for reported malignancies[1]. It is noteworthy that the number of FNA cases in this study steadily rose from 2012 (n = 357) to 2014 (n = 449). From various studies it was evident that the percentage of cases that were subjected to surgery differed widely among different institutions, reporting a range from 11.8%[19] to 45.1%[20] with an average rate of 25%[17]; the current study identified 26.2% of the study population who had surgical outcome. Each Bethesda category showed a malignancy rate ranging from 1%-10% (“benign category) to 94%-100% (“malignant” category). This comprehensive range highlights the ability of the Bethesda system to differentiate and determine the likelihood of malignancy. The results recorded in our research concurred closely with the results reported in the American Thyroid Association Management Guidelines and other studies: 25% vs 9%-32% (“non-diagnostic or unsatisfactory” category), 9.3% vs 1%-10% (“benign and non-neoplastic” category), 14.3% vs 6%-48% (AUS/FLUS), 69.2% vs 53%-97% (“suspicious for malignancy” category), and 96.7% vs 94%-100% (“malignant” category)[13,17]. Among Bethesda, category IV found 47.2% malignancy risk, a value higher than the meta-analysis results of 14%-34% (FNS/SFN), published recently by Bongiovanni et al[17]. However, many studies revealed the greatest variation in the risk of malignancy class IV, some of which are higher (malignancy rate 50%-67%) than the present values[21-23].

ry IV found 47.2% malignancy risk, a value higher than the meta-analysis results of 14%-34% (FNS/SFN), published recently by Bongiovanni et al[17]. However, many studies revealed the greatest variation in the risk of malignancy class IV, some of which are higher (malignancy rate 50%-67%) than the present values[21-23]. The current study reported PTC (89.6%) as the commonest type of thyroid cancer in the population under study. Studies also reported that overall PTC as the commonest kind of thyroid cancer represents 80% of all the thyroid malignancies and more than 90% of the differentiated thyroid cancers[13,24,25]. A spurt in the occurrence of PTC over the past decades has triggered greater interest in this disease. This is one of the fastest growing kinds of cancer recording over 20000 new cases annually. Although individuals are susceptible to papillary carcinoma irrespective of age, most patients will show the disease prior to 45 years of age[26], a fact corroborated by the current findings (42% PTC between 31-45 years of age). Unfortunately, FTC is not being diagnosed as often, although there is an increasing incidence of well-differentiated thyroid carcinomas everywhere else[27,28], concurring with the results of the current study. There are a two limitations to this study, mainly the retrospective design and performance in a single center. As the PSMMC is a tertiary center for thyroid lesions, the data of this study may not precisely reflect the general population. More research is warranted to overcome the limitations of the study.

The current study reported PTC (89.6%) as the commonest type of thyroid cancer in the population under study. Studies also reported that overall PTC as the commonest kind of thyroid cancer represents 80% of all the thyroid malignancies and more than 90% of the differentiated thyroid cancers[13,24,25]. A spurt in the occurrence of PTC over the past decades has triggered greater interest in this disease. This is one of the fastest growing kinds of cancer recording over 20000 new cases annually. Although individuals are susceptible to papillary carcinoma irrespective of age, most patients will show the disease prior to 45 years of age[26], a fact corroborated by the current findings (42% PTC between 31-45 years of age). Unfortunately, FTC is not being diagnosed as often, although there is an increasing incidence of well-differentiated thyroid carcinomas everywhere else[27,28], concurring with the results of the current study. There are a two limitations to this study, mainly the retrospective design and performance in a single center. As the PSMMC is a tertiary center for thyroid lesions, the data of this study may not precisely reflect the general population. More research is warranted to overcome the limitations of the study. In conclusion, 33.6% of the cases overall among the surgically excised nodules, showed malignancy. The malignancy values reported in our research were constant and comparable with the results of other data with respect to the risk of malignancy. For the FN/SF patients and those with suspicions of malignancy, total thyroidectomy is indicated because of the substantial risk of malignancy. It is clear, that reviewing the thyroid FNAs with the Bethesda system allowed a more precise cytological diagnosis. However, the impact of Bethesda application may vary among different institutions. Clinicians are advised to be aware of the malignancy rate in the Bethesda categories in their respective institutions to improve the investigation and decision regarding patients with thyroid nodules.

more precise cytological diagnosis. However, the impact of Bethesda application may vary among different institutions. Clinicians are advised to be aware of the malignancy rate in the Bethesda categories in their respective institutions to improve the investigation and decision regarding patients with thyroid nodules. COMMENTS Background The National Cancer Institute, United States, established guidelines employing a standardized nomenclature to interpret thyroid fine-needle aspirations (FNAs) called the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) which is now accepted as the proposed diagnostic categories for thyroid cancer. Research frontiers Compared with the developed countries, research regarding the malignancy risks in thyroid nodules is still inadequate due to lack of appropriate studies being conducted in these specified areas in Saudi Arabia. Hence, this present study attempts to stratify the malignancy risks in thyroid nodules in a tertiary care referral center in Saudi Arabia utilizing the Bethesda system. Innovations and breakthroughs The study found that there were 124 total cases of malignancy on resection, giving an overall surgical yield malignancy of 33.6%. Majority of the thyroid cancer nodules in Bethesda VI category followed by Bethesda IV. Almost 40% of the cancer nodules in 31-45 age group in both sex. Papillary thyroid carcinoma was the most common form of thyroid cancer among the study population followed by follicular thyroid carcinoma, medullary carcinoma and anaplastic carcinoma.

yroid cancer nodules in Bethesda VI category followed by Bethesda IV. Almost 40% of the cancer nodules in 31-45 age group in both sex. Papillary thyroid carcinoma was the most common form of thyroid cancer among the study population followed by follicular thyroid carcinoma, medullary carcinoma and anaplastic carcinoma. Applications Reviewing the thyroid FNAs with the Bethesda system allowed a more precise cytological diagnosis. However, the impact of Bethesda application may vary among different institutions. Clinicians are advised to be aware of the malignancy rate in the Bethesda categories in their respective institutions to improve the investigation and decision regarding patients with thyroid nodules. Terminology PTC: Papillary thyroid carcinoma; FTC: Follicular thyroid carcinoma; SCR: Saudi Cancer Registry; FNA: Fine-needle aspiration; FNAC: Fine-needle aspiration cytology; NCI: National Cancer Institute, United States; BSRTC: Bethesda System for Reporting Thyroid Cytopathology; PSMMC: Prince Sultan Military Medical City; TBSRTC: The Bethesda system of reporting thyroid cytology; UNS: Unsatisfactory; ND: Nondiagnostic; AUS/FLUS: Atypia of undetermined significance or follicular lesion of undetermined significance; US: Ultrasound; MIFTC: Minimally invasive follicular thyroid carcinoma; WIFTC: Widely Invasive follicular thyroid carcinoma; ATC: Anaplastic thyroid carcinoma.

rting thyroid cytology; UNS: Unsatisfactory; ND: Nondiagnostic; AUS/FLUS: Atypia of undetermined significance or follicular lesion of undetermined significance; US: Ultrasound; MIFTC: Minimally invasive follicular thyroid carcinoma; WIFTC: Widely Invasive follicular thyroid carcinoma; ATC: Anaplastic thyroid carcinoma. Peer-review The study shows a very exhaustive analysis of the throughput of thyroid cytopathology over a three-year period. The manuscript contains a detailed exposition of the results, including comprehensive tables and a comparison to other recent studies. In my opinion, this manuscript fulfills all the requirements to be published. Institutional review board statement: The study protocol was approved by the Research and Ethics committee of Prince Sultan Military Medical City, Riyadh, Saudi Arabia. Informed consent statement: Not applicable. Conflict-of-interest statement: Authors have no conflict of interests and the work was not supported or funded by any drug company. Data sharing statement: No data sharing as this manuscript and the data were not published elsewhere. Manuscript source: Unsolicited manuscript Specialty type: Oncology Country of origin: Saudi Arabia Peer-review report classification Grade A (Excellent): A Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: November 6, 2016 First decision: November 30, 2016 Article in press: December 29, 2016 P- Reviewer: Peters GJ, Velasco I, Xu Z S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: Recombinant human interleukin-12 (rhIL-12) is a new kind of biological agent secreted by Chinese hamster ovary cells. Study has shown that it has the advantage of promoting recovery of hematopoietic function, regulating the body’s immunity and inhibiting angiogenesis growth. At present, the research of rhIL-12 stays in the foundational realm and in animal experimentation. In our study, however, there were 100 patients with large or numerous tumors (more than two) and who received precision radiotherapy (Cyber knife or image-guided radiotherapy). The results showed that rhIL-12 can prevent radiation damage, improve hematopoietic function, regulate immunity, reduce the side effect of radiotherapy and improve the quality of life of patients. INTRODUCTION Interleukin-12 (IL-12) is an immunoregulatory protein produced by macrophages, B cells, mononuclear cells, keratinocytes and dendritic cells, and its target point lies in early undifferentiated pluripotent hematopoietic stem cells. The studies of IL-2 trace back to early in 1986. Subsequently, many studies have confirmed that IL-12 can contribute to enhancing immunity. For example, Zhang et al[1] found a cytokine which can promote secretion of cytotoxic T cells (CTLs) and lymphatic factor-activated killer cells (LAK) in synergy with IL-2. In 1989, Bellone and Trinchieri[2] found a cytokine called natural killer cell-stimulating factor (NKSF), which can stimulate the production of IFN-γ. Eventually, it became known that the two cytokines were the same substance, now known as IL-12.

) and lymphatic factor-activated killer cells (LAK) in synergy with IL-2. In 1989, Bellone and Trinchieri[2] found a cytokine called natural killer cell-stimulating factor (NKSF), which can stimulate the production of IFN-γ. Eventually, it became known that the two cytokines were the same substance, now known as IL-12. Based on subsequent research studies, IL-12 seems to serve as an immunoregulatory anti-cancer agent for oncology patients. However, the adverse events related to IL-12, including fever, chills, decreased peripheral blood cells and organ dysfunction, have limited the clinical application of IL-12[3]. Recombinant human interleukin-12 (rhIL-12) is an immunoregulatory protein produced by gene engineering technology. RhIL-12 has similar biological activity to IL-12. With the advantage of high purity (> 98%), high activity and low therapeutic dose, rhIL-12 became the only agent which could not only restore hematopoietic function but also improve immune function[4].

an immunoregulatory protein produced by gene engineering technology. RhIL-12 has similar biological activity to IL-12. With the advantage of high purity (> 98%), high activity and low therapeutic dose, rhIL-12 became the only agent which could not only restore hematopoietic function but also improve immune function[4]. The basic experimental studies have found that the recovery and reconstitution of hematopoiesis system after radiotherapy is helpful to avoid the rapid increase of single blood cells, which lead to high fever, conjunctival hemorrhage, abnormal immune response, embolism and other detrimental side effects[5]. But a large number of studies are only based on animal experiments. The aim of our study, then, was to explore the interventional effects of rhIL-12 in tumor patients receiving radiotherapy, including the complications after radiotherapy, the curative effects on hematopoietic function and immune function as well as dose-effect relationship, and to provide scientific basis for clinical application.

ur study, then, was to explore the interventional effects of rhIL-12 in tumor patients receiving radiotherapy, including the complications after radiotherapy, the curative effects on hematopoietic function and immune function as well as dose-effect relationship, and to provide scientific basis for clinical application. MATERIALS AND METHODS Objectives To observe 100 patients with mid-advanced tumors who were treated with Cyber knife or image-guided radiotherapy (IGRT) in the People’s Liberation Army 107th Hospital, from October 2014 to June 2016. Inclusion criteria were: (1) Tumor confirmed by pathology, CT or MRI diagnosis, for which the clinical staging criteria were III-IV according to the World Health Organization (WHO); (2) ECOG score of 1 to 4 points; (3) Postoperative recurrence or lymphocytes invasion and metastasis, and need for radiosurgery; and (4) Provision of written informed consent for research and therapy. Exclusion criteria were: (1) Illness combined with tuberculosis or serious failure of important organs, such as heart, liver, kidney, lung, etc.; (2) Presence of benign tumors; (3) History of organ transplantation or allergies; and (4) Pregnant or lactating women or women of childbearing age. The experimental study was approved by the hospital’s ethics committee.

with tuberculosis or serious failure of important organs, such as heart, liver, kidney, lung, etc.; (2) Presence of benign tumors; (3) History of organ transplantation or allergies; and (4) Pregnant or lactating women or women of childbearing age. The experimental study was approved by the hospital’s ethics committee. In the treatment group, 34 of the subjects (68%) were males and 16 (32%) were females; the mean age was 58.5-year-old (range: 27-83 years). Fifty subjects (30%) had lung cancers, 12 (24%) had liver cancers, 8 (16%) had head tumors, 1 (2%) had pancreatic cancer and 14 (28%) had other tumors. Solid relapse and metastasis tumor, for which size of the nidus could be assessed, accounted for 98% (n = 49) of the cases. Diffuse invasive metastatic tumor accounted for 2% (n = 1) of the cases, and the tumor diameter ranged from 3 cm to 16 cm. The patients who were classified as recurrent after the surgery or with more than 2 lesions accounted for 96% (n = 48) of the cases. In the control group, 28 of the subjects (56%) were males and 22 (44%) were females; the mean age was 57.4-year-old (range: 25-79 years). Twenty subjects (40%) had lung cancers, 15 (30%) had liver cancers and 15 (30%) had pancreatic cancers.

e surgery or with more than 2 lesions accounted for 96% (n = 48) of the cases. In the control group, 28 of the subjects (56%) were males and 22 (44%) were females; the mean age was 57.4-year-old (range: 25-79 years). Twenty subjects (40%) had lung cancers, 15 (30%) had liver cancers and 15 (30%) had pancreatic cancers. Main equipment, drugs and reagents Equipment: Cyber knife, third-generation model produced by a United States’ accuracy company; IGRT, Eiekta Synergy model produced by a Swedish medical company; Flow cytometer produced by the United States’ BD Biosciences; Enzyme-mark instrument produced by a United States’ automation company; Chemiluminescence apparatus and automatic biochemistry analyzer produced by Roche Company; Hematology analyzer produced in Japan. Drugs and reagents: RhIL-12 (for injection) produced by Qingdao Litai Kang Pharmaceutical Co. Ltd. Antibodies used in the study were purchased from BD Biosciences, including anti-human-IgG-FITC, anti-human-CD45-FITC, anti-human-CD56-PE and anti-human-CD3-PerCP-CD4-FITC-CD8-PE. Hemolysin was produced by an American research and development company.

for injection) produced by Qingdao Litai Kang Pharmaceutical Co. Ltd. Antibodies used in the study were purchased from BD Biosciences, including anti-human-IgG-FITC, anti-human-CD45-FITC, anti-human-CD56-PE and anti-human-CD3-PerCP-CD4-FITC-CD8-PE. Hemolysin was produced by an American research and development company. Methods: After being admitted to hospital, all patients’ data were recorded for the three routine( liver and kidney function, heart function, bleeding and clotting time) and the imaging examination (such as electrolytes, color Doppler, CT and IMT), as well as adverse reactions, etc. All patients signed “consent form of precise radiotherapy”, “consent form of experimental study” and “agreement about the clinical application of rhIL-12 for prevention and treatment of malignant tumor radiotherapy complications”. According to the research program, the patients were divided into 50, 100, 150, 200 and 250 ng/kg different-dose subgroups, and injected with rhIL-12 subcutaneously. Peripheral blood samples (for red blood cell (RBC), white blood cell (WBC) and platelet (PLT) assessment) and the immunophenotypes (CD4/8, CD45 and CD56) were collected before dosing (0 d) and at 12 h, 3 d, 7 d, 10 d, 14 d, 21 d, 28 d after dosing respectively. The effects on hematopoietic function and immune function were observed, as well as the dose-effect relationship. The control group used symptomatic supportive treatment.

e immunophenotypes (CD4/8, CD45 and CD56) were collected before dosing (0 d) and at 12 h, 3 d, 7 d, 10 d, 14 d, 21 d, 28 d after dosing respectively. The effects on hematopoietic function and immune function were observed, as well as the dose-effect relationship. The control group used symptomatic supportive treatment. Efficacy evaluation criteria Test evaluation: The patients who accepted high-dose and short-course of accurate radiotherapy, such as cyber knife and IGRT, could experience induction of decrease of peripheral blood cells and decline or imbalance of immune function. In this study, rhIL-12 was given to explore the interventional effects on radiation oncology surgery patients, including effects on hematopoietic function and immune function as well as dose-effect relationship and to provide scientific basis for drug development and clinical application. WHO objective evaluation criteria: Complete remission (CR) indicated all symptoms and signs disappearing for 4 wk; partial remission (PR) indicated the tumor size was estimated to have reduced by more than 50% for at least 4 wk. No change (NC) indicated the patient’s condition had no obvious change for at least 4 wk, the tumor size has increased less than 25% and decreased less than 50%. PD (worsen) indicated new lesions having appeared or lesions had increased by more than 25%.

was estimated to have reduced by more than 50% for at least 4 wk. No change (NC) indicated the patient’s condition had no obvious change for at least 4 wk, the tumor size has increased less than 25% and decreased less than 50%. PD (worsen) indicated new lesions having appeared or lesions had increased by more than 25%. Zubrod-ECOG-WHO score: 0 score stood for normal activities; 1 score stood for mild symptoms and almost normal activities; 2 score stood for the time staying in bed as less than 50% of the daytime; 3 score stood for the time staying in bed as more than 50% of the daytime; 4 score stood for completely bedridden; 5 score stood for death. Total efficiency = (CR + PR)/total number of cases × 100%. Statistical analysis SPSS16.0 software was used for statistical analysis. Continuous variables are expressed as a mean and standard deviation; the mean differences between the groups were compared by independent t-test and ANOVA, and χ2 test was used to compare classified variables. Two values of data used two distribution tests. P < 0.05 indicated that the difference was statistically significant. Charts and tables were made by Prism GraphPad 4 software. RESULTS Analysis of research subjects’ number All of the 100 patients completed the study. Results of whole blood test Treatment group: There was a transient decline of WBC and PLT within 12 h after treatment and by 3 d the lowest level was reached; the recovery rate decreased after 7 d, and the trend became stable after 21 d until the end of observation. This trend was relatively significant for WBC.

RESULTS Analysis of research subjects’ number All of the 100 patients completed the study. Results of whole blood test Treatment group: There was a transient decline of WBC and PLT within 12 h after treatment and by 3 d the lowest level was reached; the recovery rate decreased after 7 d, and the trend became stable after 21 d until the end of observation. This trend was relatively significant for WBC. Control group: The whole blood cells declined on 3 d after radiotherapy, decreased significantly after 7 d, and reached the lowest point on 14 d. The degree of decrease was related to the radiation dose and tumor size. The difference between the treatment group and the control group was statistically significant (Table 1 and Figure 1). Table 1 Results of whole blood test for the treatment group and the control group (mean ± SD, n = 10)

Control group: The whole blood cells declined on 3 d after radiotherapy, decreased significantly after 7 d, and reached the lowest point on 14 d. The degree of decrease was related to the radiation dose and tumor size. The difference between the treatment group and the control group was statistically significant (Table 1 and Figure 1). Table 1 Results of whole blood test for the treatment group and the control group (mean ± SD, n = 10) Group Indicator 0 d 12 h 3 d 7 d 14 d 21 d 28 d Control WBC (× 109/L) 7.66 ± 0.82 5.5 ± 0.67 4.2 ± 0.39 4.3 ± 0.48 4.21 ± 0.62 4.69 ± 0.38 4.89 ± 0.63 RBC (× 1012/L) 3.92 ± 0.31 4.26 ± 0.43 3.9 ± 0.41 3.93 ± 0.22 4.38 ± 0.36 3.86 ± 0.34 3.89 ± 0.28 PLT (× 109/L) 358 ± 0.43 339 ± 31.45 232 ± 20.43 258 ± 19.2 275 ± 0.31 296 ± 0.29 321 ± 0.26 50 ng/kg WBC (× 109/L) 6.31 ± 0.59 3.6 ± 0.35 4.27 ± 0.46 4.85 ± 0.35 4.52 ± 0.42 4.72 ± 0.39 5.18 ± 0.52 RBC (× 1012/L) 5.43 ± 0.54 4.92 ± 0.53 4.16 ± 0.38 4.59 ± 0.82 4.73 ± 0.32 5.26 ± 0.37 5.63 ± 0.41 PLT (× 109/L) 231 ± 20.81 185 ± 18.24 195 ± 18.97 205 ± 18 226 ± 18.2 246 ± 17.5 229 ± 19.4 100 ng/kg WBC 8.4 ± 0.45 7.7 ± 7.89 5.3 ± 10.64 6.6 ± 0.98 6.1 ± 0.64 5.73 ± 0.47 5.9 ± 0.21 RBC (× 1012/L) 6.3 ± 0.72 3.37 ± 0.43 3.2 ± 0.34 4.2 ± 0.37 4.78 ± 0.46 4.5 ± 0.42 4.6 ± 0.34 PLT (× 109/L) 231 ± 27.2 185 ± 10.7 178 ± 12.6 195 ± 12.8 166 ± 10.9 182 ± 12.5 205 ± 15.3 150 ng/kg WBC (× 109/L) 6.6 ± 0.73 5.4 ± 0.76 3.8 ± 0.35 5.2 ± 0.37 5.7 ± 0.42 6.2 ± 0.54 6.3 ± 0.54 RBC (× 1012/L) 3.4 ± 0.36 3.5 ± 0.37 3.2 ± 0.29 3.3 ± 0.28 3.6 ± 0.24 3.6 ± 0.26 3.44 ± 0.21 PLT (× 109/L) 367 ± 35.75 352 ± 32.45 306 ± 30.12 316 ± 16 357 ± 17 348 ± 26 317 ± 16 200 ng/kg WBC (× 109/L) 5.3 ± 0.8 4.2 ± 0.3 3.2 ± 0.1 3.5 ± 0.32 3.6 ± 0.27 4.3 ± 0.31 5.4 ± 0.43 RBC (× 1012/L) 4.6 ± 0.51 4.2 ± 0.41 4.1 ± 0.45 4.2 ± 0.3 4 ± 0.2 4.12 ± 0.34 4.5 ± 0.42 PLT (× 109/L) 278 ± 36 183 ± 19 149 ± 14 208 ± 22 259 ± 24 267 ± 25 271 ± 21 250 ng/kg WBC (× 109/L) 3.6 ± 3 ± 0.37 2.7 ± 0.24 3.4 ± 0.4 4.2 ± 0.3 4.5 ± 0.4 4.2 ± 0.4 RBC (× 1012/L) 3.6 ± 0.3 3.3 ± 0.2 3.1 ± 0.2 3.2 ± 0.3 3.4 ± 0.3 3.7 ± 0.2 4.1 ± 0.3 PLT (× 109/L) 364 ± 35 235 ± 21 240 ± 20 276 ± 22 314 ± 19 342 ± 21 312 ± 20 WBC: White blood cell; RBC: Red blood cell; PLT: Platelet.

271 ± 21 250 ng/kg WBC (× 109/L) 3.6 ± 3 ± 0.37 2.7 ± 0.24 3.4 ± 0.4 4.2 ± 0.3 4.5 ± 0.4 4.2 ± 0.4 RBC (× 1012/L) 3.6 ± 0.3 3.3 ± 0.2 3.1 ± 0.2 3.2 ± 0.3 3.4 ± 0.3 3.7 ± 0.2 4.1 ± 0.3 PLT (× 109/L) 364 ± 35 235 ± 21 240 ± 20 276 ± 22 314 ± 19 342 ± 21 312 ± 20 WBC: White blood cell; RBC: Red blood cell; PLT: Platelet. Figure 1 Count changes of the whole blood test. A: Control group; B: 50 ng/kg subgroup; C: 100 ng/kg subgroup; D: 150 ng/kg subgroup; E: 200 ng/kg subgroup; F: 250 ng/kg subgroup. WBC: White blood cell; RBC: Red blood cell; PLT: Platelet. Results of immunologic detection The aim was to observe the immune indexes, including CD4/8, CD45 and CD56. Treatment group: There was a transient decline of CD4/CD8 within 12 h in the 150, 200 and 250 ng/kg subgroups. There was volatility rise between 3-14 d but the level remained below the pre-medication level, and went down after 21 d. There was a transient decline of CD45 and CD56 within 12 h, which rose after 3 d and went down after 21 d. The overall recovery improvement trend was obvious. Control group: The trend of the immune indexes showed rebound on 3 d and a continuous downward trend after 7 d. There was no significant difference in these immune indexes between the other two groups (50 and 100 ng/kg subgroups) and the control group (Table 2 and Figure 2). Table 2 Results of immunologic detection for the treatment group and the control group (mean ± SD, n = 10)

Control group: The trend of the immune indexes showed rebound on 3 d and a continuous downward trend after 7 d. There was no significant difference in these immune indexes between the other two groups (50 and 100 ng/kg subgroups) and the control group (Table 2 and Figure 2). Table 2 Results of immunologic detection for the treatment group and the control group (mean ± SD, n = 10) Group Indicator 0 d 12 h 3 d 7 d 14 d 21 d 28 d Control CD4/8 20.4 ± 2.6 18 ± 1.2 17.5 ± 1.2 17.8 ± 1.3 18.4 ± 1.2 17.3 ± 1.4 16.5 ± 0.4 CD45 75.2 ± 7.5 68.1 ± 5.2 65.4 ± 4.2 83.3 ± 5.2 79.3 ± 3.7 62.4 ± 5.1 60.3 ± 3.6 CD56 12.3 ± 1.2 8.7 ± 0.6 7.6 ± 0.5 11.4 ± 1.3 10.4 ± 0.9 8.2 ± 0.6 6.7 ± 0.5 50 ng/kg CD4/8 28.2 ± 1.8 22.4 ± 1.6 19.8 ± 1.6 19.6 ± 1.6 21.4 ± 1.8 18.8 ± 1.2 16.9 ± 1.4 CD45 81.9 ± 2.4 78.8 ± 5.1 75.2 ± 3.6 82.9 ± 3.8 79.8 ± 5.6 62.4 ± 4.6 51.8 ± 4.3 CD56 27.9 ± 2.2 21.9 ± 1.8 19.2 ± 1.2 22.1 ± 1.8 19.7 ± 2.1 21.6 ± 1.3 22.4 ± 0.9 100 ng/kg CD4/8 15.4 ± 1.5 12.7 ± 1.2 11.5 ± 1.1 10.6 ± 1.2 10.1 ± 0.3 9.2 ± 0.4 8.7 ± 0.2 CD45 84.3 ± 2.6 66.1 ± 3.8 68.7 ± 3.5 67.9 ± 4.4 57.6 ± 4.6 63.1 ± 3.6 70.2 ± 3.1 CD56 14.8 ± 1.8 8.9 ± 0.6 10.8 ± 0.9 12.3 ± 1.5 10.4 ± 4.6 12.9 ± 0.3 11.6 ± 0.4 150 ng/kg CD4/8 16.5 ± 1.2 12.8 ± 1.2 11.6 ± 0.9 13.1 ± 4.6 12.6 ± 1.5 10.8 ± 0.9 7.6 ± 0.5 CD45 74.2 ± 3.6 63.2 ± 3.2 66.1 ± 4.8 82.7 ± 5.2 68.1 ± 4.6 75.2 ± 4.2 65.1 ± 4.1 CD56 6.2 ± 0.3 6.5 ± 0.3 5.9 ± 0.3 7.9 ± 0.6 8.2 ± 7.4 8.8 ± 0.9 6.5 ± 0.1 200 ng/kg CD4/8 9.6 ± 0.4 8.3 ± 0.61 7.6 ± 0.6 7.2 ± 0.9 8.6 ± 4.6 8.2 ± 1.2 6.1 ± 0.2 CD45 64.5 ± 4.2 60.7 ± 4.2 65.8 ± 4.4 76.6 ± 5.9 74.3 ± 4.6 73.2 ± 5.2 55.9 ± 5.0 CD56 3.8 ± 0.2 2.8 ± 0.2 4.4 ± 0.4 4.9 ± 0.9 6.2 ± 0.6 5.9 ± 0.3 4.5 ± 0.1 250 ng/kg CD4/8 6.4 ± 0.2 5.7 ± 0.2 4.2 ± 0.3 4.7 ± 0.3 3.5 ± 0.3 3.4 ± 0.9 3.1 ± 0.9 CD45 46.5 ± 3.9 40.2 ± 3.2 57.9 ± 4.1 66.5 ± 3.8 54.9 ± 4.2 50.4 ± 4.9 35.9 ± 3.2 CD56 4.3 ± 0.2 3.9 ± 0.2 5.4 ± 0.4 6.3 ± 0.5 6.6 ± 0.3 7.2 ± 0.6 6.4 ± 0.8 Figure 2 Changes of immune indexes. A: Control group; B: 50 ng/kg subgroup; C: 100 ng/kg subgroup; D: 150 ng/kg subgroup; E: 200 ng/kg subgroup; F: 250 ng/kg subgroup.

46.5 ± 3.9 40.2 ± 3.2 57.9 ± 4.1 66.5 ± 3.8 54.9 ± 4.2 50.4 ± 4.9 35.9 ± 3.2 CD56 4.3 ± 0.2 3.9 ± 0.2 5.4 ± 0.4 6.3 ± 0.5 6.6 ± 0.3 7.2 ± 0.6 6.4 ± 0.8 Figure 2 Changes of immune indexes. A: Control group; B: 50 ng/kg subgroup; C: 100 ng/kg subgroup; D: 150 ng/kg subgroup; E: 200 ng/kg subgroup; F: 250 ng/kg subgroup. Objective evaluation results The remission rate in the treatment group (84%) was obviously higher than that in the control group (60%), and the difference was statistically significant (P < 0.05). During the observation period, there were no recurrence, metastasis or death, and the survival time of patients was significantly prolonged. There were 2 patients in the 250 ng/kg subgroup that had low fever after administration, 1 in the 200 ng/kg subgroup and 2 in the 250 ng/kg subgroup that had mild impairment of liver function during the observation period. There was no other adverse event found (Table 3). Table 3 Results of objective evaluation for the treatment group and the control group Group n CR % PR % MR % PD % Treatment 50 12 24a 30 60 5a 10a 3a 6a Control 10 5 10 25 50 10 20 10 20 Compared with the control group, a P < 0.05. CR: Complete remission; PR: Partial remission. ECOG score results The aim was to observe the ECOG score for a period of a month after rhIL-12 intervention. Treatment group: There were 26 patients (52%) with normal activities after treatment, and the difference was statistically significant as compared with the 10 cases (20%) before treatment (P < 0.05). The life quality of patients was significantly improved.

