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Introduction Panitumumab is a high-affinity, fully human monoclonal antibody targeting epidermal growth factor receptor (EGFR) [1]. Panitumumab was first approved for the treatment of patients with EGFR-expressing metastatic colorectal cancer as monotherapy in the USA in 2006, based on the results of a multinational, open-label, randomized phase III study showing an improvement in the median progression-free survival [2].

actor receptor (EGFR) [1]. Panitumumab was first approved for the treatment of patients with EGFR-expressing metastatic colorectal cancer as monotherapy in the USA in 2006, based on the results of a multinational, open-label, randomized phase III study showing an improvement in the median progression-free survival [2]. In Japan, panitumumab was approved in 2010 for the treatment of wild-type KRAS unresectable, advanced or recurrent colorectal cancer as monotherapy, and for use in combination therapy in all-line treatment settings based on the global clinical trials and a Japanese phase II trial [3–9]. As a condition for its approval, the Japanese Ministry of Health, Labour and Welfare requested the implementation of a postmarketing all-case surveillance study to confirm the safety and efficacy of panitumumab in the clinical setting because the number of Japanese patients enrolled in the global and Japanese clinical trials was limited. Hence, the postmarketing all-case surveillance study was conducted in Japan. Following is a summary of the survey results from 3085 enrolled patients [10]: (a) the favorable toxicity profile and clinical benefit of panitumumab treatment in daily clinical practice were confirmed, and were similar to those reported in the previous clinical trials; and (b) the most common adverse drug reaction observed was skin disorders (78.4 %), including dermatitis acneiform, paronychia, dry skin, and pruritus, followed by electrolyte abnormalities (19.3 %), infusion reaction (1.5 %), interstitial lung disease (ILD) (1.3 %), and cardiac disorders (0.2 %).

ious clinical trials; and (b) the most common adverse drug reaction observed was skin disorders (78.4 %), including dermatitis acneiform, paronychia, dry skin, and pruritus, followed by electrolyte abnormalities (19.3 %), infusion reaction (1.5 %), interstitial lung disease (ILD) (1.3 %), and cardiac disorders (0.2 %). Drug-induced ILD is noted as one of the most serious adverse reactions associated with molecular targeting agents including an anti-EGFR monoclonal antibody (cetuximab) and EGFR-tyrosine kinase inhibitors (TKIs) (gefitinib and erlotinib), as it can be fatal [11–20]. Multiple studies have reported that the incidence of drug-induced ILD was higher in Japan than in other countries, and this trend was more prominently observed in the postmarketing surveillance studies of the EGFR-TKIs [13–15, 21] and anti-EGFR antibody [20].

TKIs) (gefitinib and erlotinib), as it can be fatal [11–20]. Multiple studies have reported that the incidence of drug-induced ILD was higher in Japan than in other countries, and this trend was more prominently observed in the postmarketing surveillance studies of the EGFR-TKIs [13–15, 21] and anti-EGFR antibody [20]. There were no adverse drug reaction reports of ILD in panitumumab monotherapy (1052 patients) prior to its approval in Japan; however, such reports were received in combined therapy with FOLFOX4 (fluorouracil, leucovorin, and oxaliplatin) [0.6 % (2/322 patients)] and with FOLFIRI (fluorouracil, leucovorin, and irinotecan) [0.7 % (2/302 patients)] [10]. In addition, during the course of clinical trials, a patient with non-small-cell lung cancer who had a history of pulmonary fibrosis developed ILD and died. Thereafter, patients with a history of or current ILD were excluded from clinical trials, and the experience of administration to patients with a history of ILD was limited. It is therefore important to evaluate the clinical features and risk factors of panitumumab-induced ILD to prevent the fatal outcome of ILD as well as to ensure an appropriate use of the drug. Materials and methods Patients and surveillance design This postmarketing surveillance study was planned to include all patients treated with panitumumab (Vectibix) from the start date (June 15, 2010) of its launch in Japan (ClinicalTrials.gov: NCT02089737; Japan Pharmaceutical Information Center–Clinical Trials Information: 132374) [10].

s Patients and surveillance design This postmarketing surveillance study was planned to include all patients treated with panitumumab (Vectibix) from the start date (June 15, 2010) of its launch in Japan (ClinicalTrials.gov: NCT02089737; Japan Pharmaceutical Information Center–Clinical Trials Information: 132374) [10]. To promote appropriate use and evaluate safety information, a Vectibix Appropriate Use Committee, a Vectibix Safety Evaluation Committee, and a Vectibix ILD review subcommittee were organized. The Vectibix ILD review subcommittee was established to evaluate the relationship between panitumumab and ILD, or the tendency of ILD occurrence, from the viewpoint of a third party on the basis of information on the treatment of patients in whom ILD developed or who had symptoms or disease states related to ILD after they received panitumumab. The registration period of this postmarketing surveillance study was from June 2010 to November 2010. All patients were registered by fax before their first administration of panitumumab after panitumumab was marketed. Regarding ILD risk, a letter recommending to avoid administration was sent from the Vectibix Appropriate Use Committee if the patient had a history of ILD or pulmonary fibrosis and previous or concurrent ILD with the FOLFIRI regimen. A letter recommending to reconsider the administration was sent if the patient had a history of ILD.

risk, a letter recommending to avoid administration was sent from the Vectibix Appropriate Use Committee if the patient had a history of ILD or pulmonary fibrosis and previous or concurrent ILD with the FOLFIRI regimen. A letter recommending to reconsider the administration was sent if the patient had a history of ILD. Data on patients’ demographics, clinical course, and safety information were collected using a case report form at 10 months after the start of the panitumumab therapy or at the time when the therapy was discontinued for any reason within less than 10 months. Reported terms were classified according to the Preferred Terms in the Medical Dictionary for Regulatory Activities (MedDRA), version 15.0.

llected using a case report form at 10 months after the start of the panitumumab therapy or at the time when the therapy was discontinued for any reason within less than 10 months. Reported terms were classified according to the Preferred Terms in the Medical Dictionary for Regulatory Activities (MedDRA), version 15.0. Evaluation of ILD The Vectibix ILD review subcommittee (hereafter referred to as the committee) was established before this survey was performed. The committee consisted of the following 5 external experts to assess ILD case reports appropriately: 2 experts in radiology, 2 in pulmonology, and 1 in medical oncology. ILD and potential ILD case reports were then evaluated according to the flowchart shown in Fig. 1. Radiologic images were analyzed in the current study according to the diagnostic criteria determined by the American Thoracic Society/European Thoracic Society. The committee gave not only evaluations of each individual case, but also advice on further investigations and safety measures. The committee was convened 10 times from November 2010 to December 2012.Fig. 