ECOG score results The aim was to observe the ECOG score for a period of a month after rhIL-12 intervention. Treatment group: There were 26 patients (52%) with normal activities after treatment, and the difference was statistically significant as compared with the 10 cases (20%) before treatment (P < 0.05). The life quality of patients was significantly improved. Control group: There were 20 patients (40%) with normal activities after treatment, and the difference was not statistically significant as compared with the 12 cases (24%) before treatment (P > 0.05) (Table 4). Table 4 Comparison of ECOG scores before and 1 mo after intervention for the treatment and control groups ECOG scores before treatment after treatment Treatment group (n = 50) 0 0 11a 1 10 15a 2 15 19a 3 20 5a 4 5 0a Control group (n = 50) 0 1 4 1 11 16 2 17 20 3 15 10 4 6 0 There was significant difference between the two groups, a P < 0.05. Imaging evaluation results There were two CT pictures, including 1 case of pancreatic cancer and 1 case of lung cancer in the treatment group, before and after treatment. The results showed that the original lesion was significantly reduced after treatment and no new lesions appeared (Figures 3 and 4). Figure 3 Changes of computed tomography slice before and after treatment in pancreatic cancer patients. A: Pancreatic head tumor mass (4.4 cm × 3.6 cm × 5.3 cm) accompanied by dilation of intrahepatic and extrahepatic bile duct, pancreatic duct and gallbladder before treatment; B: Most of the pancreatic head mass disappeared in 2 mo after B treatment. Low obstruction disappeared.

treatment in pancreatic cancer patients. A: Pancreatic head tumor mass (4.4 cm × 3.6 cm × 5.3 cm) accompanied by dilation of intrahepatic and extrahepatic bile duct, pancreatic duct and gallbladder before treatment; B: Most of the pancreatic head mass disappeared in 2 mo after B treatment. Low obstruction disappeared. Figure 4 Changes of computed tomography slice before and after treatment in lung cancer patients. A: Right peripheral lung tumor mass (4.5 cm × 4.8 cm × 4.0 cm) located in the right upper lobe of the right lung before treatment; B: The mass disappeared 1 year after treatment. DISCUSSION The incidence of cancer is rising, and cancer has become one of the main causes of death[6]. In recent years, radiotherapy of malignant tumors has developed rapidly, especially for accurate radiotherapy. However, the clinical curative effect for patients who have larger or more numerous tumor lesions are often reduced due to adverse reactions after radiotherapy, such as immune injury and myelosuppression. Under normal circumstances, the immune systems maintain the physiological balance and stabilization of the body. Immune cells are the first line of the anti-tumor system. Immune regulatory factors or cytokines participate in immune regulation by means of signal transduction[7]. Studies have shown that tumor cells can escape immunosurveillance through a number of special mechanisms. The immune system is critical to the body’s surveillance against cancer.

st line of the anti-tumor system. Immune regulatory factors or cytokines participate in immune regulation by means of signal transduction[7]. Studies have shown that tumor cells can escape immunosurveillance through a number of special mechanisms. The immune system is critical to the body’s surveillance against cancer. The immune function of about 86% of patients has been shown to be on the decline in the early stage of cancer, and to further decline after treatment, which is the main cause of tumor metastasis and recurrence[8]. What’s more, the complications including infection and bleeding that are caused by the decrease of peripheral blood cells counts are also common causes of death in patients with cancer. Therefore, improving immune function and reducing myelosuppression are indispensable auxiliary treatments in the process of tumor radiotherapy. At present, the treatment of cancer has entered into the era of personalized multidisciplinary treatment. The research shows that the combination of radiotherapy and immunotherapy has a unique advantage[9]. In this respect, IL-12 has received more and more attention.

ary treatments in the process of tumor radiotherapy. At present, the treatment of cancer has entered into the era of personalized multidisciplinary treatment. The research shows that the combination of radiotherapy and immunotherapy has a unique advantage[9]. In this respect, IL-12 has received more and more attention. IL-12 is an immunoregulatory protein produced by macrophages, B cells, mononuclear cells, keratinocytes and dendritic cells. IL-12 mainly functions to mediate cellular immunity, and it can induce the differentiation of T helper cell 1 (Th1), as well as promote the proliferation of NK cells and T cells, further stimulate IFN-γ secretion, and enhance the ability to kill target cells. IL-12 also can promote the formation of interferon-inducible protein-10 (IP-10). IP-10 can prevent the formation of tumor blood vessels, thereby reducing and blocking the nutrition source of tumor cells and inhibiting their growth[10,11]. It has been nearly 30 years since IL-12 was discovered, and a large amount of the related research is still at the stage of basic study and animal study. Many studies have found its abilities to improve immunity capability, adjust the immune function and inhibit the production of tumor blood vessels, but the adverse reactions such as chills and fever, nausea and vomiting, pulmonary edema and allergic reactions limit its clinical application and temper its promotion.

dy. Many studies have found its abilities to improve immunity capability, adjust the immune function and inhibit the production of tumor blood vessels, but the adverse reactions such as chills and fever, nausea and vomiting, pulmonary edema and allergic reactions limit its clinical application and temper its promotion. RhIL-12 is a new kind of biological agent secreted by Chinese hamster ovary cells through gene engineering. Its biological activity is similar to that of IL-12. The advantage of rhIL-12, however, is its high purity (> 98%), high activity (≥ 10000 IU/μg), low therapeutic dose and dosage that can be tolerated. Preliminary experimental study[12-14] found that rhIL-12 is currently the only biological agent with the advantage of comprehensive recovery of hematopoietic function, regulation of the body’s immunity, inhibition of tumor angiogenesis and inhibition of tumor growth, thereby improving the life quality of patients with cancer[15]. As an immune regulatory factor, it plays an important role in both primary and secondary immunity[16]. Especially for radiotherapy patients who present with larger or more numerous tumor targets (more than two), it has important research value.

mor growth, thereby improving the life quality of patients with cancer[15]. As an immune regulatory factor, it plays an important role in both primary and secondary immunity[16]. Especially for radiotherapy patients who present with larger or more numerous tumor targets (more than two), it has important research value. In our study, there were 100 patients who had larger or more numerous tumor s (more than two) and who received precision radiotherapy (Cyber knife or IGRT). The following conclusions are drawn. In the treatment group, the whole blood cells showed a transient decline within 12 h after treatment. The reason for this may be that the cell changes into the microcirculation or the bone marrow microenvironment, which may affect the proliferation of hematopoietic stem cells. The whole blood cells reached the lowest level at 3 d. People have always stopped treatment at this time, which represents a misunderstanding of the early research. The recovery rate decreased after 7 d, and the trend became stable after 21 d until the end of observation. This trend is relatively significant for WBC. Compared with the control group, the difference was significant, which indicated that the rhIL-12 was effective.

represents a misunderstanding of the early research. The recovery rate decreased after 7 d, and the trend became stable after 21 d until the end of observation. This trend is relatively significant for WBC. Compared with the control group, the difference was significant, which indicated that the rhIL-12 was effective. Observation of the immune indexes, including CD4/8, CD45 and CD56, showed a transient decline of CD4/CD8 within 12 h in the 150, 200 and 250 ng/kg subgroups in the treatment group, with volatile rises between 3-14 d, but remaining below the pre-medication level, and then decreasing after 21 d. There was a transient decline of CD45 and CD56 within 12 h, which rose up after 3 d and went down after 21 d. Compared with the control group, the difference was significant. The improvement tendency was obvious, which suggested that rhIL-12 could promote the immune function of the patients after radiotherapy. From our observations of the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, which could be returned to normal after physical cooling. One patient in the 200 ng/kg subgroup and 2 in the 250 ng/kg subgroup showed mild injury of liver function during the observation period, which could be returned to normal after liver-protecting therapy. The injury related to radiation or rhIL-12 needs to be further studied.

e returned to normal after physical cooling. One patient in the 200 ng/kg subgroup and 2 in the 250 ng/kg subgroup showed mild injury of liver function during the observation period, which could be returned to normal after liver-protecting therapy. The injury related to radiation or rhIL-12 needs to be further studied. In addition, this study explored the relationship between biological activity and concentration. The result showed that there was no statistical difference among the 50 ng/kg subgroup, the 100 ng/kg subgroup and the control group for immune response. What’s more, the adverse reactions were mainly concentrated in the 200 and 250 ng/kg subgroups, which suggests that the suitable clinical dosage concentration in our study is 150 ng/kg. The anti-tumor activity of rhIL-12 has been shown in clinical trials, but its toxicity to some extent limits the application. Therefore, rhIL-12 still needs to be further researched. In conclusion, rhIL-12 can prevent radiation damage, improve hematopoietic function, regulate immunity, reduce the side effect of radiotherapy and improve the quality of life of patients. It has good clinical application value and good development prospect as tumor auxiliary treatment.

In addition, this study explored the relationship between biological activity and concentration. The result showed that there was no statistical difference among the 50 ng/kg subgroup, the 100 ng/kg subgroup and the control group for immune response. What’s more, the adverse reactions were mainly concentrated in the 200 and 250 ng/kg subgroups, which suggests that the suitable clinical dosage concentration in our study is 150 ng/kg. The anti-tumor activity of rhIL-12 has been shown in clinical trials, but its toxicity to some extent limits the application. Therefore, rhIL-12 still needs to be further researched. In conclusion, rhIL-12 can prevent radiation damage, improve hematopoietic function, regulate immunity, reduce the side effect of radiotherapy and improve the quality of life of patients. It has good clinical application value and good development prospect as tumor auxiliary treatment. COMMENTS Background Interleukin-12 (IL-12) is an immunoregulatory protein produced by macrophages, B cells, mononuclear cells, keratinocytes and dendritic cells, and its target point lies in early undifferentiated pluripotent hematopoietic stem cells. However, the adverse events related to IL-12, including fever, chills, decrease of peripheral blood cells and organ dysfunction, have limited its clinical application. Recombinant human interleukin-12 (rhIL-12) is an immunoregulatory protein produced by gene engineering technology. RhIL-12 has similar biological activity to IL-12, but with the advantage of high purity (> 98%), high activity and low therapeutic dose. RhIL-12 has become the only available agent which can not only restore hematopoietic function but also improve immune function. Basic experimental study has found that the recovery of hematopoietic function after radiotherapy is helpful to avoid the rapid increase of single blood cells, which can lead to high fever, conjunctival hemorrhage, abnormal immune response, embolism and other detrimental side effects. But a large number of studies are basic in nature and based on animal experiments. The aim of the study was to explore the interventional effects of rhIL-12 on tumor patients receiving radiotherapy, including the complications after radiotherapy, the curative effects on hematopoietic function and immune function as well as dose-effect relationship, and to provide scientific basis for drug development and clinical application.

o explore the interventional effects of rhIL-12 on tumor patients receiving radiotherapy, including the complications after radiotherapy, the curative effects on hematopoietic function and immune function as well as dose-effect relationship, and to provide scientific basis for drug development and clinical application. Research frontiers Some studies have shown that rhIL-12 can stimulate various kinds of cytokines through stimulating the bone marrow microenvironment, either directly or indirectly, further promoting long-term hematopoietic reconstitution progenitor cells’ differentiation and maturation, and instigate short-term hematopoietic reconstitution progenitor cells. These help to achieve a comprehensive recovery of hematopoietic function. In addition, in the circumstances of no supportive treatments, primate experiments demonstrated that using a low dose of rhIL-12 within 24 h after lethal dose irradiation could significantly improve (4-times) the animal’s survival rate. What’s more, rhIL-12 could promote the healing of skin wounds after radiation injury. As a radiation injury prevention drug, rhIL-12 is still effective at 24 h to 48 h after radiation. At the same time, a large number of animal experiments have shown that IL-12 can significantly inhibit the growth and metastasis of malignant tumors, prolonging the survival time of tumor-bearing animals. IL-12 can enhance the natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cell response and the ability to induce production of IFN-C, which indicates that it may have anti-tumor activity. IL-12 enhances the binding ability of NK cells and K562 target cells and tumor cell monolayer, and enhances the cytotoxicity of NK cells to tumor cells. Because rhIL-12 and IL-12 have similar biological activities, some studies have shown that low dose of rhIL-12 can inhibit tumor cell growth, and rhIL-12 has synergistic anti-cancer effect on radiotherapy and chemotherapy, which needs further clinical validation.

nd enhances the cytotoxicity of NK cells to tumor cells. Because rhIL-12 and IL-12 have similar biological activities, some studies have shown that low dose of rhIL-12 can inhibit tumor cell growth, and rhIL-12 has synergistic anti-cancer effect on radiotherapy and chemotherapy, which needs further clinical validation. Innovations and breakthroughs The study found that low-dose rhIL-12 has the effect of prevention and treatment for the decrease of blood cells after radiotherapy, and could effectively improve the immune function and reduce the complications of radiotherapy. Applications RhIL-12 can prevent radiation damage, improve hematopoietic function, regulate immunity, reduce the detrimental side effects of radiotherapy and improve the quality of life of patients. Terminology IL-12: Interleukin-12 is an immunoregulatory protein produced by macrophages, B cells, mononuclear cells, keratinocytes and dendritic cells, and its target point lies in early undifferentiated pluripotent hematopoietic stem cells; RhIL-12: Recombinant human interleukin-12 is an immunoregulatory protein produced by gene engineering technology; its biological activity is similar to IL-12. Peer-review The authors conducted an interesting clinical study on rhIL-12 for the prevention and treatment of complications after radiotherapy in patients with malignant tumors. The manuscript was well written. The methodology was clear and accurate. The results section was adequate.

Terminology IL-12: Interleukin-12 is an immunoregulatory protein produced by macrophages, B cells, mononuclear cells, keratinocytes and dendritic cells, and its target point lies in early undifferentiated pluripotent hematopoietic stem cells; RhIL-12: Recombinant human interleukin-12 is an immunoregulatory protein produced by gene engineering technology; its biological activity is similar to IL-12. Peer-review The authors conducted an interesting clinical study on rhIL-12 for the prevention and treatment of complications after radiotherapy in patients with malignant tumors. The manuscript was well written. The methodology was clear and accurate. The results section was adequate. Institutional review board statement: This study was reviewed and approved by the scientific ethical committee of the Hospital. All operations were performed according to international guidelines concerning the care and treatment of cancer patients. Informed consent statement: Patients were informed of the purpose of the experiment and agreed to treatment with rhuIL-12. Informed consent was obtained in all cases, and protocols were approved by the scientific ethical committee of the Hospital. Conflict-of-interest statement: The authors declare no conflicts of interest. Data sharing statement: No additional unpublished data are available. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: China Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: October 14, 2016 First decision: November 14, 2016

Data sharing statement: No additional unpublished data are available. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: China Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: October 14, 2016 First decision: November 14, 2016 Article in press: January 4, 2017 P- Reviewer: Liu G, Ozyigit G, Xiao EH S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: Colon endoscopic surveillance and prophylactic colectomy have strongly reduced mortality due to colorectal carcinoma and have improved survival of familial adenomatous polyposis (FAP) patients, leading to the development of surveillance for extra-colonic cancers. Polyps in the duodenum and stomach are frequent findings in FAP. The timing of endoscopic and histology surveillance is currently a great challenge. Spectral estimation by fujinon intelligent color enhancement (FICE) may identify dysplasia and discriminate between adenomatous and non-adenomatous polyps. Interestingly, application of FICE to FAP patients significantly increases the detection of adenomas but does not yet change the prognosis, surveillance program and treatment strategies. INTRODUCTION Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome characterized by the development of colorectal cancer by the age of 40 years in nearly 100% of individuals[1]. Colon Endoscopic surveillance and prophylactic colectomy have strongly reduced mortality due to colorectal carcinoma and have improved survival of FAP patients with minimal consequences[2,3], leading to the introduction of surveillance strategies for the prevention of other extracolonic malignancies[4]. The duodenum is the second most common site of polyps development after colon, with a life-time risk of duodenal adenomas that approaches 100% in FAP affected individuals[5,6]. The cumulative risk of duodenal cancer or high grade of dysplasia by the age of 60 years is 4%-10%[6-8].

INTRODUCTION Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome characterized by the development of colorectal cancer by the age of 40 years in nearly 100% of individuals[1]. Colon Endoscopic surveillance and prophylactic colectomy have strongly reduced mortality due to colorectal carcinoma and have improved survival of FAP patients with minimal consequences[2,3], leading to the introduction of surveillance strategies for the prevention of other extracolonic malignancies[4]. The duodenum is the second most common site of polyps development after colon, with a life-time risk of duodenal adenomas that approaches 100% in FAP affected individuals[5,6]. The cumulative risk of duodenal cancer or high grade of dysplasia by the age of 60 years is 4%-10%[6-8]. Endoscopic surveillance and removal of neoplastic tissue is useful in the prevention of duodenal cancer[8]. However, the choice of treatment and the optimal timing of surveillance based on endoscopic and histopathology examination for each patient is currently a great challenge. Currently the surveillance of duodenum is based on the Spigelman classification of duodenal adenomatosis (Table 1); however, this staging system has low predictive values and has never been validated[6,8]. Table 1 Demographic features

Endoscopic surveillance and removal of neoplastic tissue is useful in the prevention of duodenal cancer[8]. However, the choice of treatment and the optimal timing of surveillance based on endoscopic and histopathology examination for each patient is currently a great challenge. Currently the surveillance of duodenum is based on the Spigelman classification of duodenal adenomatosis (Table 1); however, this staging system has low predictive values and has never been validated[6,8]. Table 1 Demographic features Features Patients Total 76 Age (yr) Mean 40.3 (24-64) Gender Male 41 (53.9%) Female 35 (46.1%) Prior surgery IRA 10 (13.2%) IPAA 66 (86.8%) Chemoprevention 16 (21.1%) NSAIDS intake 17 (22.4%) Tobacco exposure 21 (27.6%) PPI/anti-H2 intake 14 (18.4%) Family history of GI malignancies None 31 (40.8%) 1 member 32 (42.1%) 2 members 10 (13.2%) 3 members 3 (3.9%) Spigelman duodenal stage 0 28 (36.8%) I 7 (9.2%) II 34 (44.8%) III 7 (9.2%) IV 0 (0.0%) IRA: Total colectomy and ileo-rectal anastomosis; IPAA: Total proctocolectomy and ileopouch-anal anastomosis; NSAIDS: Non steroidal anti-inflammatory drugs; PPI: Proton pump inhibitors. Gastric polyps are also a common finding in patients with FAP: they mostly consist of FAP-associated fundic gland polyps (FGPs) reported to occur at variable rates, up to 88%[9,10], against a strongly smaller rate (0.8%-5.0%)[11,12] in non-FAP subjects who undergo an esophagogastroduodenoscopy (EGD).

Features Patients Total 76 Age (yr) Mean 40.3 (24-64) Gender Male 41 (53.9%) Female 35 (46.1%) Prior surgery IRA 10 (13.2%) IPAA 66 (86.8%) Chemoprevention 16 (21.1%) NSAIDS intake 17 (22.4%) Tobacco exposure 21 (27.6%) PPI/anti-H2 intake 14 (18.4%) Family history of GI malignancies None 31 (40.8%) 1 member 32 (42.1%) 2 members 10 (13.2%) 3 members 3 (3.9%) Spigelman duodenal stage 0 28 (36.8%) I 7 (9.2%) II 34 (44.8%) III 7 (9.2%) IV 0 (0.0%) IRA: Total colectomy and ileo-rectal anastomosis; IPAA: Total proctocolectomy and ileopouch-anal anastomosis; NSAIDS: Non steroidal anti-inflammatory drugs; PPI: Proton pump inhibitors. Gastric polyps are also a common finding in patients with FAP: they mostly consist of FAP-associated fundic gland polyps (FGPs) reported to occur at variable rates, up to 88%[9,10], against a strongly smaller rate (0.8%-5.0%)[11,12] in non-FAP subjects who undergo an esophagogastroduodenoscopy (EGD). FGPs have historically been considered non-neoplastic lesions without malignant potential[13]; however recent studies have questioned this assumption reporting high rates of low and high grade dysplasia (up to 54%)[9,14,15]. In particular, European and Asian registries of FAP patients proved the presence of gastric carcinoma arising from FGPs in FAP patients and an incidence of gastric adenocarcinoma between 0.6% and 4.0%[16-19].

dies have questioned this assumption reporting high rates of low and high grade dysplasia (up to 54%)[9,14,15]. In particular, European and Asian registries of FAP patients proved the presence of gastric carcinoma arising from FGPs in FAP patients and an incidence of gastric adenocarcinoma between 0.6% and 4.0%[16-19]. Other common types of gastric polyps are represented by adenomas (gastric foveolar or intestinal type-gastric adenomas and pyloric gland adenomas) which are reported in approximately 10% of gastric polyps in FAP patients[10,20,21] and which can arise in the gastric antrum, in the gastric body-fundus or in the context of FGP[22]. So, identification of dysplastic lesions or adenomatous tissue in these patients is often made difficult by the great number of polyps (up to hundreds) and by the patchy distribution of dysplasia. By now, dysplasia finding in this subgroup of subjects is made on the basis of random biopsies[9] which lead to a time consuming, laborious and poorly performing procedure, that can result in a high rate of missed lesions. According to that, it would be useful identifying FGPs at risk of malignant degeneration. A better characterization of patients, an optimized program of surveillance and a good survival are possible with a selective and complete asportation and with a careful histological evaluation.

By now, dysplasia finding in this subgroup of subjects is made on the basis of random biopsies[9] which lead to a time consuming, laborious and poorly performing procedure, that can result in a high rate of missed lesions. According to that, it would be useful identifying FGPs at risk of malignant degeneration. A better characterization of patients, an optimized program of surveillance and a good survival are possible with a selective and complete asportation and with a careful histological evaluation. It is well known that is possible to predict the histology of a mucosal lesion by observing the crypt orifices (the so called pit pattern) of mucosal glands[23] and the capillary pattern of the mucosa[24]. Several endoscopic imaging techniques have been proposed to enhance the details of these patterns[25]. Among these, chromoendoscopy is a widely applied staining method that uses biocompatible dye agents which accumulate within crypt orifices during endoscopy[26]. Although chromoendoscopy is effective for many applications, it still has some problems, such as difficulty in achieving complete and even coating of the mucosal surface with the dye, the extra cost of the equipment needed for dye spraying and the extra time required to perform the procedure. Moreover, traditional chromoendoscopy isn’t able to enhance the capillary pattern, whose evaluation is essential in early diagnoses of malignant lesions[24]. In attempt to resolve these problems, other endoscopic technologies have been developed. Fujinon intelligent color enhancement (FICE™, Fujinon Corp, Saitama, Japan) represents a spectral estimation technique based on arithmetically processing of a white-light image captured by a video endoscope and sent to the spectral-estimation matrix-processing circuit. The image of the white-light endoscopic observation is resolved in each color image of the red, green and blue signal. Next, each resolved image is converted into various presumed wavelength images by a pixel unit. The images of an arbitrary single wavelength are then extracted and reconstructed. Due to its variable setting functions (up to 10) it is possible to select flexibly the most suitable wavelengths required for examination. Preliminary studies suggested that FICE successfully achieved enhancements of real-time observations of mucosal and microvascular patterns[27,28].

then extracted and reconstructed. Due to its variable setting functions (up to 10) it is possible to select flexibly the most suitable wavelengths required for examination. Preliminary studies suggested that FICE successfully achieved enhancements of real-time observations of mucosal and microvascular patterns[27,28]. The light penetration into the mucosa varies according to the wavelengths: Those in the 400-500 nm range are ideal for analyzing surface structures whereas, because of the absorption properties of hemoglobin, longer wavelengths of about 550 nm are more effective for the visualization of blood vessels. FICE seems able to discriminate between adenomatous and non-adenomatous polyps and to identify the presence of dysplasia[29-32]. Few studies have been conducted to determine the efficacy of chromoendoscopy, both traditional and virtual, in the evaluation of duodenal and peri-ampullary adenomatous polyps in FAP patients[33-35]. Interestingly, FICE application in the discrimination between neoplastic and non-neoplastic gastric lesions has not been throughly investigated[36-39], and no data are available about FICE in evaluating FGPs dysplasia or application of FICE for the screening of FAP patients. In FAP cohort, the specific identification of who is at a greater risk of cancer development could be of paramount importance to assure a personalized program of surveillance or a therapeutic intervention.

FICE seems able to discriminate between adenomatous and non-adenomatous polyps and to identify the presence of dysplasia[29-32]. Few studies have been conducted to determine the efficacy of chromoendoscopy, both traditional and virtual, in the evaluation of duodenal and peri-ampullary adenomatous polyps in FAP patients[33-35]. Interestingly, FICE application in the discrimination between neoplastic and non-neoplastic gastric lesions has not been throughly investigated[36-39], and no data are available about FICE in evaluating FGPs dysplasia or application of FICE for the screening of FAP patients. In FAP cohort, the specific identification of who is at a greater risk of cancer development could be of paramount importance to assure a personalized program of surveillance or a therapeutic intervention. The primary aim of this study was to assess the capability of FICE in identifying gastric polyps with dysplastic or adenomatous tissue in comparison to traditional endoscopy and in identifying a greater number of duodenal adenomas with advanced histological features. Secondary aim was to assess the capability of FICE in identifying adenomas not seen on white light evaluation.

The primary aim of this study was to assess the capability of FICE in identifying gastric polyps with dysplastic or adenomatous tissue in comparison to traditional endoscopy and in identifying a greater number of duodenal adenomas with advanced histological features. Secondary aim was to assess the capability of FICE in identifying adenomas not seen on white light evaluation. MATERIALS AND METHODS Patients Seventy-six consecutive FAP patients, already treated by colectomy and members of sixty-five families, were enrolled. Exclusion criteria were: Uncorrectable coagulopathy, inability to give informed consent, age < 18 years, prior gastro-duodenal surgery or a personal history of gastro-duodenal cancer. All patients underwent a surveillance esophagogastroduodenoscopy (EGD) in deep sedation at the Gastroenterology U.O. of the Azienda Ospedaliero Universitaria di Careggi, Firenze, Italy. Written informed consent were obtained before EGD and sedation. Endoscopy A FICE system (EG-590ZW; Fujinon Corp, Saitama, Japan) for the upper gastro-intestinal tract with an electronic endoscope system (EPX-4400; Fujinon Corp, Saitama, Japan) was used for this study. In this system, ten channels with different predefined combinations of absorption wavelengths are available. We used channel 5, corresponding to R 500 nm, G 480 nm, B 420 nm, on the basis of previous studies.

nal tract with an electronic endoscope system (EPX-4400; Fujinon Corp, Saitama, Japan) was used for this study. In this system, ten channels with different predefined combinations of absorption wavelengths are available. We used channel 5, corresponding to R 500 nm, G 480 nm, B 420 nm, on the basis of previous studies. A standard duodenoscope was used for side-viewing examination. Because this model of duodenoscope does not support FICE system, ampullary and periampullary evaluations were not included in the analysis. All of the endoscopic procedures were performed by two experts examiners. “A” operator performed the exam on white light, while “B” operator used only FICE system for gastroduodenal visualization. Each EGD was divided into three phases. During the first phase, “A” operator observed stomach and duodenum by white light recording photographic images of suspected polyps and pointing them. We intended suspected polyps on white light those larger than 1 cm and those with irregular shape or surface features. During the second phase, “B” operator performed the exam using FICE and, like “A” operator, recorded photographic images of suspected polyps on the basis of Kudo classification[23] and capillary pattern, and pointed them. Kudo classification classifies mucosal crypt patterns into five types, with type I and II predicting non-adenomatous lesions and type III-V predicting adenomatous lesions.

During the second phase, “B” operator performed the exam using FICE and, like “A” operator, recorded photographic images of suspected polyps on the basis of Kudo classification[23] and capillary pattern, and pointed them. Kudo classification classifies mucosal crypt patterns into five types, with type I and II predicting non-adenomatous lesions and type III-V predicting adenomatous lesions. Hyperplastic polyps were suspected when the surface showed pale color with only minute thin superficial (couperose-like) vessels and round or asteroid pattern (type I and II). Adenomas were suspected in the presence of increased vascular density (darkening of the mucosal pattern or a fine meshwork of brownish/bluish vessels) and a typical tubular or gyrus-like pattern (type III-IV). Finally, type V have a non structural pattern which identifies high dysplastic or yet carcinomatous lesions. During this phase we intended suspected those lesions with a pit pattern type III-V and those with an increased capillary density. During the third phase, after the two endoscopists’ cross-evaluation, lesions which seemed suspected only by FICE, only by white light or by both methods were resected or biopsied according to Kudo class. Endoscopic resection was performed with diatermic loop for polyps ≥ 6 mm and with forceps for polyps < 6 mm. The size was estimated using on open biopsy forceps (8 mm) for comparison and recorded as smaller than 6 mm, 6 to 10 mm, 11 to 20 mm and greater than 20 mm.

During the third phase, after the two endoscopists’ cross-evaluation, lesions which seemed suspected only by FICE, only by white light or by both methods were resected or biopsied according to Kudo class. Endoscopic resection was performed with diatermic loop for polyps ≥ 6 mm and with forceps for polyps < 6 mm. The size was estimated using on open biopsy forceps (8 mm) for comparison and recorded as smaller than 6 mm, 6 to 10 mm, 11 to 20 mm and greater than 20 mm. The total number of FGPs was documented as below: 0 to 2 polyps, 3 to 20, 21 to 30 and more than 30 polyps. On the basis of location we identified: Fundus-corpus, antrum, duodenal bulb, II°/III° duodenal portion. For fundic polyps seen on white light, the number of FGPs from which a biopsy specimen was taken was based on the total number of FGPs present: 3 biopsies if 3-20 polyps, 5 biopsies if 21-30 polyps, 7 biopsies if > 30 polyps[9]. On FICE, only suspected polyps (Kudo III-V, high capillary density) were removed. For antral and bulbar polyps, all of them were removed or biopsied both on withe light than on FICE. In the second and third duodenal portion, on white light only suspected polyps were resected or biopsied, while on FICE were biopsied those with Kudo V and those with Kudo IV and high capillary density. Macroscopic classification of lesions followed the Paris classification[40] as polyp, superficially flat or depressed lesion, and lateral spreading tumor.

In the second and third duodenal portion, on white light only suspected polyps were resected or biopsied, while on FICE were biopsied those with Kudo V and those with Kudo IV and high capillary density. Macroscopic classification of lesions followed the Paris classification[40] as polyp, superficially flat or depressed lesion, and lateral spreading tumor. Histology All biopsy specimens, fixed in 10% neutral buffered formalin, were analyzed by two expert gastrointestinal pathologists blinded to size, location and number of FGPs. In the case of multiple lesions in the same patient, each lesion was identified and placed in different flasks. Lesions were histological classified in adenomatous, hyperplastic or inflammatory polyps, fundic gland polyps, and metaplastic areas. Adenomatous polyps were classified according to OMS classification: Tubular if holding more than 75% of tubular glands, villus if holding more than 75% of villus glands, tubulo-villus if not prevailing none of the two patterns. Dysplasia was classified according to Vienna criteria[41] in low grade if holding nuclear enlargement, stratification and hyperchromasia with overall preservation of nuclear polarity; high grade as above but with nuclear polarity loss and glandular crowding; indefinite for dysplasia if present mild nuclear enlargment and insufficient hypercromasia to be classified as dysplasia or if present a significant obscuring background inflammation.

hyperchromasia with overall preservation of nuclear polarity; high grade as above but with nuclear polarity loss and glandular crowding; indefinite for dysplasia if present mild nuclear enlargment and insufficient hypercromasia to be classified as dysplasia or if present a significant obscuring background inflammation. The stage of duodenal polyposis was graded according to Spigelman classification modified sec. Saurin[42], which take into account duodenal polyp number, size, histological type and grade of dysplasia. It was noted before and after FICE evaluation. Statistical analysis The diagnostic performances (sensitivities, specificities, positive and negative predictive values) of FICE and WL were determined by comparing the endoscopic diagnoses with the histo-pathological findings. To identify associations of demographic, clinical and endoscopic features with the presence of FGP dysplasia or with adenomas, the Fisher exact test was used to study univariable associations of categoric demographic and endoscopic factors with the presence of dysplasia or adenomatous tissue. The Student t test was used for continuous factors. A P value “two tailed” < 0.05 was considered statistical significant. The strength of association was calculated by odds ratio (OR). The statistical methods of this study were reviewed by S. Milani, University of Florence.

presence of dysplasia or adenomatous tissue. The Student t test was used for continuous factors. A P value “two tailed” < 0.05 was considered statistical significant. The strength of association was calculated by odds ratio (OR). The statistical methods of this study were reviewed by S. Milani, University of Florence. RESULTS Seventy-six consecutive FAP patients (41 male and 35 female; mean age 40.3 years old, range 24-64) underwent EGD. Among all patients, 69 (90.8%) had gastric polyps (34 only in the corpus-fundus, 7 only in the antrum and 28 in the whole stomach) and 52 (68.4%) in duodenum (7 in the bulb, 35 in second/third duodenal portion, 10 both in the bulb and the second portion of duodenum) (Table 2). Table 2 Stomach and duodenum polyps Patients Patients Fundus 34 (49.3%) Bulb 7 (13.5%) Antrum 7 (10.1%) Ii°/iii° portion 35 (67.3%) Fundus + antrum 28 (40.6%) Bulb+ii°/iii° 10 (19.2%) Total stomach 69 (100%) Total duodenum 52 (100%) Identification of polyps in the stomach Fundus: 62 patients had a widespread fundic polyposis (81.6%); 52 of them had more than 30 fundic polyps (68.5%), 3 between 21 and 30 (3.9%) and 7 between 5 and 20 (9.2%). On white light visualization, 397 polyps in 62 patients (6.4 polyps per patient) were removed. Three were hyperplastic polyps, 7 inflammatory while the rest were cystic fundic gland polyps. No polyp harboured dysplasia nor adenomatous foci (specificity 100%, sensitivity NV, positive predictive value NV, negative predictive value 100%, 95%CI) (Table 3). Table 3 Features of fundic polyps identified by white light endoscopy

On white light visualization, 397 polyps in 62 patients (6.4 polyps per patient) were removed. Three were hyperplastic polyps, 7 inflammatory while the rest were cystic fundic gland polyps. No polyp harboured dysplasia nor adenomatous foci (specificity 100%, sensitivity NV, positive predictive value NV, negative predictive value 100%, 95%CI) (Table 3). Table 3 Features of fundic polyps identified by white light endoscopy P1-P3 P4-P10 P11-P24 P25-P55 P56-P397 Kudo I II II II I Size (mm) 5 5 6-10 5 5 Paris CL Is Is Is Is Is Histology HYP IN FGP FGP FGP IN: Inflammatory; FGP: Fundic gland polyp; IP: Hyperplastic. After FICE evaluation, 10 polyps were removed from 10 patients on the basis of suspicious features of dysplasia or adenomatous tissue. All of them were cystic fundic gland polyps, none of them harboring dysplasia or adenomatous foci (specificity 97%; sensitivity NV; positive predictive value 0%; negative predictive value 100%) (Table 4). Table 4 Features of fundic polyps identified by fujinon intelligent color enhancement P1-P6 P7-P10 Kudo IIIS IIIL Size (mm) 5 5 Paris CL Is Is Histology FGP FGP FGP: Fundic gland polyp. Thirty-eight patients with fundic polyposis had also duodenal polyposis (61.2%), while among the 14 patients without fundic polyps, 10 had polyps in the duodenum (71.4%). Thus the presence of fundic polyps doesn’t correlate with a higher risk to develop duodenal polyps (P = 0.55; OR = 0.6).

P1-P6 P7-P10 Kudo IIIS IIIL Size (mm) 5 5 Paris CL Is Is Histology FGP FGP FGP: Fundic gland polyp. Thirty-eight patients with fundic polyposis had also duodenal polyposis (61.2%), while among the 14 patients without fundic polyps, 10 had polyps in the duodenum (71.4%). Thus the presence of fundic polyps doesn’t correlate with a higher risk to develop duodenal polyps (P = 0.55; OR = 0.6). Antrum: A total of 56 polyps were identified and removed in the antrum of 35 patients (average 1.6 polyps per patient). Twenty-four polyps were identified in 35 patients by white light endoscopy (0.7 polyps per patient); 21 of them were tubular adenomas with low grade dysplasia while 3 were inflammatory polyps (specificity 88.0%; sensitivity 67.7%; positive predictive value 87.5%; negative predictive value 68.7%) (Table 5). Table 5 Features of antral polyps identified by white light endoscopy P1-P3 P4 P5-P10 P11-P17 P18-P24 Kudo II IIIS IIIS IIIS IV Size (mm) 6-10 6-10 5 5 5 Paris CL IIa IIa + IIc Is Is Is Histology IN TA, LGD TA, LGD TA, LGD TA, LGD Spigelman 0 0 I° 0 0 IN: Inflammatory; TA: Tubular adenoma; LGD: Low grade dysplasia. Beside polyps seen with conventional endoscopy, FICE was further able to identify 32 polyps in 28 patients. They were 7 tubular adenomas with low grade dysplasia, 14 inflammatory polyps, 3 areas with low grade dysplasia in the context of flogistic mucosa, 8 metaplasic areas (specificity 12.0%; sensitivity 100%; positive predictive value 58.5%; negative predictive value 100%) (Table 6). Table 6 Features of antral polyps identified by fujinon intelligent color enhancement

Beside polyps seen with conventional endoscopy, FICE was further able to identify 32 polyps in 28 patients. They were 7 tubular adenomas with low grade dysplasia, 14 inflammatory polyps, 3 areas with low grade dysplasia in the context of flogistic mucosa, 8 metaplasic areas (specificity 12.0%; sensitivity 100%; positive predictive value 58.5%; negative predictive value 100%) (Table 6). Table 6 Features of antral polyps identified by fujinon intelligent color enhancement P25 P26 P27 P28-P30 P31 P32-P33 P34-P35 P36-P38 P39-P44 P45-P49 P50-P53 P54-P56 Kudo V V IIIS IIIL IIIL IV IV IIIL IIIL IIIS IIIS V Size (mm) 5 5 5 6-10 5 5 6-10 5 5 5 6-10 5 Paris CL IIb IIa IIb IIb IIa IIb IIa IIa IIb IIb Is IIb Histology IN LGD IN LGD IN LGD MET MET MET MET IN IN IN TA LGD TA LGD Spigelman III° II° I° II° II° 0 II° 0 0 0 I° II° IN: Inflammatory; TA: Tubular adenoma; LGD: Low grade dysplasia; MET: Metaplasia. FICE identified a higher number of polyps than traditional endoscopy (56 vs 24; P < 0.0001), showing a better, but not statistically significant, tendency to identify adenomas and displastic areas (31 vs 21; P = 0.0857). All but 4 polyps missed out by white light, were flat. Eighteen of patients with antral lesions (51.4%) had polyps also in the duodenum. There is no relationship between presence of dysplasia in antral stomach and Spigelman advanced stages (P = 1; OR = NV).

FICE identified a higher number of polyps than traditional endoscopy (56 vs 24; P < 0.0001), showing a better, but not statistically significant, tendency to identify adenomas and displastic areas (31 vs 21; P = 0.0857). All but 4 polyps missed out by white light, were flat. Eighteen of patients with antral lesions (51.4%) had polyps also in the duodenum. There is no relationship between presence of dysplasia in antral stomach and Spigelman advanced stages (P = 1; OR = NV). Identification of duodenal polyps Bulb: 21 polyps were seen in 17 patients (1.2 polyps per patient). All of them were endoscopically removed. White light endoscopy identified 14 polyps in 12 patients; 8 polyps were inflammatory, while 6 were tubular adenomas with low grade dysplasia (specificity 0%; sensitivity 46.2%; positive predictive value 42.9%; negative predictive value 0%) (Table 7). Table 7 Features of bulbal polyps identified by white light endoscopy P1-P5 P6-P8 P9 P10-P12 P13 P14 Kudo II IIIS IIIS IIIL IV IV Size (mm) 5 6-10 6-10 5 6-10 6-10 Paris CL Is Is Is IIa Is Is Histology IN TA LGD TA LGD IN TA LGD TA LGD Spigelman 0 II° I° 0 III° II° TA: Tubular adenoma; LGD: Low grade dysplasia. During FICE evaluation, beside polyps seen with conventional endoscopy, 7 more polyps in 7 patients, five of them new, were discovered. All of them were tubular adenomas with low grade dysplasia (specificity 62.5%; sensitivity 100%; positive predictive value 81.3%; negative predictive value 100%) (Table 8). Table 8 Features of bulbal polyps identified by fujinon intelligent color enhancement

During FICE evaluation, beside polyps seen with conventional endoscopy, 7 more polyps in 7 patients, five of them new, were discovered. All of them were tubular adenomas with low grade dysplasia (specificity 62.5%; sensitivity 100%; positive predictive value 81.3%; negative predictive value 100%) (Table 8). Table 8 Features of bulbal polyps identified by fujinon intelligent color enhancement P15 P16-P17 P18 P19 P20-P21 Kudo IIIS IV IIIS IIIL IIIS Size (mm) 5 6-10 6-10 5 5 Paris CL IIa IIb IIa IIb IIb Histology TA LGD TA LGD TA LGD TA LGD TA LGD Spigelman I° II° II° I° I° TA: Tubular adenoma; LGD: Low grade dysplasia. FICE was able to see further 7 polyps than traditional endoscopy, and it was able to identify a quite significant higher number of polyp in the duodenal bulb (21 vs 14; P = 0.069). FICE added a statistical significant advantage in identifying adenomas (13 vs 6; P = 0.03). All FICE identified polyps were flat lesions. All patients with bulbal polyps had also lesions in the gastric fundus and no adenoma in the antrum. All patients with bulbal adenomas had polyps in the second/third portion of duodenum, while patients with inflammatory polyps had a Spigelman’s stage 0.

FICE was able to see further 7 polyps than traditional endoscopy, and it was able to identify a quite significant higher number of polyp in the duodenal bulb (21 vs 14; P = 0.069). FICE added a statistical significant advantage in identifying adenomas (13 vs 6; P = 0.03). All FICE identified polyps were flat lesions. All patients with bulbal polyps had also lesions in the gastric fundus and no adenoma in the antrum. All patients with bulbal adenomas had polyps in the second/third portion of duodenum, while patients with inflammatory polyps had a Spigelman’s stage 0. II°/III° duodenal portion: Totally, 391 polyps in 45 patients (8.7 polyps per patient) were identified. Of them, 105 were removed or biopsied (26.5%). Conventional endoscopy identify 324 polyps in 45 patients (7.2 polyps per patient). Of them, 94 were removed or biopsied (2.1 polyps per patient) and they resulted: 80 tubular adenomas with low grade dysplasia, 10 inflammatory polyps and 4 tubulo-villous adenomas with low grade dysplasia. No case of high grade dysplasia (3 suspected). (Table 9). FICE identified further 67 polyps in 35 patients and 11 were removed or biopsied in 11 subjects. All of them were tubular adenomas with low grade dysplasia. No case of high grade dysplasia (Table 10). FICE was able to identify a higher number of polyps than traditional endoscopy (8.7 vs 7.2; P < 0.001). All polyps not seen on white light were flat lesions. Table 9 Features of duodenal polyps identified by white light endoscopy

II°/III° duodenal portion: Totally, 391 polyps in 45 patients (8.7 polyps per patient) were identified. Of them, 105 were removed or biopsied (26.5%). Conventional endoscopy identify 324 polyps in 45 patients (7.2 polyps per patient). Of them, 94 were removed or biopsied (2.1 polyps per patient) and they resulted: 80 tubular adenomas with low grade dysplasia, 10 inflammatory polyps and 4 tubulo-villous adenomas with low grade dysplasia. No case of high grade dysplasia (3 suspected). (Table 9). FICE identified further 67 polyps in 35 patients and 11 were removed or biopsied in 11 subjects. All of them were tubular adenomas with low grade dysplasia. No case of high grade dysplasia (Table 10). FICE was able to identify a higher number of polyps than traditional endoscopy (8.7 vs 7.2; P < 0.001). All polyps not seen on white light were flat lesions. Table 9 Features of duodenal polyps identified by white light endoscopy Kudo Size (mm) Paris CL Histology Spigelman P1-P4 IV 11-20 IIa TA LGD III° P5-P7 V 6-10 IIa TA LGD II° P8-P11 IV 5 IIb TA LGD II° P12-P16 IIIS 6-10 IIa TVA LGD III° P17-P27 IIIS 5 IIa TA LGD II° P28-P34 IIIS 5 IIa TA LGD I° P35-P40 IIIS 5 IIb TA LGD II° P41-P43 IIIS 5 Is TA LGD II° P44-P47 IIIS 5 Is TA LGD I° P48-P50 IIIS 11-20 IIa TA LGD II° P51-P58 IIIL 5 IIb TA LGD II° P59-P65 IIIL 5 IIa TA LGD II° P66-P68 IIIL 6-10 IIa TA LGD II° P69-P72 IIIL 6-10 IIa TA LGD III° P73-P79 IIIL 6-10 IIb TA LGD II° P80-P84 II 5 IIa TA LGD III° P85-P91 IIIL 5 IIb IN II° P92-P94 IIIS 5 IIa IN I° IN: Inflammatory; TA: Tubular adenoma; LGD: Low grade dysplasia.

IIa TA LGD II° P51-P58 IIIL 5 IIb TA LGD II° P59-P65 IIIL 5 IIa TA LGD II° P66-P68 IIIL 6-10 IIa TA LGD II° P69-P72 IIIL 6-10 IIa TA LGD III° P73-P79 IIIL 6-10 IIb TA LGD II° P80-P84 II 5 IIa TA LGD III° P85-P91 IIIL 5 IIb IN II° P92-P94 IIIS 5 IIa IN I° IN: Inflammatory; TA: Tubular adenoma; LGD: Low grade dysplasia. Table 10 Features of duodenal polyps identified by fujinon intelligent color enhancement P95-P97 P98-P100 P101-P102 P103-P105 Kudo IV IV V IV Size (mm) 5 6-10 6-10 5 Paris CL IIa IIb IIb IIb Histology TA LGD TA LGD TA LGD TA LGD Spigelman I° II° II° II° TA: Tubular adenoma; LGD: Low grade dysplasia. Thirty-five of patients with duodenal polyposis had also polyps in the fundus, 4 had adenomas and 2 dysplastic areas in the antrum, thus FICE didn’t change any Spigelman stage just defined with conventional endoscopy. DISCUSSION Duodenal adenomatous polyps are common manifestations of FAP found in 30% to 90% of patients, with a life time risk approaching 100%[5,6,43]. While rare in the general population (0.01%-0.04% of incidence at an average age of 65 years)[43], the risk of duodenal or periampullary cancer is increased several hundreds fold in FAP patients (estimated cumulative risk of 4.5% by age 57 and a median age at presentation of 52 years)[6,8]. Duodenal cancer is the second most common cause of disease-related mortality in patients with FAP, only the second to advanced and metastatic colorectal cancer. A regular and careful program of endoscopic surveillance is worthwhile in identifying early pre-malignant lesions.

edian age at presentation of 52 years)[6,8]. Duodenal cancer is the second most common cause of disease-related mortality in patients with FAP, only the second to advanced and metastatic colorectal cancer. A regular and careful program of endoscopic surveillance is worthwhile in identifying early pre-malignant lesions. Gastric polyps, particularly fundic polyps, are considered always non-neoplastic lesions, also in FAP and non-FAP patients; nonetheless high rate of their prevalence (20%-88%)[6,9-11] and several cases of dysplasia in FGPs in FAP have been recently reported, with rate of incidence up to 54%[9-11,18]. Chromoendoscopy, both traditional and virtual, has been proven to be a good tool to increase polyps identification rate and to predict their histology[29-32]. Only one study was published on the use of FICE in the evaluation of duodenal lesions[44]. This study was conducted using a double balloon enteroscopy on patients with duodenal lesions. In this study only two FAP patients were included and FICE enhanced mucosal pattern of these polyps, and it correlated with the increase of detection of more lesions. However, neither previous studies using traditional chromoendoscopy nor FICE, were conducted in evaluation of gastric polyps in FAP patients. To the best of our knowledge, our study is the first that has assessed the role of FICE in FGPs dysplasia identification and in the gastroduodenal polyps characterization in FAP subjects.

neither previous studies using traditional chromoendoscopy nor FICE, were conducted in evaluation of gastric polyps in FAP patients. To the best of our knowledge, our study is the first that has assessed the role of FICE in FGPs dysplasia identification and in the gastroduodenal polyps characterization in FAP subjects. In agreement with the literature’s data, the prevalence of gastric polyps was relatively elevated (90.8%), while duodenal polyps were diagnosed in 68.4% of patients, slightly lower than the reported literature value.

neither previous studies using traditional chromoendoscopy nor FICE, were conducted in evaluation of gastric polyps in FAP patients. To the best of our knowledge, our study is the first that has assessed the role of FICE in FGPs dysplasia identification and in the gastroduodenal polyps characterization in FAP subjects. In agreement with the literature’s data, the prevalence of gastric polyps was relatively elevated (90.8%), while duodenal polyps were diagnosed in 68.4% of patients, slightly lower than the reported literature value. In the majority of FAP subjects (62/76; 83.3%), gastric polyps were so numerous that they carpeted the fundic mucosa, making difficult identifying dysplasia by random biopsies on the basis of the total number of polyps, as indicated in a recent study conducted by Bianchi et al[9]. Consequently, having an endoscopic technique able to target fundic biopsies is important to overcome this issue. Moreover, Bianchi et al[9] reported a prevalence of dysplasia in fundic polyps of 42%, while we have found only fundic gland polyps without displastic or adenomatous areas, although we have followed their sampling method. A possible explanation to this marked mismatch, could lie in the size of the polyps removed: we did found only subcentimetric polyps, while Bianchi et al[9] have demonstrated that the risk of dysplasia correlated with polyp size. No polyps removed had suspected superficial features according to Kudo classification, while Bianchi et al[9] did not adopted any classification to describe mucosal and vascular pattern; consequently we don’t know if their removed polyps had or not a suspected pattern.

the risk of dysplasia correlated with polyp size. No polyps removed had suspected superficial features according to Kudo classification, while Bianchi et al[9] did not adopted any classification to describe mucosal and vascular pattern; consequently we don’t know if their removed polyps had or not a suspected pattern. FICE pointed our attention on 10 fundic polyps, that seemed suspected for harboring adenomatous tissue; however histological results did not confirmed this suspect and all of polyps resulted fundic gland polyps. In this case, FICE has not increased the identification rate of dysplasia or adenomatous tissue in fundic polyps. Prevalence of patients with antral adenomas was about 21.1% (16/76), more than reported in the Western World data, but consistent with Japanese findings. The use of FICE could explain our result, since it has increased the identification rate of antral adenomas compared to white light, with a difference near to statistical significance (P = 0.0857). The very low specificity of the method (12.0%) could be explained by the presence of phlogosis (in fact almost all false positive harbored flogistic areas) able to distort the mucosal and vascular pattern, specifically enhanced by virtual chromoendoscopy.

Prevalence of patients with antral adenomas was about 21.1% (16/76), more than reported in the Western World data, but consistent with Japanese findings. The use of FICE could explain our result, since it has increased the identification rate of antral adenomas compared to white light, with a difference near to statistical significance (P = 0.0857). The very low specificity of the method (12.0%) could be explained by the presence of phlogosis (in fact almost all false positive harbored flogistic areas) able to distort the mucosal and vascular pattern, specifically enhanced by virtual chromoendoscopy. Therefore, FICE allows to identify a greater number of adenomas to the detriment of a greater number of biopsies. Anyway this approach didn’t determine a different timing in the surveillance program, but changed the attention on the antral evaluation during the following endoscopies. In duodenal bulb FICE was able to identify more adenomas than traditional endoscopy (P = 0.03). Furthermore, all patients with FICE-identified adenomas had polyps in the duodenum too, thus the identification of bulbar adenomas didn’t modify surveillance timing. Taking into account also bulbar polyps, duodenal adenomas prevalence in FAP patients was 68.4%, with low Spigelman stages (9.2% stage III e 0% stage IV). In duodenum, FICE has allowed to see a greater number of adenomas than white light (P < 0.001), without no modifications of Spigelman stage neither identification of high grade dysplasia.

lso bulbar polyps, duodenal adenomas prevalence in FAP patients was 68.4%, with low Spigelman stages (9.2% stage III e 0% stage IV). In duodenum, FICE has allowed to see a greater number of adenomas than white light (P < 0.001), without no modifications of Spigelman stage neither identification of high grade dysplasia. Among FICE identified polyps, 4 lesions were suspected for high grade dysplasia, but three were inflammatory polyps at histopathological examination and one was a tubular adenoma with low grade of dysplasia. Other 7 polyps (Kudo IV) had an increased capillary density but they were tubular adenomas with low grade of dysplasia. Finally, in duodenum, FICE increased the polyps detection rate but didn’t change any Spigelman stage determined with conventional endoscopy. These data are in agreement with the little size and the absence of high grade dysplasia. Moreover this method wasn’t able to modify FAP patients’ prognosis, polyps’ surveillance program and their therapeutic management. We did not find any relationship between the presence of gastric polyps, duodenal polyposis and high Spigelman stage (P = 1).

t with the little size and the absence of high grade dysplasia. Moreover this method wasn’t able to modify FAP patients’ prognosis, polyps’ surveillance program and their therapeutic management. We did not find any relationship between the presence of gastric polyps, duodenal polyposis and high Spigelman stage (P = 1). Adenomas were 435 and 81 of them were diagnosed only by FICE that was able to identify a significative higher number of adenomas (P = 0.0062). Overall, FICE has specificity, sensitivity,positive and negative predictive values higher than traditional endoscopy referring to adenomas (96.0% vs 7.1%; 98.8% vs 80.2%; 90.3% vs 44.9%; 98.8% vs 27.6%, respectively; P < 0.0001). Conversely, it wasn’t possible to correlate for high grade dysplasia due the absence of dysplastic lesions according to the histopathological examination. The FICE identified lesions (106/468; 22.6%) were mostly flat (67.9%; P = 0.029) and small (all below 1 cm). According to already published data, FICE was particularly able to identify polyps with these features. It isn’t clear if this ability might have clinical and procedural consequences. In summary, in our study, FICE, like traditional endoscopy, could not identify any adenoma at risk of malignant transformation probably as a consequence of patients features (e.g., favorable genotype, recent EGD).

The FICE identified lesions (106/468; 22.6%) were mostly flat (67.9%; P = 0.029) and small (all below 1 cm). According to already published data, FICE was particularly able to identify polyps with these features. It isn’t clear if this ability might have clinical and procedural consequences. In summary, in our study, FICE, like traditional endoscopy, could not identify any adenoma at risk of malignant transformation probably as a consequence of patients features (e.g., favorable genotype, recent EGD). Nonetheless FICE significantly increases adenoma detection rate (P = 0.0062) but does not change any Spigelman stage and thus does not modify patient’s prognosis, surveillance program and treatment strategies. Probably a careful patient selection, an accurate histological examination, a concomitant use of lateral viewing endoscope, could make FICE gain that role who everybody expects in FAP patient.

oes not change any Spigelman stage and thus does not modify patient’s prognosis, surveillance program and treatment strategies. Probably a careful patient selection, an accurate histological examination, a concomitant use of lateral viewing endoscope, could make FICE gain that role who everybody expects in FAP patient. COMMENTS Background Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome characterized by the development of colorectal cancer by the age of 40 years in nearly 100% of individuals. The use of colon endoscopic surveillance and prophylactic colectomy have strongly reduced mortality in FAP patients leading to the introduction of surveillance strategies for the prevention of other extracolonic malignancies (e.g., in the duodenum and in the stomach). Duodenal adenomatous polyps are common manifestations of FAP found in 30% to 90% of patients. Duodenal cancer is the second most common cause of disease-related mortality in patients with FAP, only the second to advanced and metastatic colorectal cancer. Gastric polyps, particularly fundic polyps, are considered always non-neoplastic lesions, also in FAP and non-FAP patients; nonetheless high rate of their prevalence (20%-88%) and several cases of dysplasia in FGPs in FAP have been recently reported, with rate of incidence up to 54%. Research frontiers The observation of the pit and capillary patterns of the mucosal glands and the mucosa, respectively, by chromoendoscopy might predict the histology of mucosal lesions.

COMMENTS Background Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome characterized by the development of colorectal cancer by the age of 40 years in nearly 100% of individuals. The use of colon endoscopic surveillance and prophylactic colectomy have strongly reduced mortality in FAP patients leading to the introduction of surveillance strategies for the prevention of other extracolonic malignancies (e.g., in the duodenum and in the stomach). Duodenal adenomatous polyps are common manifestations of FAP found in 30% to 90% of patients. Duodenal cancer is the second most common cause of disease-related mortality in patients with FAP, only the second to advanced and metastatic colorectal cancer. Gastric polyps, particularly fundic polyps, are considered always non-neoplastic lesions, also in FAP and non-FAP patients; nonetheless high rate of their prevalence (20%-88%) and several cases of dysplasia in FGPs in FAP have been recently reported, with rate of incidence up to 54%. Research frontiers The observation of the pit and capillary patterns of the mucosal glands and the mucosa, respectively, by chromoendoscopy might predict the histology of mucosal lesions. Innovations and breakthroughs Chromoendoscopy is a staining method that uses biocompatible dye agents which accumulate within crypt orifices during endoscopy. Chromoendoscopy has difficulty in achieving complete and even coating of the mucosal surface with the dye, requires the extra cost for the of the dye spraying equipments and extra time to perform the procedure. Fujinon Intelligent Color Enhancement (FICE™, Fujinon Corp, Saitama, Japan) is a spectral estimation technique based on arithmetically processing of a white-light image captured by a video endoscope and sent to the spectral-estimation matrix-processing circuit. Preliminary studies suggested that FICE successfully achieves enhancements of real-time observations of mucosal and microvascular patterns and may discriminate between adenomatous and non-adenomatous polyps and it may identify the presence of dysplasia. In the study, FICE, like traditional endoscopy, could not identify any adenoma at risk of malignant transformation probably as a consequence of patients features. However FICE significantly increases adenoma detection without changing patient’s prognosis, surveillance program and treatment strategies. Probably a careful patient selection, an accurate histological examination, a concomitant use of lateral viewing endoscope, could make FICE gain that role who everybody expects in FAP patient.

significantly increases adenoma detection without changing patient’s prognosis, surveillance program and treatment strategies. Probably a careful patient selection, an accurate histological examination, a concomitant use of lateral viewing endoscope, could make FICE gain that role who everybody expects in FAP patient. Applications The timing of endoscopic and histology surveillance is currently a great challenge. Spectral estimation by fujinon intelligent color enhancement (FICE) may identify dysplasia and discriminate between adenomatous and non-adenomatous polyps. Terminology FAP is an autosomal dominant inherited syndrome who invariably develops to colorectal cancer by the age of 40 years in nearly 100% of individuals. Several endoscopic imaging techniques have been proposed to enhance the detail of these patterns. Among these, chromoendoscopy is a staining method applied in endoscopy that uses biocompatible dye agents which accumulate within crypt orifices. FICE is a modern endoscopic spectral estimation technique that successfully enhances the observation of mucosal and micro-vascular patterns. Peer-review The presented results, obtained with 76 FAP patients, indicate that FICE assay offers considerable advantage over traditional chromoendoscopy to discriminate between adenomatous and non-adenomatous polyps. The authors, however, caution that the application of FICE to FAP patients while helpful in prediction the histology of the mucosal lesion and significantly increases the detection of adenomas, do not change the prognosis and treatment.

nal chromoendoscopy to discriminate between adenomatous and non-adenomatous polyps. The authors, however, caution that the application of FICE to FAP patients while helpful in prediction the histology of the mucosal lesion and significantly increases the detection of adenomas, do not change the prognosis and treatment. Institutional review board statement: The study was reviewed and approved by the AOU Careggi Institutional Review Board. Informed consent statement: All study participant, or their legal guardian, provided informed written consent prior to study enrollment. Conflict-of-interest statement: The authors have nothing to disclose. Data sharing statement: No additional data are available. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Italy Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: August 27, 2016 First decision: November 19, 2016 Article in press: February 26, 2017 P- Reviewer: Caboclo JLF, Slomiany BL S- Editor: Kong JX L- Editor: A E- Editor: Lu YJ

Core tip: Watch and wait (W and W) is a term coined to indicate observation without therapy assessing the evolution of the tumor. Given that neuroendocrine tumors sometimes are radiologically stable over months since they tend to grow slowly observation has been reported as an option to be considered in several guidelines and recommendations. However it has neither validated nor specifically investigated so far. Therefore its application in clinical practice is arbitrary and it differs in terms of tumor status assessment, type and timing of imaging or other exams utilized. While undertaking W and W to delay the first-line therapy by some weeks may be justified in good performance asymptomatic patients with low-grade neuroendocrine neoplasms (NENs) in order to usefully characterize the disease and patient and thereby choose the best therapy and therapeutic strategy, it seems to be far more difficult to justify W and W with the intent of avoiding an anti-tumor treatment. It should be considered that not only do NENs tend to grow even when they have very favorable biological characteristics but also that the alternative to W and W is most commonly a low toxic and effective treatment with somatostatin analogs. INTRODUCTION Neuroendocrine neoplasms (NENs) represent a group of rare and heterogeneous malignancies, which can develop in various organ. They are classified on the basis of their level of aggressiveness into low, intermediate and high grades of malignancy.

Core tip: Watch and wait (W and W) is a term coined to indicate observation without therapy assessing the evolution of the tumor. Given that neuroendocrine tumors sometimes are radiologically stable over months since they tend to grow slowly observation has been reported as an option to be considered in several guidelines and recommendations. However it has neither validated nor specifically investigated so far. Therefore its application in clinical practice is arbitrary and it differs in terms of tumor status assessment, type and timing of imaging or other exams utilized. While undertaking W and W to delay the first-line therapy by some weeks may be justified in good performance asymptomatic patients with low-grade neuroendocrine neoplasms (NENs) in order to usefully characterize the disease and patient and thereby choose the best therapy and therapeutic strategy, it seems to be far more difficult to justify W and W with the intent of avoiding an anti-tumor treatment. It should be considered that not only do NENs tend to grow even when they have very favorable biological characteristics but also that the alternative to W and W is most commonly a low toxic and effective treatment with somatostatin analogs. INTRODUCTION Neuroendocrine neoplasms (NENs) represent a group of rare and heterogeneous malignancies, which can develop in various organ. They are classified on the basis of their level of aggressiveness into low, intermediate and high grades of malignancy. Neuroendocrine neoplasms from the digestive tract, are classified on the basis of proliferation index as G1 (≤ 2% Ki-67), G2 (3%-20% Ki-67) and G3 (> 20% Ki-67). Furthermore, based on their morphology they are named “tumors” (NETs) when they are well differentiated, whereas “carcinomas” (NECs) when they are poorly differentiated"[1]. Neuroendocrine neoplasms from the thoracic region are classified into typical carcinoid, TC (< 2 mitoses/2 mm2 with absence of necrosis), atypical carcinoid, AC (2-10 mitoses/2 mm2 with necrosis), large cell neuroendocrine carcinoma, LCNEC (> 10 mitoses with extensive necrosis) and small cell lung cancer, SCLC (> 10 mitoses with extensive necrosis)[2].

thoracic region are classified into typical carcinoid, TC (< 2 mitoses/2 mm2 with absence of necrosis), atypical carcinoid, AC (2-10 mitoses/2 mm2 with necrosis), large cell neuroendocrine carcinoma, LCNEC (> 10 mitoses with extensive necrosis) and small cell lung cancer, SCLC (> 10 mitoses with extensive necrosis)[2]. While high-grade NENs are treated with chemotherapy in the vast majority of cases when they are in advanced stage of disease, the therapeutic approach to advanced low-intermediate grade NENs varies. Somatostatin analogs (SSA), interferon (IFN), molecular targeted agents (MTAs), chemotherapy, peptide receptor radionuclide therapy (PRRT), and liver-directed treatments (LDTs), are all potentially effective therapies to propose, often in the same clinical setting. Although some of these therapies have been approved on the basis of positive regulatory phase III trials[3-7] in specific settings and several guidelines about NENs do exist[8,9], no sequencing or priority criteria about the different therapies have been validated. Furthermore, the clinical course of NETs is quite heterogeneous, with different spontaneous growth rates after diagnosis, and different degrees of sensitivity to the same therapy even when they have similar characteristics.

xist[8,9], no sequencing or priority criteria about the different therapies have been validated. Furthermore, the clinical course of NETs is quite heterogeneous, with different spontaneous growth rates after diagnosis, and different degrees of sensitivity to the same therapy even when they have similar characteristics. “Watch and wait (W and W)”, “watchful waiting”, “wait and see”, “observation” and “active surveillance” are all terms which are used to describe assessing the evolution of the tumor without an anti-tumor therapy. These terms have been applied synonymously to NETs as in rare cases they have a spontaneous very indolent clinical course. Sometimes they are also applied to a localized disease, as in the case of so-called pancreatic “incidentaloma”, namely a < 2 cm isolated nodule in the pancreas. European Neuroendocrine Tumor Society (ENETS) 2016 guidelines recommend W and W for a < 2 cm pancreatic NET, “G1 or low G2, asymptomatic, mainly in the head, with no radiological signs suspicious for malignancy”, and suggest that one also consider the patient’s attitude, age and comorbidity. It is specified that the follow-up should be performed with endoscopic ultrasound (EUS), magnetic resonance imaging (MRI) (or computed tomography, CT) “every 6 to 12 mo”. However, the length of follow-up is not specified[10].

r malignancy”, and suggest that one also consider the patient’s attitude, age and comorbidity. It is specified that the follow-up should be performed with endoscopic ultrasound (EUS), magnetic resonance imaging (MRI) (or computed tomography, CT) “every 6 to 12 mo”. However, the length of follow-up is not specified[10]. In the ENETS guidelines W and W is also recommended for advanced disease, for instance in NETs from the midgut when they are “non-functional, G1, low tumor burden, no symptoms, stable disease”. This policy is advised even for pancreatic NETs, when they are “non-functional, G1, ≤ 10% Ki-67, low tumor burden, stable disease or initial diagnosis, no symptoms”[11].

ommended for advanced disease, for instance in NETs from the midgut when they are “non-functional, G1, low tumor burden, no symptoms, stable disease”. This policy is advised even for pancreatic NETs, when they are “non-functional, G1, ≤ 10% Ki-67, low tumor burden, stable disease or initial diagnosis, no symptoms”[11]. In both midgut and pancreatic NETs the W and W policy is a possible alternative to SSA. However, SSA compared with placebo resulted effective in two phase III randomised controlled trials, with octreotide long-acting repeatable (LAR) producing a longer time to progression (TTP) in midgut NETs in the PROMID trial and lanreotide autogel significantly prolonging progression free survival (PFS) in enteropancreatic NETs in the CLARINET trial, respectively[5,6]. Notably, time to progression (TTP) was quite short in the placebo arm of the PROMID trial demonstrating that also NETs with < 3% Ki-67, as were the vast majority of the tumors included in the PROMID, will progress eventually. Interestingly, NETs included in the CLARINET trial, which resulted as having a stable disease in 96% of cases in accordance with RECIST criteria, in fact were progressing at baseline, as showed with the so-called tumor growth rate (TGR)[12].

vast majority of the tumors included in the PROMID, will progress eventually. Interestingly, NETs included in the CLARINET trial, which resulted as having a stable disease in 96% of cases in accordance with RECIST criteria, in fact were progressing at baseline, as showed with the so-called tumor growth rate (TGR)[12]. Another report indicating that NETs tend to grow early spontaneously, is a retrospective analysis of more than 200 patients with advanced pancreatic NETs showing that those patients who did not receive antitumor treatment during follow-up had a significantly shorter PFS compared to treated patients, thus confirming that anti-tumor therapy can favorably impact on the clinical course of the disease[13]. In the ENETS 2016 guidelines it is not specified whether radiological or functional imaging or both are recommended to monitor the tumor status of a low-grade NET; it is not clear whether some biochemical tests, such as chromogranin-A, should be performed periodically; timing of follow-up imaging is not specified. Furthermore no data exist about the impact of the W and W on the patient’s quality of life and costs.

In the ENETS 2016 guidelines it is not specified whether radiological or functional imaging or both are recommended to monitor the tumor status of a low-grade NET; it is not clear whether some biochemical tests, such as chromogranin-A, should be performed periodically; timing of follow-up imaging is not specified. Furthermore no data exist about the impact of the W and W on the patient’s quality of life and costs. The W and W policy is debated also in other fields of oncology. For instance in renal cancer it was investigated in a phase II trial including medical anti-tumor treatment-naive patients with advanced disease[14]. The decision to choose W and W over immediate systemic therapy was made jointly by the patient and treating physician. Therefore patients underwent homogeneous radiological and clinical follow-up and also filled in quality of life questionnaires. Median time to radiological progression, RECIST-based, was 9.4 mo (95%CI: 7.4-13.4); at progression, patients received a first-line systemic therapy; no observed adverse effects on quality of life, anxiety and depression, were recorded during the observation period. Although this study seems to indicate that in some selected patients with metastatic renal carcinoma, active surveillance might be a good approach, homogeneous criteria for selection of patients to undergo W and W, type of follow-up and timing of first-line therapy remain debatable.

orded during the observation period. Although this study seems to indicate that in some selected patients with metastatic renal carcinoma, active surveillance might be a good approach, homogeneous criteria for selection of patients to undergo W and W, type of follow-up and timing of first-line therapy remain debatable. Further while in renal cancer one of the reasons for performing W and W instead of administering treatment to patients is to avoid therapies which may well be highly toxic, in NETs the choice is almost always between W and W and SSAs, which are a very low-toxic therapy.

orded during the observation period. Although this study seems to indicate that in some selected patients with metastatic renal carcinoma, active surveillance might be a good approach, homogeneous criteria for selection of patients to undergo W and W, type of follow-up and timing of first-line therapy remain debatable. Further while in renal cancer one of the reasons for performing W and W instead of administering treatment to patients is to avoid therapies which may well be highly toxic, in NETs the choice is almost always between W and W and SSAs, which are a very low-toxic therapy. Finally, in good-performance status asymptomatic patients with advanced NETs, the diagnostic work-up, morphological and functional staging and characterization of the disease require some weeks. Luckily in most cases this time without therapy is not detrimental for the patient and it allows an assessment to be made of clinical behavior and tumor growth, a thorough understanding of tumor and patient characteristics, and the discussion of the global therapeutic strategy within a dedicated multidisciplinary team. All of this may be very helpful to patients when compared with starting a single first-line therapy right from the time of diagnosis of an advanced NET. Proposing a W and W policy after completing this initial period of observation to a patient with a metastatic NET means waiting for a tumor growth or a clinical progression. On the one hand it is arbitrary to define whether morphological (radiological), functional (receptorial? metabolic?) or biochemical progression should be considered and with which threshold; on the other hand it could be detrimental to start therapy only when tumor-related symptoms arise. Nonetheless patients should be informed that no study has specifically investigated this topic comparing W and W and anti-tumor therapy, and therefore we have no evidence either for or against. Patients will need to understand that follow-up will be life-long even with stable disease, that there are data showing that the vast majority of advanced NETs tends to grow and that SSAs can be active even when the tumor is very indolent.

and anti-tumor therapy, and therefore we have no evidence either for or against. Patients will need to understand that follow-up will be life-long even with stable disease, that there are data showing that the vast majority of advanced NETs tends to grow and that SSAs can be active even when the tumor is very indolent. In conclusion, W and W policy in advanced NENs is yet to be well-defined. First of all it should be clarified whether W and W means delaying or avoiding an anti-tumor treatment. Delaying may be justified in an asymptomatic good performance status patient with a low-grade NETs over some weeks in order to thoroughly characterize both disease and patient and so make a well-informed choice as to the best therapy and therapeutic strategy to pursue. This is a quite common clinical scenario in the field of NETs. By contrast it is hard to justify W and W with the intent to avoid treatment considering that low-grade advanced NETs tend to grow even when they have very favorable biological characteristics. Therefore, also in that case, rather than avoiding, it would mean once again delaying the first-line therapy. Of course the first-line therapy and the therapeutic strategy depend on the specific clinical context and on the goal of treatment. In other words in a patient who is a good candidate for a future absolute debulking, then the first-line treatment even more than an SSA should be applied even with a stable disease without any delay. On the other hand, in a patient with a metastatic low grade, really stable NET, when absolute debulking is not possible and the goal of the treatment is the tumor growth control over time with a systemic medical therapy, then a thoughtful analysis needs to be made. It is important to bear in mind the cost- and risk-benefit of SSA, which is the most commonly proposed therapy in such a context, and also the cost, invasiveness, impact on quality of life and possible detrimental effect of W and W.

over time with a systemic medical therapy, then a thoughtful analysis needs to be made. It is important to bear in mind the cost- and risk-benefit of SSA, which is the most commonly proposed therapy in such a context, and also the cost, invasiveness, impact on quality of life and possible detrimental effect of W and W. I would argue that given the absence of evidence and of clinical trials designed to specifically investigate this topic, as is currently the case, clinicians should consider administering treatment to all patients, whether their NETs are advanced. ACKNOWLEDGMENTS The author would like to thank William Russell for English revision. Conflict-of-interest statement: Novartis and Ipsen: Honoraria for presentations and advisory boards. Novartis: Research funds (to the institution). Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Italy Peer-review report classification Grade A (Excellent): A Grade B (Very good): B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: September 23, 2016 First decision: October 20, 2016 Article in press: January 13, 2017 P- Reviewer: Tsolakis AV, Yoshitomi H S- Editor: Kong JX L- Editor: A E- Editor: Lu YJ

Core tip: Improvements of colorectal cancer liver metastases (CRC-LM) treatment allows the down-staging of several patients. There is currently no agreement in the correct sequence of surgical resection of the primary cancer and metastatic disease. Surgical resection can be performed if the complete removal of cancer is achievable, leaving an adequate normal liver tissue. Neoadjuvant chemotherapy is widely accepted as primary therapy. Chemotherapy may lead to disease regression for unresectable CRC-LM, allowing resection and cure. The application of loco-regional therapies is increasing. They are recommended as third-line treatment for unresectable CRC-LM and have a palliative intent. INTRODUCTION Colorectal cancer (CRC) is an increasing global health issue[1,2] It is the most common gastro-intestinal tumor and the third most frequently diagnosed malignancy worldwide. It has a mortality rate of up to 10%[1,2]. Most recent epidemiological data show more than 1.4 million newly diagnosed CRC each year[1,2]. The liver is the most common site of CRC metastases with an incidence of 15%-20% at diagnosis. CRC patients have a > 50% probability of liver metastases development[3]. The majority of CRC liver metastases (CRC-LM) were defined not resectable in the past century. Surgery methods are considerably improved nowadays, resulting in cure or survival increase. CRC-LM resection rates are also increased[4]. Recent updating of resectability criteria of CRC-LM considerably improves outcomes, resulting in 5 and 10-year survival rates of 40% and 25% respectively[5,6].

e in the past century. Surgery methods are considerably improved nowadays, resulting in cure or survival increase. CRC-LM resection rates are also increased[4]. Recent updating of resectability criteria of CRC-LM considerably improves outcomes, resulting in 5 and 10-year survival rates of 40% and 25% respectively[5,6]. Notwithstanding these good outcomes, the recurrence rate one year after metastasis resection is 30% and a recent study on CRC-LM survival after resection shows a 5-year survival of 16%-71%[7]. Neoadjuvant chemotherapy allow initially unresectable CRC-LM patients to have long term survival similar to those of resectable patients[8-12]. Chemotherapy efficacy, in terms of tumor reduction, is strongly correlated to resectability[10-13]. For this reason, chemotherapy associated to biological agents is increasingly used as resectability conversion of CRC-LM from unresectable to resectable. This method can efficiently increase downsizing rates[14,15]. Candidate selection for resection is still difficult and several CRC-LM patients are never referred to hepatobiliary multidisciplinary group[10,13]. For this reason CRC-LM patients need a multidisciplinary team for treatment decision. This team should include specialists from different disciplines: Oncology, surgery, radiology and radiotherapy. The purpose of this review is to examine the current management of CRC-LM, in order to better define potential advantages and limitations of the several available treatments.

nary team for treatment decision. This team should include specialists from different disciplines: Oncology, surgery, radiology and radiotherapy. The purpose of this review is to examine the current management of CRC-LM, in order to better define potential advantages and limitations of the several available treatments. PERIOPERATIVE EVALUATION The perioperative evaluation of a patient’s global health and liver function is essential to reduce postoperative complications. A dedicated multidisciplinary team should assess co-morbidities and patient’s performance status in order to decide a future treatment plan. Complete blood examination should be performed before surgery, to assess liver function [alanine aminotransferase (ALT), glutamic-oxalacetic transaminase (AST)], coagulation profile, bilirubin, creatinine and tumor markers, such as carcinoembryonic antigen (CEA). Exclusion criteria for surgery include several factors to guarantee patient safety. They include advanced age, male gender, low serum albumin, presence of liver disease (hepatitis or alcoholic hepatitis), ascites, kidney or cardiologic impairment, bleeding syndromes, and chronic obstructive pulmonary disease[16-18]. Morbidity and mortality after liver resection is often due to inadequate function of remnant liver, leading to liver failure. Morbidity and mortality rates are around 61% and 11%, respectively[16,17].

Exclusion criteria for surgery include several factors to guarantee patient safety. They include advanced age, male gender, low serum albumin, presence of liver disease (hepatitis or alcoholic hepatitis), ascites, kidney or cardiologic impairment, bleeding syndromes, and chronic obstructive pulmonary disease[16-18]. Morbidity and mortality after liver resection is often due to inadequate function of remnant liver, leading to liver failure. Morbidity and mortality rates are around 61% and 11%, respectively[16,17]. The remnant liver cannot sustain metabolic, synthetic, and detoxifying functions if reduced below a critical liver volume[18]. Liver volume is not the best index for liver functionality assessment[16-20]. Patients with concomitant liver disease may have impaired liver regeneration capacity due to cirrhosis, steatosis, or jaundice obstruction[20]. Most chemotherapeutic agents (5-fluorouracil, irinotecan, oxaliplatin) can result in hepatic damage and modification of liver regeneration[11,19-22]. Morbidity and mortality after liver resection may be improved by measuring the intake of 99mTc mebrofenin of tumor-free liver in a pre-operative setting, in order to assess the risk of liver failure and liver failure-related mortality after partial liver resection[17].

Most chemotherapeutic agents (5-fluorouracil, irinotecan, oxaliplatin) can result in hepatic damage and modification of liver regeneration[11,19-22]. Morbidity and mortality after liver resection may be improved by measuring the intake of 99mTc mebrofenin of tumor-free liver in a pre-operative setting, in order to assess the risk of liver failure and liver failure-related mortality after partial liver resection[17]. During liver regeneration induced by partial hepatectomy, normally quiescent hepatocytes start to replicate in order to restore the original liver. Several genes are involved in liver regeneration, including cytokine, growth factor and metabolic genes[21]. Several studies show that recurrence and progression are directly proportional to the amount of liver resected[22,23]. Neoadjuvant chemotherapy may induce hepatic changes, such as steatohepatitis, hepatic sinusoidal obstruction and periportal inflammation, negatively affecting patient outcome[20,21] and increasing the risk of liver failure and death after major liver resection. A normal liver can bear an extensive resection. Severely compromised livers, on the contrary, cannot tolerate even a minor hepatectomy[8,9,19]. For this reason, monitoring the functionality of surrounding tumor-free liver needs to be highly considered for selection of surgical method.

death after major liver resection. A normal liver can bear an extensive resection. Severely compromised livers, on the contrary, cannot tolerate even a minor hepatectomy[8,9,19]. For this reason, monitoring the functionality of surrounding tumor-free liver needs to be highly considered for selection of surgical method. RADIOLOGICAL ASSESSMENT CRC-LM radiological study is necessary for assessment of surgical resectability. This can be performed using any of these main radiological methods: Magnetic resonance imaging (MRI), computed tomography (CT) and positron emission tomography (PET) scan[24]. Liver metastases can be detected as hypoattenuating lesions, when using contrast-enhanced normal or multidetector CT scans with a sensitivity rate of 85% and 90%, respectively[25]. MRI performed with liver-specific contrast agents has > 90% sensitivity in cases of underlying liver disease (steatosis, cirrhosis) or very small lesions (< 1 cm). For this reason MRI is better than CT for metastasis detection[26]. Specificity of CT, MRI and PET is very high: 95%, 93%, and 97% respectively. PET scan is useful to obtain whole body map, to identify extrahepatic disease (EHD) and to assess resectability[24]. A recent study showed that the FDG PET scan is the best radiological modality for detecting CRC-LM. It can have high false negative rates in patients recently treated with chemotherapy[17,18]. The association of CT to FDG PET scans is highly recommended because it improves the sensitivity up to 97%[24].

ectability[24]. A recent study showed that the FDG PET scan is the best radiological modality for detecting CRC-LM. It can have high false negative rates in patients recently treated with chemotherapy[17,18]. The association of CT to FDG PET scans is highly recommended because it improves the sensitivity up to 97%[24]. Nowadays, also intraoperative ultrasound (IOUS) is a mandatory surgical tool to confirm preoperative investigations by CT or MRI and for detection of missed lesions[27]. CRITERIA FOR RESECTABILITY The preferred therapy for CRC-LM is surgery, providing up to 50% survival at five-years[28]. Patient selection criteria for resectability are not standardized and still controversial in clinical practice. The American Hepato-Pancreato-Biliary Association (AHPBA) consensus on definition of resectability is currently accepted by most liver surgeons[28,29]. Main CRC-LM resection criteria of AHPBA are: Presence of disease confined to the liver as identified after surgery of primitive cancer; disease in a single hepatic lobe; < 3 nodules; the largest size of nodules < 5 cm in diameter; margin FLR > 1 cm. According to these criteria, however, less than 10% of patients would be indicated for resection.

ria of AHPBA are: Presence of disease confined to the liver as identified after surgery of primitive cancer; disease in a single hepatic lobe; < 3 nodules; the largest size of nodules < 5 cm in diameter; margin FLR > 1 cm. According to these criteria, however, less than 10% of patients would be indicated for resection. The classification of resectable disease is broader nowadays, increasing the number of resections[30]. Current guidelines generally agree that resection should be performed for liver metastases only[12,30,31], but hepatectomy and resections of concomitant extrahepatic disease are considered[32]. The remaining liver must be undamaged and at least 20% or 25% of the whole hepatic volume, and have a full functional vascular and biliary in- and out- flow. In this case also multiple resection can be performed[8,14,30]. The survival advantage of repeated resection is close to that after the surgery of primary hepatic disease[33]. Hepatic resection safety depends on: Age of patients, performance score, and concomitant hepatic impairments. Resection is contraindicated when the following are observed: Non resectable extra hepatic tumor; wide involvement of parenchyma; or patient’s poor general conditions. Possible prognostic factors of resection outcome of CRC-LM are: Age, sex, synchronous or metachronous hepatic metastases, tumor size, number and distribution of LM, primary tumor stage, extrahepatic distant metastases, surgical margin, type of primary hepatic tumor surgery and previous tumor pharmacological therapy, levels of tumor markers.

ctors of resection outcome of CRC-LM are: Age, sex, synchronous or metachronous hepatic metastases, tumor size, number and distribution of LM, primary tumor stage, extrahepatic distant metastases, surgical margin, type of primary hepatic tumor surgery and previous tumor pharmacological therapy, levels of tumor markers. Fong et al[34] report data from 1001 CRC-LM patients who were candidates for resection. These data led to the identification of seven criteria for worse prognosis prediction after resection. Five of these criteria are actually used for the Clinical Risk Score (CRS) that is a preoperative scoring system. These criteria are: Disease-free interval from primary to metastases < 12 mo; largest hepatic tumor > 5 cm in diameter; node-positivity; number of lesions > 1; and CA 19-9 > 200 ng/mL. Positive prognosis after surgery corresponds to a score < 2. Scores of 3-4 indicate that patients are candidates for resection followed by adjuvant therapy. Prognosis is poor when the score is five. The appropriateness of CRS is proved. CRS can predict patients’ response and OS[35].

esions > 1; and CA 19-9 > 200 ng/mL. Positive prognosis after surgery corresponds to a score < 2. Scores of 3-4 indicate that patients are candidates for resection followed by adjuvant therapy. Prognosis is poor when the score is five. The appropriateness of CRS is proved. CRS can predict patients’ response and OS[35]. A new method has been recently introduced in the CRC-LM resectability criteria assessment[5]. Resection criteria are different. They depend less on the size, number, and location of the metastases. They give more importance to the volume and function of the future liver remnant (FLR), which should be > 25% estimated normal liver parenchyma or 30% in the presence of impaired liver function[36]. Metastases are considered resectable if the excision of all metastatic lesions can be obtained with an adequate FLR[37] and the presence of EHD is currently no longer considered as a contraindication[5]. The new requirements for LM resection are: R0 resection achievement of intrahepatic and extra hepatic disease; adequate FRL; and > 2 adjacent liver segments to be spared with blood and bile inflow and outflow preservation[31,37]. TIMING OF COLON AND LIVER RESECTION The best sequence and timing of CRC-LM resection is still under debate and many options are available. The use of up front chemotherapy is increasing. Strong evidence is missing and there are currently no randomized controlled trials comparing the different approaches[38].

A new method has been recently introduced in the CRC-LM resectability criteria assessment[5]. Resection criteria are different. They depend less on the size, number, and location of the metastases. They give more importance to the volume and function of the future liver remnant (FLR), which should be > 25% estimated normal liver parenchyma or 30% in the presence of impaired liver function[36]. Metastases are considered resectable if the excision of all metastatic lesions can be obtained with an adequate FLR[37] and the presence of EHD is currently no longer considered as a contraindication[5]. The new requirements for LM resection are: R0 resection achievement of intrahepatic and extra hepatic disease; adequate FRL; and > 2 adjacent liver segments to be spared with blood and bile inflow and outflow preservation[31,37]. TIMING OF COLON AND LIVER RESECTION The best sequence and timing of CRC-LM resection is still under debate and many options are available. The use of up front chemotherapy is increasing. Strong evidence is missing and there are currently no randomized controlled trials comparing the different approaches[38]. The classic surgical method is “primary first”, whose suggested sequence is to firstly resect the primary CRC, then to administer the chemotherapy and after 3-6 mo to eventually resect the LM. This approach is indicated for patients with advanced or symptomatic CRC, important comorbidities, or inadequate FLR. In cases of advanced CRC, indeed, the chemotherapy may be associated with high complication rates and the insurgence of disease progression may lead to unresectability[39]. Any delay correlated to complications during surgery of CRC may also increase the risk of progression occurrence for some patients[40,41]. A possible benefit of this method can be the possibility to identify previously occult LM that may become visible during adjuvant chemotherapy. This allows avoidance of the morbidity of a liver resection.

complications during surgery of CRC may also increase the risk of progression occurrence for some patients[40,41]. A possible benefit of this method can be the possibility to identify previously occult LM that may become visible during adjuvant chemotherapy. This allows avoidance of the morbidity of a liver resection. Another surgical method is the “synchronous resection of LM and primary CRC”. This approach can avoid delays in chemotherapy treatment that can be started earlier if no complications occur after surgery. The possible disadvantage of this method is the increased postoperative morbidity and mortality because of infection resulting from bacterial contamination of the surgical field[36]. For this reason this approach is indicated for patients who can tolerate long operative times[6]. The third available surgical method is the “alternative staged liver-first” approach that firstly resect the LM, then administer 3-6 cycles of chemotherapy, and at last resect the primary CRC. Adjuvant chemotherapy can be administered in between both procedures. Recent data report that this method is indicated for selected patients with advanced CRC-LM, and when neo-adjuvant and adjuvant chemotherapy may have better results[9,12].

hen administer 3-6 cycles of chemotherapy, and at last resect the primary CRC. Adjuvant chemotherapy can be administered in between both procedures. Recent data report that this method is indicated for selected patients with advanced CRC-LM, and when neo-adjuvant and adjuvant chemotherapy may have better results[9,12]. CHEMOTHERAPY FOR RESECTABLE CRC-LM Neo-adjuvant chemotherapy The utility of neoadjuvant chemotherapy for CRC-LM is unclear even if there is the tendency to use it frequently[15]. There are many advantages of neo-adjuvant treatment such as increasing tumor sensitivity, downstaging large or multiple liver lesions, increasing resectability, and treating micrometastases[8,9,11]. This therapy also allows better planning for the date of surgical resection. On the other hand, neo-adjuvant chemotherapy can delay surgical treatment, which may be detrimental for patients, increasing the risk of disease progression[12,15]. This chemotherapy can also induce liver toxicity, such as steatohepatitis, increasing postoperative mortality. It can also mask metastases on preoperative imaging, as is observed in 5%-25% of cases[42].

n delay surgical treatment, which may be detrimental for patients, increasing the risk of disease progression[12,15]. This chemotherapy can also induce liver toxicity, such as steatohepatitis, increasing postoperative mortality. It can also mask metastases on preoperative imaging, as is observed in 5%-25% of cases[42]. Perioperative chemotherapy is widely used for patients with unresectable disease (Table 1 and Figure 1) with the purpose of reducing disease progression, which occurs in 50%-70% of patients after surgery[3]. A multicentre randomized trial compared surgery alone with perioperative chemotherapy (6 cycles of preoperative and post operative FOLFOX4) in 364 unresectable CRC-LM patients. The results of this study showed no significant differences in five-year OS for the two groups; nevertheless, progression-free survival (PFS) increased by 7.3% at 3 years in the perioperative chemotherapy group[43]. The rate of post-operative complications is also increased and is directly proportional to the length of therapy. For this reason, it is suggested that only 6 cycles of chemotherapy for no longer than 3 mo should be performed, in order to reduce toxicity[28], especially for patients who need a major hepatectomy[44]. Table 1 Recommendations for perioperative and conversion therapy (adapted from ESMO 2016[110])

Perioperative chemotherapy is widely used for patients with unresectable disease (Table 1 and Figure 1) with the purpose of reducing disease progression, which occurs in 50%-70% of patients after surgery[3]. A multicentre randomized trial compared surgery alone with perioperative chemotherapy (6 cycles of preoperative and post operative FOLFOX4) in 364 unresectable CRC-LM patients. The results of this study showed no significant differences in five-year OS for the two groups; nevertheless, progression-free survival (PFS) increased by 7.3% at 3 years in the perioperative chemotherapy group[43]. The rate of post-operative complications is also increased and is directly proportional to the length of therapy. For this reason, it is suggested that only 6 cycles of chemotherapy for no longer than 3 mo should be performed, in order to reduce toxicity[28], especially for patients who need a major hepatectomy[44]. Table 1 Recommendations for perioperative and conversion therapy (adapted from ESMO 2016[110]) Perioperative treatment It is defined by technical criteria for resection and prognostic considerations It may not be necessary in patients with clearly resectable disease and favourable prognosis, in this case upfront resection is justified It should administer FOLFOX or CAPOX to patients with resectable disease and unclear (probably unfavourable) Targeted agents should not be used in resectable patients with prognostic indication for perioperative treatment It should be considered when prognostic and resectability criteria are unclearly defined, and in patients with synchronous onset of metastases Adjuvant chemotherapy is not strongly indicated for patients with favourable oncological and surgical criteria, who did not receive any neoadjuvant chemotherapy Adjuvant chemotherapy is indicated for patients with unfavourable criteria Adjuvant treatment with FOLFOX or CAPOX is recommended for patients who have not received any previous chemotherapy, unless patients already received oxaliplatin-based adjuvant chemotherapy The choice of chemotherapy type should consider patients’ clinical conditions and therapy preferences Conversion therapy A chemotherapy regimen leading to high response rates and/or a large tumour shrinkage is recommended for potentially resectable patients The best drug combination to use is still not clear because only few trials have addressed this issue: RAS wild-type patients may benefit from a cytotoxic doublet plus an epidermal growth factor receptors agents antibody (best benefit/risk), and from the combination of FOLFOXIRI plus bevacizumab and, to a lesser extent, from a cytotoxic doublet plus bevacizumab RAS mutant patients may benefit from a cytotoxic doublet plus bevacizumab or FOLFOXIRI plus bevacizumab Patients must be re-evaluated regularly (every 2-3 mo) to prevent the overtreatment of resectable patients Figure 1 Treatment indications for fit and unfit colorectal cancer liver metastases patients. BSC: Best supportive care; CHT: Chemotherapy; EGFR: Epidermal growth factor receptors agents; mut: Mutated; FP: Fluoro pyrimidine. Adapted from ESMO 2016[110].

(every 2-3 mo) to prevent the overtreatment of resectable patients Figure 1 Treatment indications for fit and unfit colorectal cancer liver metastases patients. BSC: Best supportive care; CHT: Chemotherapy; EGFR: Epidermal growth factor receptors agents; mut: Mutated; FP: Fluoro pyrimidine. Adapted from ESMO 2016[110]. Patients with more than 3 lesions, and tumor diameter greater than 3 cm are clearly indicated for this treatment. The surgery of lesions should be done 4-8 wk after the neo-adjuvant chemotherapy. In summary, the advantages of neo-adjuvant chemotherapy outnumber the disadvantages, and we are in favor of its utilization. Adjuvant chemotherapy The ultimate dilemma after complete CRC-LM resection is the rate of recurrence that is reported as high as 60% after complete surgical excision. Several studies show the benefits of adjuvant therapy such as FOLFOX4 (folinic acid, fluorouracil, and oxaliplatin), resulting in longer disease-free-survival (DFS)[45] than liver resection alone. Adjuvant chemotherapy also increases OS when compared to surgery alone, even if the difference is not statistically significant[46,47]. The classic adjuvant chemotherapeutic drugs are: 5-fluorouracil/leucovorin (5-FU/LV), capecitabine, oxaliplatin and irinotecan[47]. New molecular-targeted agents are now available. They include anti-angiogenic drugs (bevacizumab, regorafenib and aflibercept) and anti-epidermal growth factor receptors agents (anti-EGFR), such as cetuximab and panitumumab. These agents are widely used as adjuvant treatment without any evidence of clinical benefit[48].

ew molecular-targeted agents are now available. They include anti-angiogenic drugs (bevacizumab, regorafenib and aflibercept) and anti-epidermal growth factor receptors agents (anti-EGFR), such as cetuximab and panitumumab. These agents are widely used as adjuvant treatment without any evidence of clinical benefit[48]. Adjuvant chemotherapy after metastasectomy is generally recommended by clinicians, even if the best regimen protocol is still unclear, and should be considered in a patient dependent manner[24]. There, efficacy of adjuvant chemotherapy on OS for resectable CRC-LM is still under discussion[45]. The National Comprehensive Cancer Network (NCCN) guidelines suggests the use of more than one chemotherapy line[48]. Most study agree that 5-FU/LV with or without oxaliplatin should always be used as first-line[47]. More recently, however, the use of combination therapy is increasing, and several combinations have emerged. A recent study on FOLFIRI (5-FU/LV and irinotecan) vs 5FU/LV after R0 (complete resection) of CRC-LM does not report any difference in OS and median DFS. FOLFIRI improves DFS, but causes more frequent grade 3/4 toxic adverse events (47% vs 30%)[49]. We suggest the use of adjuvant chemotherapy in patients with multiple lesions that are found in more than 3 liver segments, where the surgery, even if radical, may not be able to remove undetected tumor deposits. CHEMOTHERAPY FOR UNRESECTABLE CRC-LM Patients with unreserctable CRC-LM from diagnosis should receive chemotherapy in order to downstage the disease and allow the surgery (Figure 1 and Table 2).

We suggest the use of adjuvant chemotherapy in patients with multiple lesions that are found in more than 3 liver segments, where the surgery, even if radical, may not be able to remove undetected tumor deposits. CHEMOTHERAPY FOR UNRESECTABLE CRC-LM Patients with unreserctable CRC-LM from diagnosis should receive chemotherapy in order to downstage the disease and allow the surgery (Figure 1 and Table 2). Table 2 Conversion rates in colorectal cancer liver metastases after perioperative chemotherapy

We suggest the use of adjuvant chemotherapy in patients with multiple lesions that are found in more than 3 liver segments, where the surgery, even if radical, may not be able to remove undetected tumor deposits. CHEMOTHERAPY FOR UNRESECTABLE CRC-LM Patients with unreserctable CRC-LM from diagnosis should receive chemotherapy in order to downstage the disease and allow the surgery (Figure 1 and Table 2). Table 2 Conversion rates in colorectal cancer liver metastases after perioperative chemotherapy Trial name Chemotherapy type Control n KRAS status Overall response Conversion to resection R0 resection BEAT[61] FOLFOX/XELOX/FOLFIRI or fluoropyrimidines + bevacizumab No 1914 Not selected NA 11.80% NA First BEAT[62] FOLFOX/XELOX + bevacizumab Placebo 1914 Not selected 38% 11.80% 6.3% vs 4.9% OPUS[70] FOLFOX + cetuximab FOLFOX 233 Wilde type 61% vs 37% 9% 4.7% vs 2.4% POCHER[72] Chr IFLO + cetuximab No 43 Wild type 79% 60% 25.70% PRIME[77] FOLFOX + panitumumab FOLFOX 591 Wild type 57% vs 48% 31% vs 22% 29% vs 17% CELIM[11] FOLFOX6 + cetuximab FOLFIRI + cetuximab 106 Wild type 68% vs 57% 43% 38% vs 30% BOXER[63] CAPOX + bevacizumab No 47 Not selected 78% 40% NA Loupakis et al[55] FOLFOXIRI + bevacizumab FOLFIRI + bevacizumab 508 Not selected 65% vs 53% 15% vs 12% NA Ye et al[73] FOLFIRI + cetuximab FOLFOX + cetuximab 177 Wild type 57% vs 29% 26% vs 7% NA CRYSTAL[71] FOLFIRI + cetuximab FOLFIRI 599 Wilde type 47% vs 39% 16% 4.8% vs 1.7% OLIVIA[79] FOLFOXIRI + bevacizumab FOLFOX + bevacizumab 80 Not selected 81% vs 62% 61% vs 49% 49% vs 23% CAPOX, XELOX: Capecitabine-oxaliplatin; NA: Not available; Chr IFLO: Chronomodulated irinotecan, 5-fluorouracil, leucovorin, and oxalipaltin; FOLFIRI: 5-fluorouracil, leucovorin and irinotecan; FOLFOX: 5-fluorouracil, leucovorin, and oxalipaltin; FOLFOXIRI: 5-fluorouracil, leucovorin, oxalipaltin and irinotecan.

vs 23% CAPOX, XELOX: Capecitabine-oxaliplatin; NA: Not available; Chr IFLO: Chronomodulated irinotecan, 5-fluorouracil, leucovorin, and oxalipaltin; FOLFIRI: 5-fluorouracil, leucovorin and irinotecan; FOLFOX: 5-fluorouracil, leucovorin, and oxalipaltin; FOLFOXIRI: 5-fluorouracil, leucovorin, oxalipaltin and irinotecan. About 70% of patients with CRLM are unresectable at diagnosis[4]. They have a complicated disease, often requiring a combination of loco-regional therapy (chemoembolization, hepatic arterial infusion, ablation or radiation). Perioperative chemotherapy is widely used also for unresectable CRC-LM, even if there is no proof of OS improvement[50]. Systemic chemotherapy remains the first-line therapy. FOLFOXIRI followed by surgical resection has a 70.4% response rate, and 19% of patients obtain R0. OS at 5 and 8 years are 42% and 33% respectively, and 29% of patients are disease free at 5 years[51].

ectable CRC-LM, even if there is no proof of OS improvement[50]. Systemic chemotherapy remains the first-line therapy. FOLFOXIRI followed by surgical resection has a 70.4% response rate, and 19% of patients obtain R0. OS at 5 and 8 years are 42% and 33% respectively, and 29% of patients are disease free at 5 years[51]. Downstaging of unresectable CRC-LM ranges from 5% to 38%. This is due to multiple factors including disease extension, type and duration of chemotherapy[51]. The purpose of the “conversion chemotherapy” in unresectable CRC-LM patients is to convert their disease to resectable, and is often the first line treatment. Standard regimens include FOLFIRI or FOLFOX that induce downstaging in 7%-40% of patients[12]. Giacchetti’s group reports that FOLFOX reduces the LM dimension by more than 50% in 59% of non-resectable CRC-LM, resulting in 38% of CR[52]. FOLFOXIRI allows 36% of R0 in LM patients[53]. The METHEP trial reports that FOLFIRINOX seems to be better therapy for CRC-LM than the others, bringing to resection 67% of cases with a survival > 48 mo. These results confirm that OS is greater for patients after R0 or R1 surgery, 65.2 mo vs 18.3 mo of not-operated or R2 patients[54].

s 36% of R0 in LM patients[53]. The METHEP trial reports that FOLFIRINOX seems to be better therapy for CRC-LM than the others, bringing to resection 67% of cases with a survival > 48 mo. These results confirm that OS is greater for patients after R0 or R1 surgery, 65.2 mo vs 18.3 mo of not-operated or R2 patients[54]. The use of bevacizumab is increasing for unresectable CRC-LM[55,56], even if the benefits are extremely limited. A slight gain in response rate is observed when bevacizumab is associated with FOLFOXIRI as first line chemotherapy. The association of bevacizumab to first and second line chemotherapy for CRC-LM improves PFS[57-60] and OS in some studies[59,60]. Available data on the efficacy of bevacizumab associated to perioperative chemotherapy are limited. This may be due to concerns about possible complications in wound healing after resection[61,62]. The Bevacizumab Expanded Access Trial reports good feasibility of LM surgery after first-line chemotherapy associated to bevacizumab, resulting in resection rates of 11.8% and 6% of R0[63]. Bevacizumab association with FOLFOX, however, obtains higher resection rates (16.1%) than with FOLFIRI (9.7%), and higher R0 (6.3%) than FOLFOX plus placebo (4.9%) (P = 0.24)[62]. Neoadjuvant capecitabine and oxaliplatin (CAPOX) plus bevacizumab resulted in 40% of CRC-LM resectability conversion[63]. Loupakis et al[55] report 64% of tumor response and 15% of rate of resection of CRC-LM after FOLFOXIRI plus bevacizumab, vs 53% and 12% respectively after FOLFIRI/bevacizumab.

(4.9%) (P = 0.24)[62]. Neoadjuvant capecitabine and oxaliplatin (CAPOX) plus bevacizumab resulted in 40% of CRC-LM resectability conversion[63]. Loupakis et al[55] report 64% of tumor response and 15% of rate of resection of CRC-LM after FOLFOXIRI plus bevacizumab, vs 53% and 12% respectively after FOLFIRI/bevacizumab. Transarterial chemoembolization with irinotecan combined with FOLFOX plus bevacizumab chemotherapy results in a response rate of 78%, and allows resection of 35% of non resectable CRC-LM, offering a new cure option to these patients[64]. A recent report by Stremitzer et al[65] shows that mutated BRAF/RAS are correlated to a poor outcome after CRC-LM surgery. This is in agreement with the results of other 3 studies[66-69]. These important evidences support the application of newer methods for the therapy of liver metastases, associating biological molecular aspects (biological resectability) to the other clinical and pathological indexes for the selection of good surgical candidates and the prediction of their outcomes.

3 studies[66-69]. These important evidences support the application of newer methods for the therapy of liver metastases, associating biological molecular aspects (biological resectability) to the other clinical and pathological indexes for the selection of good surgical candidates and the prediction of their outcomes. Anti-EFGR agents such as cetuximab and panitumumab are effective alone as well as in association with chemotherapy in CRC-LM that are RAS (both KRAS and NRAS) wild type[69]. Some randomized trials report the effects of cetuximab for the therapy of unresectable CRC-LM. The OPUS trial[70] showed that the association of FOLFOX-4 plus cetuximab as up front therapy doubled R0 (4.7%). The CRYSTAL study[71] showed that the association of FOLFIRI plus cetuximab as up front therapy increased the R0 resection rate from 3.7% to 7.0%. The CELIM trial[11] reported that neoadjuvant treatment with FOLFIRI plus cetuximab or FOLFOX6 resulted in 34% of R0 resections. Other studies also report that chemotherapy containing cetuximab significantly improves R0 in unresectable CRC-LM with KRAS wild-type[72,73]. There are differences in resection rates among the above studies. Overall response rate is in the range 60%-79%, however, resection rates after chemotherapy/cetuximab are very variable (Table 2). These discrepancies may be due to the fact that the resection rate is defined and determined by clinical conditions of the patients and not by specialist oncologists in CRYSTAL and OPUS studies. Resection evaluation is done by a multidisciplinary team in the other trials.

otherapy/cetuximab are very variable (Table 2). These discrepancies may be due to the fact that the resection rate is defined and determined by clinical conditions of the patients and not by specialist oncologists in CRYSTAL and OPUS studies. Resection evaluation is done by a multidisciplinary team in the other trials. The COIN[74] and NORDIC VII[75] trials report no advantage for the association of oxaliplatin based chemotherapy/cetuximab in first-line treatment of CRC-LM, independently from K-RAS status. Resection rates of first-line FOLFIRI/panitumumab treatment of CRC-LM are 15% and 7% in the KRAS wild type (WT) and mutant groups respectively[76]. FOLFOX4 plus panitumumab results in 32% of R0 resections vs 28% of those receiving only FOLFOX4[77]. A post hoc analysis of the PRIME study on RAS WT (KRAS, NRAS) shows that panitumumab/FOLFOX can convert to resection 31% of initially unresectable CRC-LM patients and lead to 29% of R0 (Table 2)[78]. A further analysis of PRIME trial also shows that NRAS mutations are indications of non-response to panitumumab[77]. For this reason, it is extremely important to analyze other types of mutations in the RAS gene to improve patient selection for anti EGFR therapy. The OLIVIA trial studies FOLFOXIRI + bevacizumab vs mFOLFOX-6 + bevacizumab and reports an overall resection rate of 61% vs 49%, with R0 resection rates of 49% vs 23%[79].

Resection rates of first-line FOLFIRI/panitumumab treatment of CRC-LM are 15% and 7% in the KRAS wild type (WT) and mutant groups respectively[76]. FOLFOX4 plus panitumumab results in 32% of R0 resections vs 28% of those receiving only FOLFOX4[77]. A post hoc analysis of the PRIME study on RAS WT (KRAS, NRAS) shows that panitumumab/FOLFOX can convert to resection 31% of initially unresectable CRC-LM patients and lead to 29% of R0 (Table 2)[78]. A further analysis of PRIME trial also shows that NRAS mutations are indications of non-response to panitumumab[77]. For this reason, it is extremely important to analyze other types of mutations in the RAS gene to improve patient selection for anti EGFR therapy. The OLIVIA trial studies FOLFOXIRI + bevacizumab vs mFOLFOX-6 + bevacizumab and reports an overall resection rate of 61% vs 49%, with R0 resection rates of 49% vs 23%[79]. In conclusion “biologically directed” chemotherapy reduces the number and size of unresectable lesions. It also allows rescue of 15%-35% of patients, bringing them to surgery. These therapies are increasingly used worldwide. EXTRA HEPATIC DISEASE Extra hepatic disease (EHD) has a poor prognosis[28]. Most common sites of EHD from CRC are lymph nodes, lungs, peritoneum, brain and bone. EHD is currently no longer a contraindication to metastasis resection, and patients after surgery have[5] longer DFS and five-year-survival rates compared to those receiving only chemotherapy[5,6].

ease (EHD) has a poor prognosis[28]. Most common sites of EHD from CRC are lymph nodes, lungs, peritoneum, brain and bone. EHD is currently no longer a contraindication to metastasis resection, and patients after surgery have[5] longer DFS and five-year-survival rates compared to those receiving only chemotherapy[5,6]. OS after lymph node resection is different according to their site and number[80]. Celiac or aorto-caval lymph node resections are associated with a worse outcome when compared to hepatic pedicle nodes, and mediastinal lymph nodes have a worse median survival than intra-thoracic ones[80]. A high number of lymph nodes positive for metastases have also a poor outcome[80]. In conclusion, the treatment of EHD is substantially palliative, aiming to improve the quality of life[81]. LOCO-REGIONAL THERAPIES Loco-regional therapies (Figure 2) are indicated for patients that are elderly, have a poor performance status, refusing surgery or chemotherapy, or refractory to chemotherapy. They also allow chemo-holidays with suspension of chemotherapy, and prolong the non-treatment period in between different chemotherapy lines. This reduces the treatment costs in respect to systemic chemotherapy. Figure 2 Liver directed treatments. TACE: Trans-arterial chemoembolization. In the last years new strategies have been developed in order to overcome several problems: High percentage of unresectable CRC-LM at diagnosis, high recurrence rates and presence of extensive disease. These methods increase the number of patients indicated for non surgical procedures.

Figure 2 Liver directed treatments. TACE: Trans-arterial chemoembolization. In the last years new strategies have been developed in order to overcome several problems: High percentage of unresectable CRC-LM at diagnosis, high recurrence rates and presence of extensive disease. These methods increase the number of patients indicated for non surgical procedures. Ablation techniques include radiofrequency ablation (RFA), Microwave ablation and external beam radiotherapy (EBRT). RFA is widely used and allows the application of extreme temperature to ablate the lesion with minimal toxicity (< 1%) in the surrounding liver tissue. RFA results in mortality and morbidity < 10% independently from the administration route[82]. The “heat sink effect” is however a major disadvantage of RFA and may cause important hepatic or vascular injury. For this reason, RFA is not indicated for unresectable tumors, lesions near blood vessels or the diaphragm because of the high risk of perforation. Another disadvantage of RFA is the recurrence rate that is higher when the tumor is > 3 cm or when treatment is delivered percutaneously[82,83]. Microwave ablation uses high frequency microwave radiation to induce coagulation with necrosis of lesions. This method, however, is not well known and there are several concerns about its feasibility[84]. Available data on this method show a 6% local recurrence rate[85].

Ablation techniques include radiofrequency ablation (RFA), Microwave ablation and external beam radiotherapy (EBRT). RFA is widely used and allows the application of extreme temperature to ablate the lesion with minimal toxicity (< 1%) in the surrounding liver tissue. RFA results in mortality and morbidity < 10% independently from the administration route[82]. The “heat sink effect” is however a major disadvantage of RFA and may cause important hepatic or vascular injury. For this reason, RFA is not indicated for unresectable tumors, lesions near blood vessels or the diaphragm because of the high risk of perforation. Another disadvantage of RFA is the recurrence rate that is higher when the tumor is > 3 cm or when treatment is delivered percutaneously[82,83]. Microwave ablation uses high frequency microwave radiation to induce coagulation with necrosis of lesions. This method, however, is not well known and there are several concerns about its feasibility[84]. Available data on this method show a 6% local recurrence rate[85]. Improvements in imaging methods have increased the use of EBRT[86]; that, however, has a low therapeutic window, and toxicity is still a major issue. EBRT is safe (at 60 Gy) and effective for liver tumors in general and in selected patients[87,88]. Intra-arterial therapies: Hepatic artery infusion Hepatic artery infusion (HAI) is indicated for patients with unresectable lesions when physicians want to associate an intra-arterial with an endovenous treatment.

or, posttreatment axial image shows normal, two-layered rectal wall (arrow). This is an example for false-negative MR assessment of complete tumor regression; C: In ypT0 rectal tumor, posttreatment axial image shows thick, fibrotic low signal intensity scar (arrow) in pretreatment T3 tumor area. CRT: Chemoradiotherapy. Figure 18 Post-chemoradiotherapy restaging using diffusion-weighted imaging in ypT3 rectal tumor. On T2-weighted (A), DW (B) and ADC (C) images in the same patient, baseline and post-CRT images are shown on upper and lower series, respectively. A: Posttreatment T2-weighted axial image shows semiannular infiltrating tumor, compatible with a residual T3 tumor (arrow); B: Posttreatment DW; C: ADC images delineate high and low signal-intensity corresponding to the tumor, respectively (arrow). Pre- and post-treatment mean ADC values are 0.68-0.72, 1.22-1.44 × 10-3 mm²/s, respectively, in the tumor area. Post-therapy ADC increase is compatible with therapy response. CRT: Chemoradiotherapy.

Improvements in imaging methods have increased the use of EBRT[86]; that, however, has a low therapeutic window, and toxicity is still a major issue. EBRT is safe (at 60 Gy) and effective for liver tumors in general and in selected patients[87,88]. Intra-arterial therapies: Hepatic artery infusion Hepatic artery infusion (HAI) is indicated for patients with unresectable lesions when physicians want to associate an intra-arterial with an endovenous treatment. The advantage of HAI is to minimize the toxicity to normal liver tissue, because the chemotherapeutic agents are injected directly to the tumor[89]. Potential risks of this method are treatable complications related to catheter and pump placement, or life-threatening complications such as biliary sclerosis, hepatotoxicity and systemic toxicity. For this reason it should be performed by experienced hospitals[89-91]. Intravenously 5-FU and intra hepatic artery oxaliplatin are successfully used[92] for unresectable CRC-LM. Best results concerning survival and response rates are obtained with floxuridine based HAI[93]. The comparison of OS between HAI therapy and systemic therapy alone (15.9 mo vs 12.4 mo) does not show any difference, however, there was a great response rate in favor of HIA (43% vs 18%)[94]. In conclusion, HAI has interesting results; however it is a cumbersome method because it requires the implantation of an infusion pump.

The comparison of OS between HAI therapy and systemic therapy alone (15.9 mo vs 12.4 mo) does not show any difference, however, there was a great response rate in favor of HIA (43% vs 18%)[94]. In conclusion, HAI has interesting results; however it is a cumbersome method because it requires the implantation of an infusion pump. Chemoembolization Trans-arterial chemoembolization (TACE) is increasingly used for unresectable CRC-LM, improving survival and tumor response[95]. TACE is indicated for unresectable CRC-LM as third line therapy, and allows the attainment of important palliative results. The use of drug-eluting beads for TACE increases efficacy, while reducing adverse events due to systemic drug leakage or liver toxicity[95-98]. The advantage of these beads is the direct delivery of toxic drugs inside the arterial capillary bed of the tumor, releasing the drug in a controlled manner. In this way the systemic exposure to toxic drugs is reduced, their local concentration is increased and a greater tissue necrosis than classic TACE with lipiodol is obtained[99,100]. The indication for TACE is presence of multinodular LM, absence of extra hepatic disease, refractory to systemic chemotherapy[101].

The use of drug-eluting beads for TACE increases efficacy, while reducing adverse events due to systemic drug leakage or liver toxicity[95-98]. The advantage of these beads is the direct delivery of toxic drugs inside the arterial capillary bed of the tumor, releasing the drug in a controlled manner. In this way the systemic exposure to toxic drugs is reduced, their local concentration is increased and a greater tissue necrosis than classic TACE with lipiodol is obtained[99,100]. The indication for TACE is presence of multinodular LM, absence of extra hepatic disease, refractory to systemic chemotherapy[101]. Recent reports show that TACE with irinotecan (DEBIRI) for the treatment of CRC-LM is effective, feasible and has limited side effects[95-101]. Systemic chemotherapy (FOLFIRI) is compared to DEBIRI for the therapy of refractory CRC-LM in some studies. This comparison shows that DEBIRI is statistically better than FOLFIRI in terms of OS, PFS, time to extra-hepatic progression, and quality of life[95]. The association of cetuximab and TACE with irinotecan is an improvement in the treatment of CRC-LM, because these drugs are efficacious and have acceptable, and not cumulative, toxicities[102].

Recent reports show that TACE with irinotecan (DEBIRI) for the treatment of CRC-LM is effective, feasible and has limited side effects[95-101]. Systemic chemotherapy (FOLFIRI) is compared to DEBIRI for the therapy of refractory CRC-LM in some studies. This comparison shows that DEBIRI is statistically better than FOLFIRI in terms of OS, PFS, time to extra-hepatic progression, and quality of life[95]. The association of cetuximab and TACE with irinotecan is an improvement in the treatment of CRC-LM, because these drugs are efficacious and have acceptable, and not cumulative, toxicities[102]. The TACE methodology is constantly improving, in particular, the last innovation is the introduction of new embolics for drug delivery. Among the new types of microspheres there are polyethylene glycol (PEG) microspheres (LifePearls, Terumo), that are more resistant to stress and attrition. The advantages of these embolics are increased suspension time, better catheter deliverability and drug retention and release[103].

new embolics for drug delivery. Among the new types of microspheres there are polyethylene glycol (PEG) microspheres (LifePearls, Terumo), that are more resistant to stress and attrition. The advantages of these embolics are increased suspension time, better catheter deliverability and drug retention and release[103]. In a recent study we show the data of TACE with PEG embolics for the treatment of 20 cases of non resectable liver tumors and metastases from colorectal carcinoma, breast cancer and uveal melanoma. Irinotecan and doxorubicin are used for PLC and LM respectively. More than 80% of cases respond to TACE patients. We observe 63% of CR, and 37% PR. The chemoembolization procedure is well tolerated by all the patients with only mild or moderate adverse events. These results indicate that PEG embolics-TACE is effective and tolerable for the therapy of hepatic primary and metastatic cancer[103]. Radioembolization In the last decade radioembolization (RE) with Yttrium 90 (Y90) has been widely used for the treatment of CRC-LM that are refractory to chemotherapy[104]. Objective tumor response rates of RE are 33%-48% in second line[105,106] and 10%-48% in third line[107-109]. Survival and progression free survival are also improved after RE application as third line[109]. RE with Y90 has, however, a low recommendation in the last ESMO guidelines[110].

refractory to chemotherapy[104]. Objective tumor response rates of RE are 33%-48% in second line[105,106] and 10%-48% in third line[107-109]. Survival and progression free survival are also improved after RE application as third line[109]. RE with Y90 has, however, a low recommendation in the last ESMO guidelines[110]. The treatment decision is very challenging for CRC-LM patients that are refractory to chemotherapy. Several patients are unfit and have a biologically unfavorable progression often associated to comorbidities. Palliative care with chemo- or radio-embolization is indicated in these cases, in order to avoid too aggressive therapies. MULTIDISPLINARY TEAM The involvement of a multidisciplinary approach should be promoted in order to obtain the best CRC-LM management and outcomes, and to reduce peri-operative morbidity and mortality, prolonging OS and rising resection rates[110,111]. For this reason, the multidisciplinary team management of CRC-LM is growing in most Western countries[112]. The team includes different types of specialists including: Liver surgeons; interventional radiologists specialized in hepatobiliary disease; an oncologist; a pathologist; and a case manager nurse. They have to discuss each case to ensure resectability appropriateness and lead to down-staging wherever possible. The team should be consulted about the choice of chemotherapy combination and type of targeted agents and care to be used, timing of chemo-holidays, and follow up.

a pathologist; and a case manager nurse. They have to discuss each case to ensure resectability appropriateness and lead to down-staging wherever possible. The team should be consulted about the choice of chemotherapy combination and type of targeted agents and care to be used, timing of chemo-holidays, and follow up. Medical oncologists select the most active treatment for the shortest time combining chemotherapy to targeted drugs, in order to reduce tumor size without damaging the normal liver. The definition of the acceptable FRL should be performed by a radiologist and a liver surgeon. Repeating the resection is safe and effective, obtaining survival rates close to those after first resection[112,113]. Finally the case manager nurse or the practitioner are important in patient’s management, because they provide indications on the follow up and assistance. CONCLUSION Recent improvements of CRC-LM treatment allows the down-staging of several patients, resulting in increased number of patients cured or living with longer disease control. There is currently no agreement about the correct sequence of surgical resection of the primary cancer and metastatic disease, however, the neoadjuvant chemotherapy is widely accepted as up front treatment. Surgical resection can be performed if the complete removal of cancer is achievable leaving an adequate FRL. The use of adjuvant chemotherapy is highly suggested, even if standardized protocols are still unclear. The use of chemotherapy may lead to disease regression for unresectable CRC-LM, allowing resection and cure.

Surgical resection can be performed if the complete removal of cancer is achievable leaving an adequate FRL. The use of adjuvant chemotherapy is highly suggested, even if standardized protocols are still unclear. The use of chemotherapy may lead to disease regression for unresectable CRC-LM, allowing resection and cure. The application of loco-regional therapies is increasing, resulting in high tumor response, however, they are not recommended as first-line treatment in case of unresectable CRC-LM. Conflict-of-interest statement: Authors declare no conflict of interests for this article. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Italy Peer-review report classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): E, E Peer-review started: January 20, 2017 First decision: March 27, 2017 Article in press: May 4, 2017 P- Reviewer: Kai K, Ooi LLPJ, Rege RV, Zhong JH S- Editor: Song XX L- Editor: A E- Editor: Lu YJ

Core tip: Accurate staging and circumferential resection margin evaluation significantly impacts determining optimal treatment scheme. Preoperative magnetic resonance imaging (MRI) is highly accurate; however, it has yet to be proved as effective in re-staging. The adding of diffusion-weighted sequences to standard T2-weighted MRI can positively affect its diagnostic accuracy. INTRODUCTION Multimodal treatment of rectal cancer, with the combination of preoperative (neoadjuvant) chemoradiotherapy (CRT) followed by surgery increases local control in locally advanced cancers and has become the standard approach to such rectal cancers[1-5]. High-resolution pelvic magnetic resonance imaging (MRI) is the primary method for evaluation in rectal cancer[6-10]. When applied according to the optimal protocols, high-resolution MRI accurately determining patients regarding neoadjuvant CRT requirement[11]. Moreover, assessing treatment response in tumors using MRI also predicts probable survival outcomes, and could be used in the future to further adjust treatment according to the patients’ response[12]. In recurrent rectal cancer, MRI enables the depiction of the extent of tumor growth, and can establish the resectability of disease[13,14]. MRI has not met expectations in re-staging, especially in complete response evaluation after neoadjuvant CRT because of post-therapeutic fibrosis and inflammation[15-19]. However, adding functional MR sequences such as dynamic contrast-enhanced and diffusion-weighted sequences to the standard approach can improve diagnostic accuracy of MRI[20-23].

in re-staging, especially in complete response evaluation after neoadjuvant CRT because of post-therapeutic fibrosis and inflammation[15-19]. However, adding functional MR sequences such as dynamic contrast-enhanced and diffusion-weighted sequences to the standard approach can improve diagnostic accuracy of MRI[20-23]. In this pictorial review, we present a synopsis of the current standing of MRI in the staging and re-staging of rectal cancer. We also present an experience- and literature-based discussion of limitations and difficulties in interpretation. MRI TECHNIQUE Rectal MRI should be performed with pelvic phased-array coils. Rectal MRI using this technique provides overall assessment of the rectal wall layers with high-spatial-resolution and benefits from a large field of view[15,24]. PATIENT PREPARATION Routine rectal filling using endoluminal contrast agents such as ultrasonography gel is discouraged[24] because this can distend of the rectum and compress the mesorectal fat, which may result in overestimation of fascial involvement and interfere with assessment of mesorectal nodes[25]. Bowel preparation is generally not necessary before the examination, but spasmolytics can be used when excessive fecal matter is visible on the planning images[15,24]. For this purpose, a dose of 40 mg butylscopolamine is used intramuscularly unless contraindicated, immediately prior to placing the patient on the MRI table.

preparation is generally not necessary before the examination, but spasmolytics can be used when excessive fecal matter is visible on the planning images[15,24]. For this purpose, a dose of 40 mg butylscopolamine is used intramuscularly unless contraindicated, immediately prior to placing the patient on the MRI table. IMAGING PROTOCOL Standard MR rectal protocols must at least include 2D T2-weighted sequences in sagittal, axial, and oblique coronal planes with 1-3 mm slice thicknesses. Sagittal sequences are used to identify the longitudinal tumor axis such that axial and coronal planes may be angled as perpendicular and parallel to the tumor axis as possible, respectively. Coronal planes must be angled in line with the anal canal for low tumors in order to evaluate the relation to the anal complex and pelvic floor muscles[15,24,26] (Figure 1). Axial images are useful for evaluation of the tumor and its relationship with the intestinal wall, mesorectal fascia (MRF), and the adjacent pelvic tissue. Sagittal images are useful for the assessment of the tumor height and length and its relationship with peritoneum and other adjacent tissue.

s[15,24,26] (Figure 1). Axial images are useful for evaluation of the tumor and its relationship with the intestinal wall, mesorectal fascia (MRF), and the adjacent pelvic tissue. Sagittal images are useful for the assessment of the tumor height and length and its relationship with peritoneum and other adjacent tissue. Figure 1 Magnetic resonance imaging planes. T2-weighted sagittal images are used to determine the longitudinal tumor axis in order to angle the axial and coronal planes. A: Oblique axial plane is obtained perpendicular to the rectal wall at the level of the rectal mass; B: Oblique axial plane is angled perpendicular to the pelvic floor, used to cover lymph node drainage territory; C: Coronal plane is angled parallel to the anal canal for imaging of low rectal tumors. Rectal tumor is indicated by arrows. In addition to T2-weighted sequences, diffusion-weighted imaging (DWI) sequences are recommended for inclusion in restaging protocols. DWI provides no additional benefit in primary staging; however, evidence is accumulating suggesting that it increases the diagnostic capability of MRI in the assessment of therapy response (yT-stage) after CRT[24]). DWI also helps T2-weighted fast-spinecho (FSE) sequences to distinguish patients having good vs poor response[20-23]. However, there is not adequate proof for supporting the usage of DWI for primary T-staging and lymph node assessment[27].

pability of MRI in the assessment of therapy response (yT-stage) after CRT[24]). DWI also helps T2-weighted fast-spinecho (FSE) sequences to distinguish patients having good vs poor response[20-23]. However, there is not adequate proof for supporting the usage of DWI for primary T-staging and lymph node assessment[27]. ANATOMIC LANDMARKS The rectum is approximately 15 cm in length from the anal verge, which is the lowest part of the anal canal. The rectum has traditionally been divided into three segments according to the distance from the anal verge: Upper (> 10 cm), middle (5-10 cm), and lower (< 5 cm)[27,28] (Figure 2). Figure 2 Rectal segments. T2-weighted sagittal image shows rectal segments: Lower, < 5 cm; middle, 5-10 cm; upper, > 10 cm from the anal verge. The upper and middle rectal walls consist of three separate layers that can be distinguished in MRI. T2-weighted MRI sequences are the best for visualizing rectal wall anatomy. The internal hyperintense layer represents the mucosa and submucosa (no distinction is possible between in two layers); the medial hypointense layer and external hyperintense area represent the muscularis propria and the mesorectum, respectively[15,29] (Figure 3).

ences are the best for visualizing rectal wall anatomy. The internal hyperintense layer represents the mucosa and submucosa (no distinction is possible between in two layers); the medial hypointense layer and external hyperintense area represent the muscularis propria and the mesorectum, respectively[15,29] (Figure 3). Figure 3 Normal rectal wall anatomy of higher and middle rectum. Schematic (A) and T2-weighted axial magnetic resonance imaging (B) presentation. The internal hyperintense layer represents the mucosa and submucosa (no distinction is possible between in two layers); the medial hypointense layer and external hyperintense area represent the muscularis propria and the mesorectum, respectively. Mesorectal fascia is seen thin hypointense layer enveloping the mesorectum (arrows). The puborectal sling constitutes the upper limit of the anal canal. The inner muscular wall of the anal canal comprises the internal sphincter, which is the direct continuation of the circular layer of the muscularis propria of the rectum. The outer muscular wall of the anal canal is cranially composed of the puborectal muscle and caudally of the external sphincter[15,26] (Figure 4). Figure 4 Normal anatomy of lower rectum. Schematic (A) and coronal plane T2-weighted (B) magnetic resonance imaging presentation. Puborectal sling, the upper portion of the puborectal muscle displaying the uppermost portion of the anal canal (intermittent line). Anal verge is the lowermost portion of the anal canal (line). LA: Levator ani muscle; IS: Internal sphincter; ES: External sphincter.

weighted (B) magnetic resonance imaging presentation. Puborectal sling, the upper portion of the puborectal muscle displaying the uppermost portion of the anal canal (intermittent line). Anal verge is the lowermost portion of the anal canal (line). LA: Levator ani muscle; IS: Internal sphincter; ES: External sphincter. The puborectal sling constitutes the upper limit of the anal canal. The internal sphincter (the internal muscular wall) of the anal canal is consisted of the direct continuity of the muscularis propria circular layer of the rectum. The external muscular wall of the anal canal is formed by the puborectal muscle in cranially and the external sphincter in caudally[15,26] (Figure 4). The peritoneal reflection covers the anterior wall of the upper rectum; the risk of peritoneal perforation in upper rectal tumors is high[27]. The peritoneal reflection can be easily displayed on sagittal and axial high-resolution T2-weighted images. In sagittal images, it can be depicted whereon upper pole of the seminal vesicles in men and at the uterocervical angle in women[15]. The evaluation of the peritoneal invasion is very important in staging, because rectal tumor is staged as T4a in the presence of peritoneal invasion (Figure 5).

ution T2-weighted images. In sagittal images, it can be depicted whereon upper pole of the seminal vesicles in men and at the uterocervical angle in women[15]. The evaluation of the peritoneal invasion is very important in staging, because rectal tumor is staged as T4a in the presence of peritoneal invasion (Figure 5). Figure 5 Periton invasion in female (A and B) and male (C and D) patients with T4a rectal tumors. On sagittal T2-weighted images, periton is seen as a hypointense linear structure in front of the tumor (arrows in A, C). On axial T2-weighted images, the peritoneum has a V shape and attaches onto the anterior aspect of the rectal cancer (arrows in B and D). T: Tumor; U: Uterus; P: Prostate. The middle rectum, which lies below the peritoneal reflection, is completely surrounded by mesorectal fatty tissue which is called the mesorectum. Mesorectum is encircled by the MRF which is constitutes the circumferential resection margin (CRM)[26-29]. The MRF can be seen as a thin, low-signal intensity envelop which surrounds the rectum and mesorectum (Figure 6). MRF tapers downward at the lower rectal level[26]. The MRF is easily seen in posterolateral views, although it is difficult to distinguish it from Denonvilliers’ fascia in the anterior wall[30]. Figure 6 Magnetic resonance imaging anatomy of mesorectum and mesorectal fascia. On T2-weighted (A) axial and (B) coronal plane magnetic resonance images, mesorectal fascia (arrows) is seen as a thin, low-signal intensity layer enveloping the mesorectal fatty tissue (*) and rectum in a male patient with rectal carcinoma.

Figure 6 Magnetic resonance imaging anatomy of mesorectum and mesorectal fascia. On T2-weighted (A) axial and (B) coronal plane magnetic resonance images, mesorectal fascia (arrows) is seen as a thin, low-signal intensity layer enveloping the mesorectal fatty tissue (*) and rectum in a male patient with rectal carcinoma. PRIMARY STAGING OF RECTAL CANCER Tumor height and length Tumor height and length should be routinely reported because outcomes and surgical management are affected by the location of the tumor[24]. The distance and length are measured on a line drawn on the sagittal MR images. For tumor localization, the distance of the lowest portion of the tumor from the anal verge is measured. Rectal tumors are classified as high, middle or low when their most caudal border is > 10 cm, 5-10 cm, or < 5 cm from the anal verge, respectively[15] (Figure 7). Figure 7 Rectal tumor levels. T2-weighted sagittal images in different patients with rectal carcinoma show distance from the anal verge (double-headed arrows) in (A) low rectal, (B) midrectal, and (C) upper rectal tumors (low rectal tumor, < 5 cm; midrectal, 5-10 cm; upper rectal, > 10 cm). T staging for middle and high tumors On T2-weighted imaging, the muscularis propria is seen as a hypointense line between the hyperintense mesorectal fat and the inner submucosa and mucosa, which show intermediate to mild hyperintensity. The signal intensity of a rectal tumor on T2-weighted images is typically intermediate between the signal intensity of the muscularis propria and mucosa (Figure 8).

a is seen as a hypointense line between the hyperintense mesorectal fat and the inner submucosa and mucosa, which show intermediate to mild hyperintensity. The signal intensity of a rectal tumor on T2-weighted images is typically intermediate between the signal intensity of the muscularis propria and mucosa (Figure 8). Figure 8 Rectal tumor T staging. The American Joint Committee on Cancer suggested an optional stratification of T3 tumors based on the extramural depth of invasion: Less than 5 mm, T3a; 5-10 mm, T3b; and more than 10 mm, T3c (adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344). T1 tumors are confined to the submucosa; T2 tumors extend into, but not beyond, the muscularis propria. The differentiation of T1 tumors from T2 tumors on MRI is usually not reliable without an endorectal coil or endorectal ultrasound, and tumors should generally be staged as T1/T2[15]. A tumor is staged as T3 when it extends beyond the muscularis propria and strands the mesorectal fat. Disruption of the muscularis propria because of penetrating vessels should not be overstaged as T3 (Figures 8 and 9).

Posttreatment DW; C: ADC images delineate high and low signal-intensity corresponding to the tumor, respectively (arrow). Pre- and post-treatment mean ADC values are 0.68-0.72, 1.22-1.44 × 10-3 mm²/s, respectively, in the tumor area. Post-therapy ADC increase is compatible with therapy response. CRT: Chemoradiotherapy. In addition to morphologic findings, DWI can provide functional information that can be correlated with changes at the cellular level in response to treatment. After CRT, the decrease in cellularity and development of fibrosis or necrosis in responders results in an increase in diffusion, which decreases diffusion signal intensity in diffusion-weighted images and increases ADC values and ADC signal intensity in ADC images[20,23] (Figures 18 and 19). Although DWI can differentiate viable tumor from fibrosis and good and bad response, it does not allow for predicting complete response[19] (Figure 20). Moreover, the response of mucinous tumors to CRT cannot be assessed using DWI because they exhibit ADC hyperintensity even before treatment (Figure 21).

out an endorectal coil or endorectal ultrasound, and tumors should generally be staged as T1/T2[15]. A tumor is staged as T3 when it extends beyond the muscularis propria and strands the mesorectal fat. Disruption of the muscularis propria because of penetrating vessels should not be overstaged as T3 (Figures 8 and 9). Figure 9 Rectal cancer T staging on magnetic resonance imaging. T2-weighted axial images showing rectal carcinomas with different T stages. A: T1 tumor is confined to the submucosa, has not entered the muscularis propria (arrowheads); B: T2 tumor extends into, but not beyond, the muscularis propria (arrowheads); C: T3 tumor extends beyond the muscularis propria and strands into mesorectal fat (arrowheads); D: T4a tumor invades the visceral peritoneum (arrowheads). T: Tumor. The extramural depth of invasion refers to extension of tumor beyond the muscularis propria[31]. The American Joint Committee on Cancer suggested an optional stratification of T3 tumors based on the extramural depth of invasion: Less than 5 mm, T3a; 5-10 mm, T3b; and more than 10 mm, T3c[32]. An extramural depth of invasion of less than 5 mm presents a significantly higher survival rate, and these early T3 tumors may be adequately managed with surgery alone and have a prognosis comparable to that of tumors characterized as T1/T2[33]. T4 tumors extend onto the surface of the visceral peritoneum or an adjacent structure (Table 1, Figures 8 and 10). Table 1 Staging systems for rectal cancer

The extramural depth of invasion refers to extension of tumor beyond the muscularis propria[31]. The American Joint Committee on Cancer suggested an optional stratification of T3 tumors based on the extramural depth of invasion: Less than 5 mm, T3a; 5-10 mm, T3b; and more than 10 mm, T3c[32]. An extramural depth of invasion of less than 5 mm presents a significantly higher survival rate, and these early T3 tumors may be adequately managed with surgery alone and have a prognosis comparable to that of tumors characterized as T1/T2[33]. T4 tumors extend onto the surface of the visceral peritoneum or an adjacent structure (Table 1, Figures 8 and 10). Table 1 Staging systems for rectal cancer Stage MRI findings T stage for middle and high tumors1 T1 Tumor signal intensity is confined to the submucosal layer T2 Tumor signal intensity extends into the muscle layer, with loss of the interface between the submucosa and circular muscle layer T3 Tumor signal intensity extends through the muscle layer into the perirectal fat, with obliteration of the interface between muscle and perirectal fat T3a Tumor < 5 mm into the perirectal fat T3b Tumor 5-10 mm into the perirectal fat T3c Tumor > 10 mm into the perirectal fat T4a Tumor signal intensity extends to surface of visceral peritoneum T4b Tumor signal intensity extends into an adjacent structure or viscus T stage for low tumors2 T1 Tumor signal intensity confined to bowel wall, outer muscle coat intact T2 Tumor signal intensity replaces muscle coat but does not enter intersphincteric plane T3 Tumor signal intensity extends intersphincteric plane or lies within 1 mm of levator muscle T4 Tumor signal intensity extends external anal sphincter or is within1 mm or beyond levator muscle with/without adjacent organ invasion N stage Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in 1-3 regional lymph nodes N2 Metastasis in > 3 regional lymph nodes 1 Adapted from ref. [32]: Edge SB, Byrd DR, Compton CC. AJCC cancer staging handbook: from the AJCC cancer staging manual, 7th ed. New York, NY: Springer, 2010: 718;

al lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in 1-3 regional lymph nodes N2 Metastasis in > 3 regional lymph nodes 1 Adapted from ref. [32]: Edge SB, Byrd DR, Compton CC. AJCC cancer staging handbook: from the AJCC cancer staging manual, 7th ed. New York, NY: Springer, 2010: 718; 2 Adapted from ref. [40]: Taylor FG, Swift RI, Blomqvist L, Brown G. A systematic approach to the interpretation of preoperative staging MRI for rectal cancer. AJR 2008; 191: 1827-1835. MRI: Magnetic resonance imaging. Figure 10 Stratification of T3 tumors on magnetic resonance imaging. T2-weighted axial magnetic resonance images in different patients with T3 rectal carcinoma showing extension of the tumor beyond the muscularis propria (double-headed arrows). The distance A: Less than 5 mm, T3a; B: 5-10 mm, T3b; and C: More than 10 mm, T3c. Distance to the mesorectal fascia For T3 tumors, the shortest distance between the most penetrating parts of the tumor and the MRF should be measured[34,35]. The distance to the MRF is a critical local prognostic factor for rectal cancer[36,37]. A tumor-MRF distance of more than 1 mm is a reliable predictor for negative margins after TME[38]. In the presence of satellite nodules such as tumor deposits, lymph nodes or extramural vascular invasion (EMVI), the shortest distance between the nodules and the MRF should also be reported[15] (Figures 11 and 12).

,37]. A tumor-MRF distance of more than 1 mm is a reliable predictor for negative margins after TME[38]. In the presence of satellite nodules such as tumor deposits, lymph nodes or extramural vascular invasion (EMVI), the shortest distance between the nodules and the MRF should also be reported[15] (Figures 11 and 12). Figure 11 Schematic representation of positive resection margin. For T3 tumors, the shortest distance between the most penetrating parts of the tumor and the MRF is measured (double-headed arrows). A tumor mesorectal fascia distance of more than 1 mm is a reliable predictor for negative margins. In the presence of satellite nodules such as tumor deposits, lymph nodes or EMVI the shortest distance between the nodules and the MRF should also be reported (Adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344). EMVI: Extramural vascular invasion; MRF: Mesorectal fascia.

F should also be reported (Adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344). EMVI: Extramural vascular invasion; MRF: Mesorectal fascia. Figure 12 Distance to mesorectal fascia and mesorectal fascia invasion in different patients on T2-weighted axial images. A: T3a tumor is far away from the mesorectal fascia (double-headed arrow); B: T4a tumor (white arrowhead) and a suspicious mesorectal lymph node (arrow) are abutting the mesorectal fascia; C: Rectal tumor is lying > 1 mm from the mesorectal fascia; however, a suspicious lymph node, located out of the mesorectal fascia, is lying within < 1 mm of the mesorectal fascia (arrow). Mesorectal fascia is indicated with black arrowheads. EMVI EMVI is associated with local and distant recurrence and poor survival[39]. It is defined as the presence of malignant cells within blood vessels located beyond the muscularis propria in the mesorectal fat. EMVI is suggested when vessels close to the tumor are obviously irregular or expanded by tumoral signal intensity[39] (Figure 13). Figure 13 Extramural vascular invasion. T2-weighted (A) coronal and (B and C) serial axial magnetic resonance images in the same patient with T4a rectal cancer showing an irregular and expanded vessel insert to the tumor with tumoral signal intensity (circles).

EMVI EMVI is associated with local and distant recurrence and poor survival[39]. It is defined as the presence of malignant cells within blood vessels located beyond the muscularis propria in the mesorectal fat. EMVI is suggested when vessels close to the tumor are obviously irregular or expanded by tumoral signal intensity[39] (Figure 13). Figure 13 Extramural vascular invasion. T2-weighted (A) coronal and (B and C) serial axial magnetic resonance images in the same patient with T4a rectal cancer showing an irregular and expanded vessel insert to the tumor with tumoral signal intensity (circles). The assessment of EMVI is a routine component of MR evaluation for primary staging; however, for restaging, there is no agreement as to whether evaluation of EMVI remains beneficial[24]. T staging for low tumors A specific T staging system is used to identify tumors and its circumferential resection margin (CRM)[40] (Table 1, Figures 14 and 15). Figure 14 Schematic and high-spatial-resolution coronal T2-weighted magnetic resonance images for each stage according to the low rectal cancer. Rectal tumors in different patients are indicated with arrows on magnetic resonance images (Adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344).

l tumors in different patients are indicated with arrows on magnetic resonance images (Adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344). Figure 15 Stage 4 low rectal cancer. On T2-weighted (A) coronal (B, C) serial axial magnetic resonance images, rectal cancer showing invasion of levator ani (red arrowheads) and mesorectal fascia (white arrowhead). LA: Levator ani; PR: Puborectal; MRF: Mesorectal fascia; BL: Bladder; V: Vagina. N-staging Staging of nodes is very important for planning preoperative treatment[41]. In the TNM system, disease involving only the regional nodes, including the mesorectal and internal iliac nodes, accounts for the N stage (Table 1); involvement of other nodes is regarded as metastasis[38]. Mesorectal nodes are often the first and the most commonly involved group of nodes. Nodal metastases are usually within the proximal 5 cm of the tumor[41]. Extramesorectal nodes (iliac, superior rectal or inferior mesenteric nodes) are generally involved in locally advanced cancers[42]. Low rectal tumors can also spread superficial inguinal nodes and imply poor prognosis[43].

Mesorectal nodes are often the first and the most commonly involved group of nodes. Nodal metastases are usually within the proximal 5 cm of the tumor[41]. Extramesorectal nodes (iliac, superior rectal or inferior mesenteric nodes) are generally involved in locally advanced cancers[42]. Low rectal tumors can also spread superficial inguinal nodes and imply poor prognosis[43]. Node size is the usual criterion in nodal staging using MRI. Lymph nodes are usually considered pathologic when their short axis is longer than 0.5 cm; however, no optimal cut-off threshold exists for involved nodes[24]. The inclusion of morphologic features such as round shape, irregular contour, and nonhomogeneous signal intensity to a size cutoff increases the accuracy of MR[44]. Although DW MRI is not accurate enough for characterizing nodes, it may be useful for locating them[45] (Figure 16). Figure 16 Mesorectal and extramesorectal lymph node involvement in rectal cancer. A: T2-weighted; B: T1-weighted contrast-enhanced axial MR images; C: 18F-FDG PET-CT; D: DWI showing suspicious lymph nodes in mesorectal (red arrows) and extramesorectal areas (white areas). On DWI, extramesorectal lymph node is more remarkable than T2W and contrast-enhanced T1W sequences. DWI: Diffusion-weighted imaging; 18F-FDG PET-CT: 18F-fluorodeoxyglucosepositron emission tomography-computedtomography.

I showing suspicious lymph nodes in mesorectal (red arrows) and extramesorectal areas (white areas). On DWI, extramesorectal lymph node is more remarkable than T2W and contrast-enhanced T1W sequences. DWI: Diffusion-weighted imaging; 18F-FDG PET-CT: 18F-fluorodeoxyglucosepositron emission tomography-computedtomography. RESTAGING AFTER NEOADJUVANT TREATMENT Neoadjuvant CRT provides downstaging and downsizing along with improvement in less extensive surgery, decreased local recurrence, and general survival[12,46]. Tumor restaging involves correlating the posttreatment images with the pretreatment images with respect to all the elements assessed in the initial staging, and necessitates image acquisition with almost the same protocol and on the same planes. T staging Post-CRT restaging using conventional MR sequences is less accurate than primary staging, especially when confirming complete response (yT0), mostly because it is difficult to distinguish fibrosis, edema and normal mucosa from small foci of residual tumor[46-48]. As such, a normal, two-layered rectal wall after CRT is indicative of complete response, whereas residual fibrosis indicates either residual tumor or complete response (Figure 17). In practice, areas of fibrosis have very low signal intensity on post-CRT T2-weighted MRI, in contrast, areas of residual tumor have intermediate signal-intensity[46]. Careful review of high-resolution images and DWI can enable distinction of small residual tumor within fibrosis (Figure 18).

plete response (Figure 17). In practice, areas of fibrosis have very low signal intensity on post-CRT T2-weighted MRI, in contrast, areas of residual tumor have intermediate signal-intensity[46]. Careful review of high-resolution images and DWI can enable distinction of small residual tumor within fibrosis (Figure 18). Figure 17 Tumor restaging after neoadjuvant chemoradiotherapy. On T2-weighted MR images in different patients showing baseline and post-CRT images on upper and lower series, respectively. A: In ypT0 rectal tumor, posttreatment axial image shows a normal, two-layered rectal wall (arrow), corresponding to complete response; B: In ypT3 rectal tumor, posttreatment axial image shows normal, two-layered rectal wall (arrow). This is an example for false-negative MR assessment of complete tumor regression; C: In ypT0 rectal tumor, posttreatment axial image shows thick, fibrotic low signal intensity scar (arrow) in pretreatment T3 tumor area. CRT: Chemoradiotherapy.

es 18 and 19). Although DWI can differentiate viable tumor from fibrosis and good and bad response, it does not allow for predicting complete response[19] (Figure 20). Moreover, the response of mucinous tumors to CRT cannot be assessed using DWI because they exhibit ADC hyperintensity even before treatment (Figure 21). Figure 19 Post-chemoradiotherapy restaging using diffusion-weighted imaging in ypT0 rectal tumor. On T2-weighted (A), DW (B) and ADC (C) images in the same patient, baseline and post-CRT images are shown on upper and lower series, respectively. A: Posttreatment T2-weighted axial image shows a thick wall of low-signal-intensity fibrosis in the previous rectal tumor area (arrow). It is difficult to determine whether this area contains tumor cells or completely devoid of tumor cells (complete response); B: On posttreatment DW image (B-800), there is no diffusion signal in previous tumor area (arrows), compatible with complete response. In this case, DWI allows the correct differentiation of viable tumor from fibrosis; C: ADC images show post-therapy mean ADC increase (0.70 × 10-3 mm²/s vs 1.40 × 10-3 mm²/s) compatible with therapy response, but does not allow prediction of complete response. DWI: Diffusion-weighted imaging; CRT: Chemoradiotherapy.

e. In this case, DWI allows the correct differentiation of viable tumor from fibrosis; C: ADC images show post-therapy mean ADC increase (0.70 × 10-3 mm²/s vs 1.40 × 10-3 mm²/s) compatible with therapy response, but does not allow prediction of complete response. DWI: Diffusion-weighted imaging; CRT: Chemoradiotherapy. Figure 20 Post-chemoradiotherapy restaging using diffusion-weighted imaging in ypT0 rectal tumor. On T2-weighted (A), DW (B) and ADC (C) images in the same patient, baseline and post-CRT images are shown on upper and lower series, respectively. A: Posttreatment T2-weighted axial image shows a thick wall of low-signal-intensity fibrosis and areas suspicious for residual tumor have intermediate signal-intensity in the previous rectal tumor area (long arrow); B: Posttreatment DW images delinate a small foci of intermediate and low signal-intensity, respectively, compatible with residual tumor (long arrow); C: ADC images show post-therapy mean ADC increase (1.05 × 10-3 mm²/s vs 1.80 × 10-3 mm²/s), compatible with therapy response, but not with complete response. The suspicious mesorectal lymph node (arrowheads) is invisible on T2 and DWI after CRT, but the other two are still visible (short arrows). This case is an example for false-positive tumor and lymph node response evaluation of DWI. DWI: Diffusion-weighted imaging; CRT: Chemoradiotherapy.

but not with complete response. The suspicious mesorectal lymph node (arrowheads) is invisible on T2 and DWI after CRT, but the other two are still visible (short arrows). This case is an example for false-positive tumor and lymph node response evaluation of DWI. DWI: Diffusion-weighted imaging; CRT: Chemoradiotherapy. Figure 21 Mucinous adenocarcinoma. A: T2; B: Diffusion-weighted; C: ADC images in the same patient, baseline and post-CRT images are shown on upper and lower series, respectively. The mucinous tumor exhibits hyperintensity on T2, diffusion, and ADC images before and after treatment regardless of their response to treatment. Pre-and post-treatment ADC values are 1.70 × 10-3 mm²/s and 2.10 × 10-3 mm²/s, respectively. Their response to CRT cannot be assessed using diffusion-weighted imaging. CRT: Chemoradiotherapy. DISTANCE TO THE MESORECTAL FASCIA CRM is considered uninvolved if a tumor free margin is seen at least 1 mm from MRF after CRT. This finding has strong negative predictive value (98%) of MR imaging for CRM involvement, whereas it has low positive predictive value[49]. In some rectal tumor, however, CRT results in a markedly reduction tumor volume, but also in retraction of pre-existing contacts with MRF. It is difficult to determine whether this area contains tumor cells or completely devoid of tumor cells[50] (Figures 22 and 23).

t, whereas it has low positive predictive value[49]. In some rectal tumor, however, CRT results in a markedly reduction tumor volume, but also in retraction of pre-existing contacts with MRF. It is difficult to determine whether this area contains tumor cells or completely devoid of tumor cells[50] (Figures 22 and 23). Figure 22 Schematic representation of effects of chemoradiotherapy on a rectal tumor and circumferential resection margins. Adapted from ref. [27]: Nougaret S, Reinhold C, Mikhael HW, Rouanet P, Bibeau F, Brown G. The use of MR imaging in treatment planning for patients with rectal carcinoma: have you checked the “DISTANCE”? Radiology 2013; 268: 330-344. Figure 23 The effects of chemoradiotherapy on a rectal tumor and circumferential resection margins. T2-weighted axial magnetic resonance images in different patients show baseline and post-CRT images on upper and lower series, respectively. A: Overstaging due to thick, hypointense tissue infiltration at the mesorectal fascia (arrow) in ypT2 rectal tumor with no MRF invasion; B: In ypT3 rectal tumor with no MRF invasion, thick fibrous retractions of the tumor, suspicious for CRM positivity (arrow); C: Rectal mass is markedly shrunken with low-signal-intensity tissue infiltration at the mesorectal fascia (arrow). At surgery, there was tumor invasion of the mesorectal fascia. CRM: Circumferential resection margins; MRF: Mesorectal fascia; CRT: Chemoradiotherapy.

carcinoma (PDAC) is one of the few cancers for which incidence and mortality rates have changed very little over the past three decades. Surgery is the only potentially curative treatment for prolongation of survival and the use of adjuvant therapy following curative surgery significantly improves 5-year survival[2-4]. Pathologic stage of the tumor is the major determinant of survival after curative resection for PDAC[1]. Serum levels of cancer antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) correlate with the extent of disease and are predictive of survival[5-13]. Serum CA 19-9 correlates with tumor burden, resectability and overall survival. Low preoperative serum CA 19-9, postoperative decline and level < 200 U/mL are independent predictors of survival[5]. Identification of novel diagnostic and prognostic biomarkers in the serum, tissue, bile and pancreatic juice of patients with PDAC may improve early diagnosis and selection of patients for adjuvant therapy.

of the tumor, suspicious for CRM positivity (arrow); C: Rectal mass is markedly shrunken with low-signal-intensity tissue infiltration at the mesorectal fascia (arrow). At surgery, there was tumor invasion of the mesorectal fascia. CRM: Circumferential resection margins; MRF: Mesorectal fascia; CRT: Chemoradiotherapy. N-staging After CRT, nodal size (short axis diameter) is more reliable for nodal re-staging. It is difficult to differentiate a metastatic lymph node from a healthy lymph node with irradiation changes using morphologic criteria or DWI; therefore, lymph node restaging often results in overstaging[27,50] (Figures 24 and 25). Figure 24 On diffusion-weighted imaging, false-positive mesorectal lymph node evaluation after chemoradiotherapy in ypT0N0 rectal cancer. A: T2-weighted axial magnetic resonance images show significant diminution in nodal size after chemoradiotherapy, compatible with negative lymph node (arrows); B: Diffusion-weighted images, high diffusion signal continues after treatment in the perirectal lymph node, compatible with positive lymph node (arrows). CRT: Chemoradiotherapy. Figure 25 On diffusion-weighted imaging, false-positive mesorectal lymph node after chemoradiotherapy in ypT0N0 rectal cancer. A: T2-weighted axial images show significant diminution in nodal size, compatible with complete response; B: The contuniation of high diffusion signal intensity on residual fibrotic lymph node incorrectly corresponds to a metastatic lymph node (arrows). CRT: Chemoradiotherapy.

chemoradiotherapy in ypT0N0 rectal cancer. A: T2-weighted axial images show significant diminution in nodal size, compatible with complete response; B: The contuniation of high diffusion signal intensity on residual fibrotic lymph node incorrectly corresponds to a metastatic lymph node (arrows). CRT: Chemoradiotherapy. The accuracy of MRI for restaging is generally lower than the accuracy of MRI for initial staging, mainly owing to overstaging of nodal disease, failure to differentiate tumoral infiltration or residual tumor from desmoplastic reaction or radiation fibrosis[50]. According to recent meta-analysis results, MRI accuracy was variable for restaging rectal cancer after neoadjuvant treatment; however, significantly better results were achieved when DWI was used or with experienced observers. The authors also reported that MRI could be used for evaluating CRM staging, but nodal staging remained a challenge[51]. CONCLUSION Using high-resolution MRI, standardizing image acquisition techniques and interpretation of images, comparative evaluation of pre- and post-CRT MR images, adding DWI to the standard approach, and importantly, experience and awareness of the limitations can improve diagnostic accuracy of MRI for re-staging. Conflict-of-interest statement: Authors declare no conflict of interests for this article. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Turkey Peer-review report classification Grade A (Excellent): A Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: February 10, 2017

Conflict-of-interest statement: Authors declare no conflict of interests for this article. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: Turkey Peer-review report classification Grade A (Excellent): A Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: February 10, 2017 First decision: March 27, 2017 Article in press: May 20, 2017 P- Reviewer: Kim HS, Koda K, Li XX S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: Pancreatic cancer is the third leading cause of cancer mortality in both men and women in the United States. Pancreatic cancer is one of nonimmunogenic cancers that lacks of optimal treatments especially from immunotherapy prospective. Therefore, combining immune checkpoint therapies with other treatment modalities in pancreatic cancer will be the best strategy to achieve better and more durable clinical responses by enhancing cytotoxic T-cell responses. INTRODUCTION Pancreatic cancer is the third leading cause of cancer mortality in both men and women in the United States[1]. The vast majority of patients with pancreatic cancer are diagnosed with advanced disease, and there has been a lack of optimal treatment option as the cancer is highly refractory to standard chemotherapy. Recently, two chemotherapy regimens, FOLFIRINOX and gemcitabine plus albumin-bound paclitaxel (nab-paclitaxel), have emerged as the standard of care for metastatic pancreatic cancer. These two regimens showed improved overall and progression-free survival (PFS) compared to gemcitabine alone in two phase III randomized controlled trials[2,3]. Nevertheless, only up to 30% of patients showed response to either of these two regimens. The median PFS and overall survival (OS) remain poor, under 6 and 12 mo, respectively. Thus, there is still an urgent need to develop therapies that deliver more effective and durable clinical responses.

ed controlled trials[2,3]. Nevertheless, only up to 30% of patients showed response to either of these two regimens. The median PFS and overall survival (OS) remain poor, under 6 and 12 mo, respectively. Thus, there is still an urgent need to develop therapies that deliver more effective and durable clinical responses. RELEVANCE OF IMMUNITY TO PANCREATIC CANCER Observations in human disease and murine modeling has suggested that pancreatic cancer is almost invariably associated with a robust inflammatory infiltrate which can have divergent influences on disease progression by either combating cancer growth via antigen-restricted tumoricidal immune responses or by promoting tumor progression via induction of immune suppression (Figure 1)[4-6]. For example, cluster of differentiation 8 (CD8+) and T-helper type 1 cells (Th1)-polarized cluster of differentiation 4 (CD4+) T cells mediate antitumor effects in murine models of pancreatic cancer and are associated with increased survival in patients with pancreatic cancer[7-10]. Conversely, we recently reported that T- helper type 2 cells (Th2)-polarized CD4+ T cells promote pancreatic cancer progression in mice and intra-tumoral CD4+ Th2 cells infiltrates correlate with reduced survival in human disease[7-9,11-13]. Similarly, Foxp3+ T-regulatory cells (Tregs) facilitate tumor immune escape in pancreatic cancer[14]. Myeloid cells can influence T cells differentiation and cytotoxicity in pancreatic cancer. We reported that tumor-infiltrating myeloid-derived suppressor cells (MDSCs) negate cytotoxic CD8+ T cells anti-tumor responses, accelerates pancreatic cancer growth and metastasis[8,15-17]. Similar to T cells, macrophages also have cell types with different properties such as classically activated (M1) macrophages induce immunogenic responses, whereas alternatively activated (M2) macrophages have permissive influences on tumor growth by recruiting Tregs and Th2 cells[18]. However, the drivers of immunosuppressive cell differentiation in pancreatic cancer are based on comprehensive understanding of regulation of the balance between immunogenic and immune-suppressive T cell populations.

ated (M2) macrophages have permissive influences on tumor growth by recruiting Tregs and Th2 cells[18]. However, the drivers of immunosuppressive cell differentiation in pancreatic cancer are based on comprehensive understanding of regulation of the balance between immunogenic and immune-suppressive T cell populations. Figure 1 Anti-tumor and pro-tumor factors. Anti-tumor factors: M1 (classically activated macrophages), Th1-CD4+ T cells (T-helper type 1-cluster differentiation 4 T cells), CD8+ T cells, DC (dendritic cells), NKG2D (natural killer group 2 member). Pro-tumor factors: M2 (alternatively activated macrophages), Th2-CD4+ T cells (T-helper type 2-cluster differentiation 4 T cells) Th2, Tregs (T-regulatory cells), and MDSCs (myeloid-derived suppressor cells).

-cluster differentiation 4 T cells), CD8+ T cells, DC (dendritic cells), NKG2D (natural killer group 2 member). Pro-tumor factors: M2 (alternatively activated macrophages), Th2-CD4+ T cells (T-helper type 2-cluster differentiation 4 T cells) Th2, Tregs (T-regulatory cells), and MDSCs (myeloid-derived suppressor cells). THE EMERGENCE OF CHECKPOINT IMMUNOTHERAPY The last few years witnessed a paradigm shift in cancer treatment strategy incorporating immunotherapy. Unprecedented clinical success has been observed for therapies targeting two major checkpoints of T cell response (Figure 2): Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1). Both checkpoints are expressed on activated T cells, but they act in distinct pathways. CTLA-4 blocks the essential cluster differentiation 28 (CD28) costimulation by competing and depleting the ligand of CD28 (B7-1 and B7-2) on antigen presenting cells (APCs). On the other hand, PD-1 interferes with the signaling pathways mediated by the T cell receptor and serves as a more distal block of T cell response by binding to its ligands (programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) which are present on many cell types including tumors cells[19].

(APCs). On the other hand, PD-1 interferes with the signaling pathways mediated by the T cell receptor and serves as a more distal block of T cell response by binding to its ligands (programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) which are present on many cell types including tumors cells[19]. Figure 2 Immunotherapy basics. Anti-PD-L1 inhibit PD-L1 (programmed cell death-ligand 1) binding to PD-1 (Programmed cell death protein-1). Anti-PD-1 inhibit PD-1 on T-cell that binds to PD-L1 or PD-L2 (programmed cell death ligand-2) on APC (antigen presenting cell). Anti-CTLA-4 (anti-cytotoxic T lymphocyte antigen 4) inhibit CD28 (cluster differentiation 28) on T cell that binds to B7-1 or B7-2 (ligand of CD28) on APC. Monoclonal antibodies targeting CTLA-4 or PD-1 have shown durable clinical responses and prolonged OS in patients with melanoma, a highly immunogenic cancer. While single agent PD-1/PD-L1 inhibitors demonstrate impressive clinical benefits in many cancers such as non small cell lung cancer (NSCLC), renal cell carcinoma, bladder cancer, and Hodgkin’s lymphoma[20-29]. These results have led to FDA approval of lpilimumab (anti-CTLA-4) in 2011 in melanoma[30]. PD-1 inhibitors such as pembrolizumab and nivolumab were approved later in melanoma as well[23,28,29]. PD-1 inhibitors (nivolumab and pembrolizumab), along with PD-L1 inhibitors such as atezolizumab have been approved in NSCLC, another example of immunogenic cancer[21,22,24,29]. The activity of CTLA-4 and PD-L1 inhibitors are being explored in pancreatic cancer as well[22,31].

proved later in melanoma as well[23,28,29]. PD-1 inhibitors (nivolumab and pembrolizumab), along with PD-L1 inhibitors such as atezolizumab have been approved in NSCLC, another example of immunogenic cancer[21,22,24,29]. The activity of CTLA-4 and PD-L1 inhibitors are being explored in pancreatic cancer as well[22,31]. EVIDENCE OF MINIMAL ACTIVITY OF SINGLE AGENT CHECKPOINT IMMUNOTHERAPY IN PANCREATIC CANCER In early clinical trials single agent therapy with anti-CTLA-4 or anti-PD-1/anti-PD-1 pathway (anti-PD-L1) alone were largely ineffective in pancreatic cancer[22,31,32]. In a single-arm phase II study, lpilimumab failed to induce tumor response in patients with advanced pancreatic cancer[32]. Similarly, single agent BMS-936559, an anti-PD-L1 monoclonal antibody, did not show any activity in 14 patients with advanced pancreatic cancer in a phase I study[22].

pancreatic cancer[22,31,32]. In a single-arm phase II study, lpilimumab failed to induce tumor response in patients with advanced pancreatic cancer[32]. Similarly, single agent BMS-936559, an anti-PD-L1 monoclonal antibody, did not show any activity in 14 patients with advanced pancreatic cancer in a phase I study[22]. POTENTIAL BARRIERS THAT HINDER EFFICACY OF IMMUNOTHERAPY The efficacy of immunotherapy in pancreatic cancer is handicapped by small number of cumulative mutational load that can lead to expression of non-self-antigens, or “neoantigens” which are recognized by the immune system as foreign. Cancers with higher number of mutational load are associated with more neoantigens that are easier to be recognized by the immune system, compared to cancer with lower number of mutational load[33-35]. There are 3 major barriers for the utility of immunotherapy in pancreatic cancer. First, the mutational load in pancreatic cancer is very low as compared with melanoma and lung cancers[36,37]. Second, pancreatic cancer features a largely immunosuppressive microenvironment, characterized by a dense desmoplastic reaction with prominent infiltration of tumorigenic macrophages and myeloid derived suppressor cells (MDSCs)[38]. Third, there are very few infiltrating T cells in the microenvironment of pancreatic cancer, therefore could not provide sufficient T cell responses. Pancreatic cancer creates a nonimmunogenic (or “cold”) tumor microenvironment, limiting the activity of immune checkpoint therapies[31].

ved suppressor cells (MDSCs)[38]. Third, there are very few infiltrating T cells in the microenvironment of pancreatic cancer, therefore could not provide sufficient T cell responses. Pancreatic cancer creates a nonimmunogenic (or “cold”) tumor microenvironment, limiting the activity of immune checkpoint therapies[31]. EVIDENCE OF T CELL IMMUNITY On the other hand, there is still evidence of T cell-mediated immunity in pancreatic cancer. An analysis of resected surgical samples of pancreatic cancer patients has shown that higher levels of CD4+ and CD8+ tumor infiltrating T cells are associated with better prognosis[10]. In addition, since immunosuppression occurs early during tumorigenesis as shown in Pdx1Cre;KrasG12D;Tp53R172H (KPC) mouse model, the tumor cells may have been shielded from immune pressure, thus preserving their sensitivity to T cell attack[38].

CD8+ tumor infiltrating T cells are associated with better prognosis[10]. In addition, since immunosuppression occurs early during tumorigenesis as shown in Pdx1Cre;KrasG12D;Tp53R172H (KPC) mouse model, the tumor cells may have been shielded from immune pressure, thus preserving their sensitivity to T cell attack[38]. In addition, downstream signals are also critical in the T cell immune responses. Interferon-gamma (IFN-γ) promotes inhibition of melanoma cell growth and induces apoptosis of tumor cells by regulating T-cells responses[39-44]. Immune checkpoint inhibitors increase production of IFN-γ from T-cell[45-50]. However its effect will be suboptimal if there is a defect in the IFN-γ pathway[51]. Studies in patients with melanoma showed that a defect in the IFN-γ pathway can lead to resistance to anti-CTLA4 and anti-PD-1 therapies[51,52]. Several genomic biomarkers of IFN-γ pathways such as interferon gamma receptor 1 (IFNGR1), janus kinase 1 (JAK1), and JAK2 have been identified in melanoma patients with good response to immune checkpoint therapies[41-43,51,52]. On the other hand, genes such as suppressor of cytokine signaling 1 (SOCS1) and protein inhibitor of activated signal transducer and activator of transcription 4 (PIAS4) have demonstrated the opposite effects by inhibiting IFN-γ signaling pathway[51,53,54].

ith good response to immune checkpoint therapies[41-43,51,52]. On the other hand, genes such as suppressor of cytokine signaling 1 (SOCS1) and protein inhibitor of activated signal transducer and activator of transcription 4 (PIAS4) have demonstrated the opposite effects by inhibiting IFN-γ signaling pathway[51,53,54]. STRATEGIES OF TURNING ON THE ACTIVITY OF IMMUNOTHERAPY Thus, the incorporation of additional therapies that can turn a “cold” tumor microenvironment into a “hot” one presents an important strategy to elicit clinical activity of immune checkpoint therapies. These additional therapies mainly fall into three categories (Figure 3): First, therapies that enhance tumor antigen presentation to help T cell priming/activation; second, therapies that modulate tumor microenvironment to relieve immunosuppression. Third, therapies which breakdown the desmoplastic barrier surrounding pancreatic cancer to bring infiltrating T cells. Below we will summarize the combination therapies that have already been assessed clinically and provide future directions of new combinations that may hold promise. Figure 3 Searching for the optimal combination to maximize the potential of immune checkpoint blockade for the treatment of pancreatic cancer. CTLA-4: Cytotoxic T lymphocyte antigen-4; PD-1: Programmed cell death protein-1; PD-L1: Programmed death ligand-1; CD40: Cluster differentiation 40; CAR T cells: Chimeric antigen receptor T cells; PI3K: Phosphoinositide-3-kinase; BTK: Bruton tyrosine kinase; JAK: Janus kinase; PEGPH20: Pegylated hyaluronidase.

cancer. CTLA-4: Cytotoxic T lymphocyte antigen-4; PD-1: Programmed cell death protein-1; PD-L1: Programmed death ligand-1; CD40: Cluster differentiation 40; CAR T cells: Chimeric antigen receptor T cells; PI3K: Phosphoinositide-3-kinase; BTK: Bruton tyrosine kinase; JAK: Janus kinase; PEGPH20: Pegylated hyaluronidase. FIRST (ENHANCE T CELL ACTIVATION) Immune checkpoint therapy + chemotherapy Gemcitabine is one of the backbone chemotherapy agents for the treatment of pancreatic cancer. It has been suggested that gemcitabine is not immunosuppressive in pancreatic cancer patients and may be able to enhance naïve T cells activation[55]. Combination of gemcitabine and immune checkpoint blockade has been evaluated for their potential synergistic activity. Gemcitabine plus CTLA-4 blockade: A phase I clinical study evaluated the combination of gemcitabine and an anti-CTLA-4 antibody (tremelimumab) in treatment naive patients with metastatic pancreatic cancer. This combination showed a tolerable side effect. Among 28 out of 34 evaluable patients, 2 achieved partial response (PR) and 7 showed stable disease (SD) for > 10 wk[4]. In another ongoing phase Ib study of unresectable pancreatic cancer, preliminary results showed that, among 11 evaluable patients (out of 13 enrolled), ipilimumab and gemcitabine resulted in 2 PR and 5 SD[56,57].

of 34 evaluable patients, 2 achieved partial response (PR) and 7 showed stable disease (SD) for > 10 wk[4]. In another ongoing phase Ib study of unresectable pancreatic cancer, preliminary results showed that, among 11 evaluable patients (out of 13 enrolled), ipilimumab and gemcitabine resulted in 2 PR and 5 SD[56,57]. Gemcitabine plus PD-1/PD-L1 blockade: An immunohistochemistry analysis has shown that positive PD-L1 expression in resected pancreatic cancer was correlated with worse OS[58]. In a mouse model of pancreatic cancer, combining gemcitabine with either anti-PD-1 or anti-PD-L1 antibody enhanced tumor infiltration of CD8+ T cells and resulted in complete responses in treated mice[58]. A clinical pilot study of combination of gemcitabine and anti-PD-1 antibody has closed to enrollment (NCT01313416).

n a mouse model of pancreatic cancer, combining gemcitabine with either anti-PD-1 or anti-PD-L1 antibody enhanced tumor infiltration of CD8+ T cells and resulted in complete responses in treated mice[58]. A clinical pilot study of combination of gemcitabine and anti-PD-1 antibody has closed to enrollment (NCT01313416). Immune checkpoint therapy + cancer vaccines The most extensively studied pancreatic cancer vaccine is GVAX. GVAX is a whole cell vaccine composed of irradiated, allogeneic pancreatic tumor cells genetically engineered to secret granulocyte macrophage-colony stimulating factor (GM-CSF), a cytokine that stimulates dendritic cell activation and T cell priming. When used as part of adjuvant therapy in the post-resection setting, GVAX was able to induce pancreatic cancer specific CD8+ T cell expansion as shown in a phase II study[59]. Also, when used as neoadjuvant and adjuvant therapy, GVAX and low dose cyclophosphamide (an alkylating agent with an ability to deplete Tregs) resulted in formation of intratumoral tertiary lymphoid aggregates and T cell infiltration, suggesting the ability of GVAX in the conversion of pancreatic cancer from a “non-immunogenic” into an “immunogenic” state[60].

t therapy, GVAX and low dose cyclophosphamide (an alkylating agent with an ability to deplete Tregs) resulted in formation of intratumoral tertiary lymphoid aggregates and T cell infiltration, suggesting the ability of GVAX in the conversion of pancreatic cancer from a “non-immunogenic” into an “immunogenic” state[60]. GVAX plus CTLA-4 blockade: In a small phase Ib study, GVAX in combination with anti-CTLA-4 antibody ipilimumab was evaluated in 30 patients with advanced, refractory pancreatic cancer that were previously treated with gemcitabine-based chemotherapy. Compared to ipilimumab alone, the combination therapy resulted in improved survival (27% vs 7% at 1 year). Also, a longer survival was associated with an increase in peak mesothelin-specific T cells and a larger T cell repertoire (the percentage of mesothelin peptides for which enhanced T-cell responses were measured), indicating a positive role of T cell response[61].

py resulted in improved survival (27% vs 7% at 1 year). Also, a longer survival was associated with an increase in peak mesothelin-specific T cells and a larger T cell repertoire (the percentage of mesothelin peptides for which enhanced T-cell responses were measured), indicating a positive role of T cell response[61]. GVAX plus PD-1/PD-L1 blockade: Detailed analysis of lymphoid aggregates formed after GVAX therapy revealed elevated expression of PD-L1 on monocytes/macrophages[60,62], suggesting the potential benefit of targeting PD-1/PD-L1 checkpoint. This concept was supported by experiments in a pancreatic cancer mouse model, where the combination of GVAX and an anti-PD-1 antibody resulted in better survival than anti-PD-1 antibody alone, and this activity was correlated with increased CD8+ T cells and elevated IFN-γ production in the tumor microenvironment[62]. Currently, a randomized clinical study (NCT02451982) is ongoing to evaluating GVAX with or without anti-PD-1 antibody (nivolumab) as neoadjuvant and adjuvant treatment in patients with resectable pancreatic cancer.

s correlated with increased CD8+ T cells and elevated IFN-γ production in the tumor microenvironment[62]. Currently, a randomized clinical study (NCT02451982) is ongoing to evaluating GVAX with or without anti-PD-1 antibody (nivolumab) as neoadjuvant and adjuvant treatment in patients with resectable pancreatic cancer. GVAX and CRS-207 plus PD-1/PD-L1 blockade: CRS-207 is a bacterial vaccine composed of live-attenuated, double deleted Listeria monocytogenes expressing human mesothelin, an antigen commonly overexpressed in pancreatic cancer cells. CRS-207 can induce robust innate as well as mesothelin-specific adaptive immune response, therefore allowing for a “boost” to the immune response initiated by GVAX. In a randomized, phase II study, GVAX prime followed by CRS-207 boost resulted in prolonged OS compared to GVAX alone in patients with metastatic, refractory pancreatic cancer. This study also showed that mesothelin-specific CD8+ T cell response was correlated with better survival[63,64]. On the basis of these findings, a randomized phase II study (NCT02243371) was to evaluate whether adding anti-PD-1 therapy (nivolumab) will further enhance the activity of this prime-boost strategy[65]. This study has closed to enrollment.

thelin-specific CD8+ T cell response was correlated with better survival[63,64]. On the basis of these findings, a randomized phase II study (NCT02243371) was to evaluate whether adding anti-PD-1 therapy (nivolumab) will further enhance the activity of this prime-boost strategy[65]. This study has closed to enrollment. In a phase IIb study (NCT02004262) in refractory and metastatic pancreatic cancer, 303 patients were randomized between GVAX and CRS-207 (arm A), only CRS-207 (arm B), and single agent chemotherapy (arm C)[66]. No OS advantage was seen in arm A when compared to arm C[66]. A large number of patient drop out prior to treatment was observed in both arm A and C (40% versus 60%, respectively), indicating the challenge of therapeutic benefit in refractory pancreatic cancer. It also hints that these patients in the refractory setting may be too sick to benefit from immunotherapy due to rapid deterioration of disease.

t drop out prior to treatment was observed in both arm A and C (40% versus 60%, respectively), indicating the challenge of therapeutic benefit in refractory pancreatic cancer. It also hints that these patients in the refractory setting may be too sick to benefit from immunotherapy due to rapid deterioration of disease. Immune checkpoint therapy + agents enhancing T cell immunity CD40 agonist: CD40 is a member of the tumor necrosis factor receptor family. Ligation of CD40 can occur on dendritic or B cells, or at CD40 ligand (CD154) on activated T cells, such effect can enhance T cell immunity[67]. In a 22 patients series with unresectable pancreatic cancer, a CD40 agonist (CP-870, 893) and gemcitabine led to an encouraging clinical response[7,11]. Rather unexpectedly, it showed that tumor infiltration by macrophages played a larger role for depletion of tumor stroma and killing of tumor cells[7]. In a more recent study in the KPC mouse model, however, the use of CD40 agonist monoclonal antibody (mAb) with gemcitabine and nab-paclitaxel induced macrophage-independent T cell immunity. This study also found that CD40 agonist in addition to chemotherapy was able to sensitize the tumors to anti-CTLA-4 and/or anti-PD-1 therapies, leading to tumor regression and improved survival[31]. A recent study using an orthotopic pancreatic cancer mouse model also demonstrated tumor regression and enhanced immune response with the combination of CD40 agonist antibody with gemcitabine/Nab-paclitaxel[68]. It is yet to be seen whether these pre-clinical results can translate into clinical benefits.

survival[31]. A recent study using an orthotopic pancreatic cancer mouse model also demonstrated tumor regression and enhanced immune response with the combination of CD40 agonist antibody with gemcitabine/Nab-paclitaxel[68]. It is yet to be seen whether these pre-clinical results can translate into clinical benefits. CAR T cells: Autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) have been developed to trigger cancer-specific T cell immunity and have shown impressive activity in acute lymphoblastic leukemia[69]. For the treatment of pancreatic cancer, the CARs are engineered to recognize mesothelin, a specific membrane protein antigen overexpressed on pancreatic cancer cells. Mesothelin-specific CAR T cells are currently under phase I clinical evaluation, with preliminary results suggesting acceptable safety profiles and potential clinical activity against advanced pancreatic cancer. This study demonstrated that 2 out of 6 patients achieved SD and one patient with liver metastasis at baseline showed no fluorodeoxyglucose (FDG) uptake within 1 mo of treatment[12,70,71]. Therefore, CAR T cells represent another treatment modality to combine with immune checkpoint therapies.

dvanced pancreatic cancer. This study demonstrated that 2 out of 6 patients achieved SD and one patient with liver metastasis at baseline showed no fluorodeoxyglucose (FDG) uptake within 1 mo of treatment[12,70,71]. Therefore, CAR T cells represent another treatment modality to combine with immune checkpoint therapies. Immune checkpoint therapy + radiotherapy The effects of radiotherapy (RT) on the immunology of pancreatic cancer have not been intensively studied. However, work in other cancers has suggested that RT should be considered an immune adjuvant as evidenced by radiotherapy (RT) induced enhancement of both innate and adaptive immunity. For example, the immunogenicity of dendritic cells (DCs) is reportedly improved by RT-induced necrotic tumor cell release of high mobility group box 1 protein (HMGB1) which ligates toll-like receptor 4 (TLR4) and toll-like receptor 9 (TLR9) on DCs. Such events promote DCs’ cellular maturation and enhance their antigen processing capabilities[72]. Another consequence of RT-induced necrotic cell death is the translocation of calreticulin from the endoplasmic reticulum to the plasma membrane which facilitates assembly of major histocompatibility-1 (MHC I)-peptide complexes. Calreticulin also enhances DCs cross presentation of antigens to cytotoxic T lymphocytes. In addition to upregulating the antigen-presentation machinery in DCs, RT can reportedly enhance immunogenicity by inducing the release of tumor antigens, upregulating the expression of T-cell co-activating ligands, and sensitizing tumor cells to antigen-independent cell death via the Fas receptor[72]. RT is further thought to augment diverse aspects of T cell immunity via adenosine triphosphate release from apoptotic cells which induces secretion of Interleukin-1-beta (IL-1β). A consequence of this cascade is T helper1 (Th1) polarization of antigen-restricted CD4+ T cell responses and activation of cytotoxic T cells. Additionally, activation of cytotoxic T cells can be further activated by irradiation, via natural killer group 2 member D (NKG2D) receptor on cytotoxic T cells. NKG2D receptor can be induced in a stress event such as DNA damage which can be achieved by RT[72]. Therefore, ionizing radiation can result in “immunogenic cell death’, in which the dying tumor cells trigger “danger signals” (a signal of releasing HMGB1 and binding to TLR4 and TLR9 on DCs to process the antigen) to boost T cell activation[72,73].

in a stress event such as DNA damage which can be achieved by RT[72]. Therefore, ionizing radiation can result in “immunogenic cell death’, in which the dying tumor cells trigger “danger signals” (a signal of releasing HMGB1 and binding to TLR4 and TLR9 on DCs to process the antigen) to boost T cell activation[72,73]. SECOND (TARGETING IMMUNOSUPPRESSIVE MICROENVIRONMENT) As described earlier, an important barrier to the success of immunotherapy in pancreatic cancer is an immunosuppressive tumor microenvironment, enriched with immunosuppressive cells such as tumor associated macrophages (TAMs) and MDSCs. In animal models of pancreatic cancer, blockade of immunosuppressive MDSCs could promote antitumor T-cell responses and block protumor macrophage responses[6,74-76]. Therefore, drugs that block these immunosuppressive cells in the tumor microenvironment represent attractive strategies to sensitize pancreatic cancer to immune checkpoint therapies. Immune checkpoint therapy + radiotherapy RT’s theoretical potential ability to convert the tumor microenvironment from a “cold” to a “hot” state suggests the opportunity of RT combination with immune checkpoint therapy. In the KPC pancreatic mouse model, any combination of immune checkpoint inhibitor with RT substantially increased OS, when compared to anti-CTLA-4 antibody or anti-PD-L1 antibody alone without RT. In particular, the triple therapy (RT + CTLA-4 antibody + PD-1 antibody) resulted in the highest response rate and longest OS among any of the immunotherapy group as single therapy or in combinations[77].

with RT substantially increased OS, when compared to anti-CTLA-4 antibody or anti-PD-L1 antibody alone without RT. In particular, the triple therapy (RT + CTLA-4 antibody + PD-1 antibody) resulted in the highest response rate and longest OS among any of the immunotherapy group as single therapy or in combinations[77]. However, our recent preclinical studies on RT in pancreatic cancer suggest caution as we found that RT induced the programming and recruitment of immuno-suppressive M2-like macrophages which lead to the expansion of tumor promoting Th2-polarized CD4+ T cells and Tregs. We also found that combining RT with either macrophage neutralization or M-CSF blockade resulted in synergistic efficacy in mice model, suggesting another treatment strategy for pancreatic cancer utilizing RT combining with colony stimulating factor-1 receptor inhibitor[76,78]. So far there have been no published clinical results on RT plus checkpoint blockade for the treatment of pancreatic cancer. Currently, an open-label, three-cohort, multi-institutional phase Ib study is ongoing at New York University (NCT02868632) to assess stereotactic body radiation therapy (SBRT) in combination with either MEDI4736 (an anti-PD-L1 antibody) alone, tremelimumab (an anti-CTLA4 antibody) alone, or the combination of MEDI4736 and tremelimumab in patients with unresectable/locally advanced previously untreated pancreatic cancer. A study with similar design that tests the combination of radiation with checkpoint blockade in second line setting is also ongoing (NCT02311361).

imumab (an anti-CTLA4 antibody) alone, or the combination of MEDI4736 and tremelimumab in patients with unresectable/locally advanced previously untreated pancreatic cancer. A study with similar design that tests the combination of radiation with checkpoint blockade in second line setting is also ongoing (NCT02311361). Immune checkpoint therapy + therapies targeting immunosuppressive microenvironment JAK inhibitors: The Janus kinase (JAK) and its downstream factor signal transducer and activator of transcription (STAT) are important mediators of signaling pathways initiated from cytokine and growth factor receptors. Excessive JAK/STAT signaling can lead to production and release of inflammatory cytokines, promote recruitment, expansion of MDSCs and Tregs which induce an immunosuppressive tumor microenvironment[79]. Also, JAK/STAT pathway has been shown to induce the expression of PD-L1 on cells in the tumor microenvironment[14,80]. In pre-clinical studies, JAK inhibitors led to decreased numbers of Tregs, TAMs and MDSCs, with enhanced number of activity of CD4+ and CD8+ T cells[18]. The study of JAK inhibitor Ruxolitinib and capecitabine for the treatment of advanced pancreatic cancer has closed to enrollment (JANUS study; NCT02117479)[81].

,80]. In pre-clinical studies, JAK inhibitors led to decreased numbers of Tregs, TAMs and MDSCs, with enhanced number of activity of CD4+ and CD8+ T cells[18]. The study of JAK inhibitor Ruxolitinib and capecitabine for the treatment of advanced pancreatic cancer has closed to enrollment (JANUS study; NCT02117479)[81]. PI3K inhibitors: Phosphoinositide-3-kinase (PI3K) is a family of lipid kinases that catalyze the production of second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3), which leads to activation of downstream kinases. PI3K was known to play an important role in signaling pathways in B cells, which were found to contribute to an immunosuppressive microenvironment that dampens T cell immunity[82]. Inactivation of PI3K was associated with a decrease in Tregs and MDSCs and an increase in CD8+ cytotoxic T cell activity, indicating a role of PI3K in regulating tumor microenvironment[5]. PI3K inhibitors could shift immunosuppressive microenvironment in pancreatic cancer into a more immunogenic one. Therefore PI3K inhibitors could help potentiate the activity of immune checkpoint inhibitors.

an increase in CD8+ cytotoxic T cell activity, indicating a role of PI3K in regulating tumor microenvironment[5]. PI3K inhibitors could shift immunosuppressive microenvironment in pancreatic cancer into a more immunogenic one. Therefore PI3K inhibitors could help potentiate the activity of immune checkpoint inhibitors. BTK inhibitors: BTK is a cytoplasmic, Tec family tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. In pancreatic cancer, the BTK inhibitor (ibrutinib) was shown to inhibit mast cells, and as a result, to reduce fibrosis in the tumor microenvironment both in a KPC mouse model and patient-derived xenograft[83]. Ibrutinib was also known to inhibit interleukin-2-inducible T-cell kinase (ITK), an important enzyme for the survival of Th2 cells; thus ibrutinib may be able to shift the balance away from the Th2 cells protumor response and toward the Th1 cells antitumor immune responses. A phase I/II clinical study assessing ibrutinib in combination with anti-PD-L1 antibody MEDI4736 in relapsed or refractory solid tumors, including pancreatic cancer has closed to enrollment (NCT02403271)[84].

hift the balance away from the Th2 cells protumor response and toward the Th1 cells antitumor immune responses. A phase I/II clinical study assessing ibrutinib in combination with anti-PD-L1 antibody MEDI4736 in relapsed or refractory solid tumors, including pancreatic cancer has closed to enrollment (NCT02403271)[84]. THIRD APPROACH (BREAKDOWN DESMOPLASTIC BARRIER) Strategy that targets the desmoplastic stroma PEGPH20: In pancreatic cancer, high levels of hyaluronan in the extracellular matrix contribute to a high interstitial pressure in the tumor stroma, leading to vascular compression and hypoperfusion. Pegylated hyaluronidase PEGPH20 is an enzyme that can degrade hyaluronan, and has been shown in a KPC mouse model to deplete hyaluronan in the tumor stroma and enhance the activity of gemcitabine[85]. In a phase I (28 patients) and a phase II (135 patients) studies, patients with previously untreated advanced pancreatic cancer, PEGPH20 along with chemotherapy (gemcitabine, or gemcitabine/nab-paclitaxel) resulted in good tumor response and PFS, but only in patients with high levels of hyaluronan[15,86]. Therefore, in pancreatic cancers with high levels of hyaluoran, PEGPH20 therapy may allow more effective T cell infiltration and enhance the activity of immune checkpoint therapies.

abine, or gemcitabine/nab-paclitaxel) resulted in good tumor response and PFS, but only in patients with high levels of hyaluronan[15,86]. Therefore, in pancreatic cancers with high levels of hyaluoran, PEGPH20 therapy may allow more effective T cell infiltration and enhance the activity of immune checkpoint therapies. CONCLUSION Both challenges and opportunities exist for the development of effective immunotherapy for pancreatic cancer. Given that single agent therapies against CLTA-4 or PD-1 or PD-L1 immune checkpoint were largely ineffective in pancreatic cancer, ongoing investigations and future directions lie in the field of combination therapies, where additional treatment modalities may unleash durable anti-tumor immune responses by enhancing tumor-specific T cell activation and antagonizing the immunosuppressive microenvironment in pancreatic cancer. Conflict-of-interest statement: There is no conflict of interest associated with any of the senior author or other coauthors contributed their efforts in this manuscript. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): A Grade B (Very good): 0 Grade C (Good): C, C, C Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: February 14, 2017 First decision: March 7, 2017 Article in press: April 20, 2017 P- Reviewer: Aung W, Avci E, Peters GJ, Takao S S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ

Core tip: Field of fertility preservation has experienced remarkable advances within the last 20 years. As a result, young cancer survivors have numerous options to maintain an important aspect of their quality of life, fertility. In this article we review the current state and controversies in fertility preservation. The article should be an important resource for professionals who take care of young women with breast cancer and other malignancies. INTRODUCTION Breast cancer is the most common malignancy in women and on average more than 25000 women are diagnosed with breast cancer before reaching the age of 45 years, each year in the United States[1]. Early diagnosis by virtue of significant advances in detection, and newly developed treatment strategies have remarkably improved the course of breast malignancies. According to the National Cancer Institute, 5-year-survival rate for the women under age 45 was estimated to be as high as 88%-98.5% in 2011[2]. While survivorship rates have dramatically increased in women with breast cancer, an important issue related to reproductive function has emerged. Most women with breast cancer are likely to undergo systemic adjuvant or neo-adjuvant chemotherapy with gonadotoxic side effects. As a consequence, preserving fertility potential has become an essential concept in the management of young cancer survivors. Fertility preservation has emerged from this concept as a new and dynamic discipline where oncology and reproductive medicine intersect.

neo-adjuvant chemotherapy with gonadotoxic side effects. As a consequence, preserving fertility potential has become an essential concept in the management of young cancer survivors. Fertility preservation has emerged from this concept as a new and dynamic discipline where oncology and reproductive medicine intersect. In this review, we aimed to highlight the importance of fertility preservation as a part of routine care for breast cancer patients of childbearing age and outline the key fertility preservation options along with still experimental but promising therapeutic procedures. COUNSELING FOR FERTILITY PRESERVATION Because of the trend for having children in later reproductive ages, the number of women who experience breast cancer before completing childbearing is growing. Coupled with the increased survival rates and the growing healthy survivor population, fertility preservation has become an important component of cancer care and the maintenance of quality of life for survivors[3].

uctive ages, the number of women who experience breast cancer before completing childbearing is growing. Coupled with the increased survival rates and the growing healthy survivor population, fertility preservation has become an important component of cancer care and the maintenance of quality of life for survivors[3]. American Society of Clinical Oncology (ASCO) and American Society of Reproductive Medicine (ASRM) guidelines for fertility preservation in cancer patients strongly recommend that oncologist should inform their patients about the potential negative effects of chemotherapy on fertility before the initiation of the planned treatment and promptly refer patients to reproductive specialist to discuss the risk of ovarian damage and currently available fertility preservation options[4,5]. However, less than half of the oncologists in the United States always or often refer their cancer patients with fertility-related questions to fertility preservation specialist[6]. It should be stressed that providing timely and accurate information for women of reproductive age with breast cancer is critical for the preservation of future fertility chances before complete loss of the limited and irreplaceable ovarian reserve due to chemotherapy. We have previously shown that early referral of breast cancer patients, especially before breast surgery results in larger number of oocytes and embryos being cryopreserved and less time to the initiation of chemotherapy[7].

nces before complete loss of the limited and irreplaceable ovarian reserve due to chemotherapy. We have previously shown that early referral of breast cancer patients, especially before breast surgery results in larger number of oocytes and embryos being cryopreserved and less time to the initiation of chemotherapy[7]. IMPACT OF CANCER TREATMENT ON OVARIAN RESERVE Modern chemotherapeutic agents that are in use for breast cancer treatment can have a spectrum of ovarian toxicity, depending on the class of the agent, age of the patient, and the cumulative dose[8]. We have shown that the most gonadotoxic agents are those that mainly target oocyte genome causing DNA double strand breaks (DBSs)[9]. Under normal circumstances, DNA repair mechanisms are capable of maintaining genomic integrity, however, at the level of severe DNA damage due to genotoxic agents, those repair mechanisms remain insufficient. The severe DNA damage consequently leads to apoptotic death[9]. Ovarian reserve is made up of about 1 million primordial follicle oocytes at birth, and this number is reduced to approximately 500000 at the onset of puberty. These numbers are reduced to about 25000 at age 37 and nearly exhausted at menopause. Because primordial follicles cannot be regenerated, any chemotherapeutic agent that induces DNA breaks in primordial follicle oocyte will result in apoptotic death and cause irreversible reduction in ovarian reserve[9].

et of puberty. These numbers are reduced to about 25000 at age 37 and nearly exhausted at menopause. Because primordial follicles cannot be regenerated, any chemotherapeutic agent that induces DNA breaks in primordial follicle oocyte will result in apoptotic death and cause irreversible reduction in ovarian reserve[9]. Among all gonadotoxic agents, those belong to the alkylating category such as cyclophosphamide, are the most gonadotoxic agents[10]. Because alkylating agents are non cell-cycle specific chemical compounds and hence can target and damage resting primordial follicles that constitute ovarian reserve[9,10].

et of puberty. These numbers are reduced to about 25000 at age 37 and nearly exhausted at menopause. Because primordial follicles cannot be regenerated, any chemotherapeutic agent that induces DNA breaks in primordial follicle oocyte will result in apoptotic death and cause irreversible reduction in ovarian reserve[9]. Among all gonadotoxic agents, those belong to the alkylating category such as cyclophosphamide, are the most gonadotoxic agents[10]. Because alkylating agents are non cell-cycle specific chemical compounds and hence can target and damage resting primordial follicles that constitute ovarian reserve[9,10]. The risk of chemotherapy-induced ovarian damage has been investigated in numerous clinical studies. Unfortunately, menstruation was used as the surrogate for ovarian function and fertility in the majority of the past studies[11]. However, return of menses is a poor surrogate for reproductive potential, and ovarian reserve might be severely diminished despite the resumption of regular menses[12,13]. In this context, it is reported that after treatment with CMF protocol (cyclophosphamide/methotrexate/5-fluorouracil) 20%- 70% of women younger than age 40 experienced amenorrhea[14]. Comparing CMF protocol to the AC protocol (doxorubicin/cyclophosphamide), significantly lower rates of amenorrhea (69% vs 34%, respectively) have been reported with the AC protocol[15]. This finding is most likely related to a lower cumulative dose of cyclophosphamide reached with AC regimen. When a taxane administered in combination with AC (AC-T), it did not significantly increase the risk of amenorrhea compared with standard AC regimen[16]. Tables 1 and 2 summarize chemotherapeutic agents that are commonly used in breast cancer treatment and their potential impact on ovarian function[15-19].

AC regimen. When a taxane administered in combination with AC (AC-T), it did not significantly increase the risk of amenorrhea compared with standard AC regimen[16]. Tables 1 and 2 summarize chemotherapeutic agents that are commonly used in breast cancer treatment and their potential impact on ovarian function[15-19]. Table 1 The risk of infertility and mechanism of damage associated with chemotherapeutic agents that are commonly used in breast cancer treatment

AC regimen. When a taxane administered in combination with AC (AC-T), it did not significantly increase the risk of amenorrhea compared with standard AC regimen[16]. Tables 1 and 2 summarize chemotherapeutic agents that are commonly used in breast cancer treatment and their potential impact on ovarian function[15-19]. Table 1 The risk of infertility and mechanism of damage associated with chemotherapeutic agents that are commonly used in breast cancer treatment Chemotherapeutic agent Class Mechanism of action Cell cycle effect Risk of infertility Cyclophosphamide Alkylating agent DNA cross-link formation and double strand breaks that result in inhibition of DNA function and synthesis leading to cellular apoptosis Cell cycle non-specific High risk Doxorubicin Epirubicin Anthracyclines Inhibition of DNA synthesis and function due to inactivation of DNA topoisomerase II, free oxygen radical formation and induction of DNA double-strand breaks Cell cycle non-specific Medium risk Carboplatin Platinum analog Inhibition of DNA synthesis and function via intra- and interstrand DNA cross-link formation by covalent binding to genome Cell cycle non-specific Medium risk Paclitaxel Docetaxel Taxanes Inhibition of mitotic division by binding to microtubules with enhancement of tubulin polymerization M phase Low risk Methotrexate Antimetabolites Inhibition de novo purine nucleotide synthesis by inactivation of dihydrofolate reductase S phase Low risk 5-fluorouracil Inhibition of DNA synthesis and function via inactivation of Thymidylate synthase and alteration in RNA processing S phase Low risk Trastuzumab Monoclonal antibodies Blockage of Human epidermal growth factor receptor 2 subdomain IV, antibody dependent cellular toxicity NA Low or no risk Pertuzumab Blockage of Human epidermal growth factor receptor 2 subdomain II, antibody dependent cellular toxicity Table 2 Common adjuvant chemotherapy regimens for breast cancer and their impact of fertility

f Human epidermal growth factor receptor 2 subdomain IV, antibody dependent cellular toxicity NA Low or no risk Pertuzumab Blockage of Human epidermal growth factor receptor 2 subdomain II, antibody dependent cellular toxicity Table 2 Common adjuvant chemotherapy regimens for breast cancer and their impact of fertility Chemotherapy regimen Risk of amenorrhea or infertility Age ≤ 35 yr Age > 35 yr CMF 4%-40% 80%-100% CEF 47% 80%-100% CAF No data 30% AC 13.90% 68.20% AC-T 9%-13% 65%-67% AC-TH 0-14% 56%-67% A: Doxorubicin; C: Cyclophosphamide; E: Epirubicin; F: 5-Fluorouracil; H: Trastuzumab; M: Methotrexate; T: Paclitaxel.

f Human epidermal growth factor receptor 2 subdomain IV, antibody dependent cellular toxicity NA Low or no risk Pertuzumab Blockage of Human epidermal growth factor receptor 2 subdomain II, antibody dependent cellular toxicity Table 2 Common adjuvant chemotherapy regimens for breast cancer and their impact of fertility Chemotherapy regimen Risk of amenorrhea or infertility Age ≤ 35 yr Age > 35 yr CMF 4%-40% 80%-100% CEF 47% 80%-100% CAF No data 30% AC 13.90% 68.20% AC-T 9%-13% 65%-67% AC-TH 0-14% 56%-67% A: Doxorubicin; C: Cyclophosphamide; E: Epirubicin; F: 5-Fluorouracil; H: Trastuzumab; M: Methotrexate; T: Paclitaxel. Patient age at the time of chemotherapy inversely correlates with the likelihood of post-chemotherapy amenorrhea. In women with breast cancer, while the incidence of chemotherapy-induced amenorrhea was reported as 15%-40% under the age of 30, this incidence dramatically increases to 49%-100% for women older than 40 years of age[20]. The reason for this age-related difference is the fact that younger women have a larger ovarian reserve. Our previous studies indicated that on average, gonadotoxic chemotherapy regimens result in the loss of approximately 10 years worth of ovarian reserve[21]. Though both younger and older women would lose follicles, gonadotoxic chemotherapy is more likely to push older women over the threshold for menopause as they have lower reserve to begin with. However, regardless of age, females of all ages, including children, are expected to experience early menopause after exposure to gonadotoxic chemotherapy agents. Therefore fertility preservation and completion of family building as early as possible, is critical regardless of the age at chemotherapy exposure in most instances[22].

rdless of age, females of all ages, including children, are expected to experience early menopause after exposure to gonadotoxic chemotherapy agents. Therefore fertility preservation and completion of family building as early as possible, is critical regardless of the age at chemotherapy exposure in most instances[22]. GONADOTROPIN-RELEASING HORMONE ANALOGS AND OVARIAN PROTECTION There has been an ongoing controversy regarding the role of ovarian suppression in cancer patients using gonadotropin-releasing hormone (GnRH) analogs in order to protect ovaries from chemotherapy-induced damage[23]. The biggest concern regarding the effectiveness of ovarian suppression is that primordial follicles that constitute the ovarian reserve are quiescent and do not express gonadotropin or GnRH receptors[24,25]. Thus, any change in gonadotropin or GnRH serum levels has no plausible direct or indirect effect on primordial follicles (Figure 1). Furthermore, we have shown that gonadotoxic agents induce primordial follicle death via inducing DNA double strand breaks in oocytes in a non-cell cycle dependent fashion, hence there is no mechanism for ovarian suppression by GnRHa to prevent chemotherapy-induced DNA damage[9,26]. It should be recognized that GnRHa induces a hormonal state similar to prepubertal stage, and if ovarian suppression were to be protective, children of prepubertal age would be resistant to gonadotoxic effects of chemotherapy, which is shown to be not to be the case[27].

o prevent chemotherapy-induced DNA damage[9,26]. It should be recognized that GnRHa induces a hormonal state similar to prepubertal stage, and if ovarian suppression were to be protective, children of prepubertal age would be resistant to gonadotoxic effects of chemotherapy, which is shown to be not to be the case[27]. Figure 1 Impact of gonadotoxic chemotherapy and gonadotropin-releasing hormone analog on ovarian reserve and function. Gonadotoxic chemotherapy reduces ovarian reserve, which is made up of resting and hormone-insensitive primordial follicles, by induction of DNA damage and apoptotic death. GnRHa reduces pituitary GnRH production and, as a result, blocks the release of FSH and LH from the pituitary, which in turn results in the cessation of late-stage follicle development. Because primordial follicles do not have FSH, LH, or GnRH receptors, GnRHa cannot have a direct influence on ovarian reserve. AMH: Anti-Müllerian hormone; FSH: Follicle-stimulating hormone; FSHr: FSH receptor; LH: Luteinizing hormone; LHr: LH receptor; GnRH: Gonadotropin-releasing hormone; GnRHr: GnRH receptor. Oktay et al. J Clin Oncol 2016; 34: 2563-2565, used with permission.

eptors, GnRHa cannot have a direct influence on ovarian reserve. AMH: Anti-Müllerian hormone; FSH: Follicle-stimulating hormone; FSHr: FSH receptor; LH: Luteinizing hormone; LHr: LH receptor; GnRH: Gonadotropin-releasing hormone; GnRHr: GnRH receptor. Oktay et al. J Clin Oncol 2016; 34: 2563-2565, used with permission. While some studies in women with breast cancer, which used menstruation as a marker, suggested some benefit in restoration of menstruation post-chemotherapy, these studies were marred by numerous weaknesses[28-30]. These include the utility of self-reported menstrual status, a highly unreliable surrogate for fertility, lack of placebo control (instead of GnRHa) or blinding, and lack of correction for the difference in desire to conceive between study and control groups[31]. Use of amenorrhea as the sign of ovarian failure is also key weakness in trials of GnRHa for ovarian protection. Especially for breast cancer patients, chemotherapy often induces occult ovarian insufficiency that most frequently presents as irregular or even normal appearing periods rather than amenorrhea. When the serum anti-Müllerian Hormone (AMH), which is the most reliable quantitative biomarker for ovarian reserve or appropriate criteria with serum FSH levels for defining ovarian failure was used, none of the studies showed fertility preservation benefit from GnRHa treatment[32-34].

g periods rather than amenorrhea. When the serum anti-Müllerian Hormone (AMH), which is the most reliable quantitative biomarker for ovarian reserve or appropriate criteria with serum FSH levels for defining ovarian failure was used, none of the studies showed fertility preservation benefit from GnRHa treatment[32-34]. Given the contradictory results and ovarian biological facts, the use of GnRHa for the prevention of ovaries from chemotherapy damage is still controversial and cannot be recommended as an effective method of fertility preservation.

g periods rather than amenorrhea. When the serum anti-Müllerian Hormone (AMH), which is the most reliable quantitative biomarker for ovarian reserve or appropriate criteria with serum FSH levels for defining ovarian failure was used, none of the studies showed fertility preservation benefit from GnRHa treatment[32-34]. Given the contradictory results and ovarian biological facts, the use of GnRHa for the prevention of ovaries from chemotherapy damage is still controversial and cannot be recommended as an effective method of fertility preservation. OVARIAN RESERVE IN WOMEN WITH BRCA MUTATIONS Most hereditary breast cancers are associated with germline mutations in BRCA1 and BRCA2 genes. BRCA genes are members of the ataxia-telangiectasia-mutated (ATM)-mediated DNA damage signaling pathway and are essential for DNA double-strand break (DSB) repair[35]. In addition to the increased risk for multiple malignancies, several clinical and experimental studies showed an association between BRCA mutations and diminished ovarian reserve[26,36-41]. While performing ovarian stimulation in women with breast cancer by using aromatase inhibitors for fertility preservation, we found significantly lower ovarian response rates in BRCA mutation carries particularly, among those with BRCA1 mutations[36]. In another important study, authors reported that unaffected women with BRCA mutation experience menopause 3-4 years earlier than healthy controls[38]. Recently, our laboratory showed that in BRCA1 mutant mice there is increased age-related accumulation of DNA double strand breaks in primordial follicle oocytes and the ovarian reserve is significantly lower. These BRCA1 mutant mice also showed reduced litter size and poor embryo development. These findings clearly indicate a biological connection between BRCA mutations, DNA repair and reproductive function. In the same study, we also showed that affected women with BRCA1 mutations had lower serum AMH levels compared to controls. Interestingly we did not find these differences in either BRCA2 mutant mice or affected women with BRCA mutations[26]. Confirming our findings in a prospective study, Philips et al[41] found 25% lower AMH concentrations on average in BRCA1 carriers compared to non-carriers. There was no significant association between the BRCA2 mutation status and the AMH levels.

es in either BRCA2 mutant mice or affected women with BRCA mutations[26]. Confirming our findings in a prospective study, Philips et al[41] found 25% lower AMH concentrations on average in BRCA1 carriers compared to non-carriers. There was no significant association between the BRCA2 mutation status and the AMH levels. Given the accumulating evidence that the ovarian reserve may be lower in women with BRCA mutations, it is possible that these women are more prone to chemotherapy-induced loss of ovarian reserve and ovarian insufficiency. However this is yet to be shown in prospective clinical trials. Nevertheless, while counseling women with BRCA mutations on fertility preservation, the possibility of higher risk of chemo-induced infertility should not be omitted.

are more prone to chemotherapy-induced loss of ovarian reserve and ovarian insufficiency. However this is yet to be shown in prospective clinical trials. Nevertheless, while counseling women with BRCA mutations on fertility preservation, the possibility of higher risk of chemo-induced infertility should not be omitted. FERTILITY PRESERVATION OPTIONS FOR BREAST CANCER PATIENTS Embryo cryopreservation after in vitro fertilization (IVF) is currently considered as an established fertility preservation option, which offers the best chance of livebirth for women with a partner or single women who elect to use donor sperm. Numerous studies have demonstrated up to 60% clinical pregnancy rates and around 34% livebirth rates after transfer of frozen-thawed embryos in infertility patients with mean age of 35.1 ± 4.03, which is comparable to fresh embryo transfer[42,43]. When preimplantation genetic screening utilized, the livebirth rates can increase up to 77% after transfer of euploid frozen-thawed embryos[44]. In women with breast cancer with the mean age of 35.8 ± 4.1, we have shown a livebirth rate of 45%, which appeared to be superior to those undergoing frozen embryo transfer for infertility[45].

on genetic screening utilized, the livebirth rates can increase up to 77% after transfer of euploid frozen-thawed embryos[44]. In women with breast cancer with the mean age of 35.8 ± 4.1, we have shown a livebirth rate of 45%, which appeared to be superior to those undergoing frozen embryo transfer for infertility[45]. Cryopreservation of mature or immature oocytes is another fertility preservation option for women without a partner and those not wishing to use donor sperm due to legal, ethical or religious considerations. Mature oocytes can be effectively cryopreserved using a vitrification method and the success rates of post-thaw fertilization and pregnancy rates have approached those with fresh oocytes in young patients, though success rates with frozen embryos may still be better[46,47]. Oocyte cryopreservation success rates vary depending on age, number of oocytes frozen and the freezing protocol. In a recent individual patient data meta-analysis we calculated these success rates[48] (An interactive online success rate estimator can be found online at http://fertilitypreservation.org/index.php/probability-calc). Based on an individual patient meta-analysis encompassing thaw cycles with frozen oocytes, we have calculated the age-based success rates for oocyte cryopreservation. An interactive online egg freezing success rate estimator can be found at this link: http://fertilitypreservation.org/index.php/probability-calc, and can be useful in patient counseling.

ta-analysis encompassing thaw cycles with frozen oocytes, we have calculated the age-based success rates for oocyte cryopreservation. An interactive online egg freezing success rate estimator can be found at this link: http://fertilitypreservation.org/index.php/probability-calc, and can be useful in patient counseling. Immature oocytes can be obtained from patients without undergoing ovarian stimulation due to dearth of time and also at the time of ovarian tissue harvesting for fertility preservation. After retrieval, immature oocyte may be cryopreserved before or after undergoing in vitro maturation (IVM) process[49]. Lee et al[50] suggested performing IVM for immature oocytes before cryopreservation rather than post-thaw as they observed significantly higher maturation and survival rates with that approach. Although IVM is still an experimental fertility preservation method and limited to a number of fertility centers, this method has recently resulted in live births[51]. Embryo and oocyte cryopreservation methods are widely used and currently considered as established methods of fertility preservation. However, typically 10-14 d of controlled ovarian stimulation is needed to obtain mature oocytes (Table 3). Table 3 Fertility Preservation options for reproductive age women with breast cancer

Embryo and oocyte cryopreservation methods are widely used and currently considered as established methods of fertility preservation. However, typically 10-14 d of controlled ovarian stimulation is needed to obtain mature oocytes (Table 3). Table 3 Fertility Preservation options for reproductive age women with breast cancer Fertility preservation option Current status Advantages Disadvantages Embryo Cryopreservation Established Highest cumulative pregnancy rates Requires about two weeks delay in the initiation of cancer treatment Requires hormonal stimulation for oocyte retrieval Requires in vitro fertilization with male partner or donor sperm Oocyte Cryopreservation Established No need for male partner or sperm donor Requires about two weeks delay in the initiation of cancer treatment Requires hormonal stimulation for oocyte retrieval Ovarian Tissue Cryopreservation and Transplantation Currently experimental, may change as success rates are rising No need for hormonal stimulation Requires outpatient laparoscopic surgery for ovarian tissue harvesting and subsequent transplantation No need to significantly delay in the initiation of chemotherapy No need for male partner or sperm donor When there is insufficient time for ovarian stimulation, the only available strategy other than immature oocyte retrieval and IVM for women with breast cancer is ovarian tissue harvesting and cryopreservation for future transplantation. Since the first report of ovarian transplantation with cryopreserved tissue by our group, there have been more than 80 livebirths with over 30% of livebirth rate after ovarian transplantation[52,53]. Some have raised the concern of reintroducing malignant cells back into the body along with ovarian tissue. However, studies showed no evidence of malignant cells in cryopreserved ovarian tissues from non-metastatic breast cancer patients and those with bone and soft tissue tumors[54-56].

varian transplantation[52,53]. Some have raised the concern of reintroducing malignant cells back into the body along with ovarian tissue. However, studies showed no evidence of malignant cells in cryopreserved ovarian tissues from non-metastatic breast cancer patients and those with bone and soft tissue tumors[54-56]. CONTROLLED OVARIAN STIMULATION PROTOCOLS The major issue associated with the conventional ovarian stimulation protocols is elevated circulating estradiol levels due to the development of large number of follicle at once. Therefore, conventional stimulation protocols are considered unsafe in women with estrogen-sensitive breast cancer. Although oocytes can be retrieved from ovaries without performing ovarian stimulation (natural cycle IVF), this strategy typically does not provide more than one oocyte per cycle and yield an embryo in only 60% of cycles[57]. On the other hand, use of tamoxifen alone for ovulation induction showed better results in mature oocyte and embryo yield compared to natural cycle IVF[58]. Tamoxifen may also be used in combination with low dose gonadotropins for IVF, resulting in increase multiple mature oocytes and embryos[59].

ly 60% of cycles[57]. On the other hand, use of tamoxifen alone for ovulation induction showed better results in mature oocyte and embryo yield compared to natural cycle IVF[58]. Tamoxifen may also be used in combination with low dose gonadotropins for IVF, resulting in increase multiple mature oocytes and embryos[59]. While reducing the circulating estrogen levels, aromatase inhibitors induce the secretion of endogenous FSH by releasing the hypothalamic-pituitary axis from estrogenic negative feedback[60]. We showed that letrozole in combination with gonadotropins can produce comparable outcomes to conventional IVF while providing significantly lower estradiol levels and decreased gonadotropin requirements[45]. We also showed that pregnancy outcomes after ovarian stimulation with letrozole protocol in premenopausal breast cancer patients before adjuvant chemotherapy were similar to a non-cancer population[60]. Moreover, after short and mid-term follow up letrozole-gonadotropin protocol was associated with disease free survival rates[61]. One of the concerns related with ovarian stimulation before adjuvant or neo-adjuvant chemotherapy is the delay in the initiation of breast cancer treatment. However, studies have shown that initiation of chemotherapy can be delayed up to 12 wk after breast surgery without any adverse effect on survival and recurrence rates[62,63].

oncerns related with ovarian stimulation before adjuvant or neo-adjuvant chemotherapy is the delay in the initiation of breast cancer treatment. However, studies have shown that initiation of chemotherapy can be delayed up to 12 wk after breast surgery without any adverse effect on survival and recurrence rates[62,63]. Another concern is that letrozole protocol is that it is a teratogenic agent if used during pregnancy. However, in the setting of fertility preservation, embryos are never exposed to letrozole as the fertilization takes place in vitro and the resultant embryos are cryopreserved for later use. Additionally, it has been reported that there was no difference in congenital malformation and chromosomal abnormality rates among children born after ovarian stimulation with clomiphene or letrozole for infertility[64].

s the fertilization takes place in vitro and the resultant embryos are cryopreserved for later use. Additionally, it has been reported that there was no difference in congenital malformation and chromosomal abnormality rates among children born after ovarian stimulation with clomiphene or letrozole for infertility[64]. PREGNANCY AFTER BREAST CANCER Patients in the decision process for fertility preservation treatments frequently question the safety of pregnancy after completion of cancer treatment. Based on the current evidence, pregnancy after breast cancer is not associated with increased risk of adverse outcomes[65]. In general, patients are advised to delay pregnancy at least 2 years after diagnosis, as the risk of recurrence is highest in this time frame. In the case of ER-positive breast cancer, pregnancy is contraindicated during tamoxifen treatment because of teratogenicity. For breast cancer survivors who do not want to delay childbearing for the completion of tamoxifen treatment or for those with other medical contraindications, gestational surrogacy may be a suitable option to utilize their frozen eggs or embryos in the future[10,65].

uring tamoxifen treatment because of teratogenicity. For breast cancer survivors who do not want to delay childbearing for the completion of tamoxifen treatment or for those with other medical contraindications, gestational surrogacy may be a suitable option to utilize their frozen eggs or embryos in the future[10,65]. CONCLUSION Fertility preservation has become a crucial part of survivorship and an important aspect of comprehensive cancer care. Fortunately, there are several well-established treatment options including embryo and oocyte cryopreservation and safer ovarian stimulation protocols. Moreover, there are emerging experimental methods such as ovarian tissue cryopreservation and transplantation and IVM, which are showing promise. To maximize the utility of these available options and avoid significant delays in the initiation of chemotherapy, timely referral to fertility preservation counseling should be an integral part of the care of young women with breast cancer. Conflict-of-interest statement: No potential conflicts of interest. Manuscript source: Invited manuscript Specialty type: Oncology Country of origin: United States Peer-review report classification Grade A (Excellent): A Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 Peer-review started: February 17, 2017 First decision: April 14, 2017 Article in press: May 15, 2017 P- Reviewer: Khajehei M, Voutsadakis IA, Wang L S- Editor: Song XX L- Editor: A E- Editor: Lu YJ

Core tip: Current literature regarding the use of biological mesh reconstruction after pelvic exenteration and extralevator abdominoperineal excision is scarce. However, it does suggest that the use of biological mesh has a lower short-term perineal hernia rate, but is probably not superior to other approaches with regards to perineal wound complications. INTRODUCTION Pelvic exenteration (PE) and extralevator abdominoperineal excision (ELAPE) are mutilating operations, leaving a large perineal incision. ELAPE for low rectal cancer was introduced to decrease the rate of positive resection margins and specimen perforation occurring during conventional abdominoperineal resection (cAPR)[1,2]. In a recent retrospective study, Stelzner et al[3] showed that the 5-year recurrence rate was 5.9% in the ELAPE group vs 18.2% in the cAPR group (P = 0.153). However, other units have not been able to reproduce such results[4], nor could they demonstrate a statistically significant superiority of ELAPE in terms of CRM positivity and bowel perforation. Furthermore, they reported comparable perineal complication rates for the two APR approaches.

in the cAPR group (P = 0.153). However, other units have not been able to reproduce such results[4], nor could they demonstrate a statistically significant superiority of ELAPE in terms of CRM positivity and bowel perforation. Furthermore, they reported comparable perineal complication rates for the two APR approaches. Vivid discussions continue to fuel the debate regarding the pros and cons of ELAPE. Overall, it is well accepted that larger wounds are independent risk factors for perineal wound complications. The combination of neoadjuvant chemoradiotherapy and ELAPE almost doubles the rate of perineal wound complications (31% for ELAPE vs 18% for cAPR)[5]. While new techniques and approaches have attempted to reduce the size of the perineal incision (and therefore reduce the risk of wound complications)[6], optimal management of perineal defects is still under investigation. The options include primary closure, myocutaneous flaps, and mesh reconstruction, including the use of a biological mesh. We aimed to evaluate the outcomes of perineal reconstruction with biological mesh following ELAPE and PE in our center and to review the current literature. CURRENT STATUS Perineal wound complications are a major concern following PE and ELAPE leading to increased morbidity, longer hospital stay, and delayed chemotherapy. Different reconstruction methods are currently used in practice with the aim of reducing the rates of wound complications and avoiding perineal herniation.

CURRENT STATUS Perineal wound complications are a major concern following PE and ELAPE leading to increased morbidity, longer hospital stay, and delayed chemotherapy. Different reconstruction methods are currently used in practice with the aim of reducing the rates of wound complications and avoiding perineal herniation. Risk factors for major perineal wound complications following APR are well known: Preoperative radiotherapy, patients with anal cancer, flap reconstruction, tumor size, obesity, and diabetes[7]. Minor wound complications appear more commonly in patients with inflammatory bowel disease or anal cancer than in those with rectal cancer[8]. Most patients with locally advanced rectal cancer, recurrent rectal cancer, and recurrent or persistent squamous cell carcinoma receive neoadjuvant radiochemotherapy or radiotherapy alone[9,10]. The poor healing ability of irradiated wounds has been attributed to local endarteritis and damaged fibroblasts[11]. It has been clearly demonstrated that preoperative radiotherapy increases the rate of major wound complications[5,12]. For example, Aldulaymi et al[13] reported a significantly increased risk of major perineal wound complications in patients undergoing APR for rectal cancer with primary closure of the perineum (26% in non-irradiated vs 71% in irradiated patients). Chadwick et al[14] found that the risk of developing a wound complication was 10 times higher after previous irradiation. This substantial problem with wound healing calls for the need to consider alternative closure techniques of the perineum.

sure of the perineum (26% in non-irradiated vs 71% in irradiated patients). Chadwick et al[14] found that the risk of developing a wound complication was 10 times higher after previous irradiation. This substantial problem with wound healing calls for the need to consider alternative closure techniques of the perineum. Different methods have been described ranging from direct/primary closure to mesh reconstruction, gluteal and rectus abdominis flaps or combinations of these techniques. Currently, there is no consensus on which is the most ideal technique[15]. The vertical rectus abdominis flap (VRAM) is indicated to bring non-irradiated tissue into the perineal defect[16]. After VRAM, perineal wound complications have been reported to range from 0% to 28%[17-20]. The use of laparoscopy for the abdominal part of the resection is almost impossible because of the donor site. In addition, in cases of PE (with a right sided urostomy and left sided end colostomy), VRAM is often contra-indicated. A potential solution is the use of a wet double-barreled colostomy[21]. Other myocutaneous flaps can potentially be used, such as the gracilis flap and the gluteus maximus flap, which have a perineal wound complication rate of 12%[22] and 10%[2] respectively. However, these flaps are typically smaller than the VRAM flap and unlikely to provide adequate cover of large defects.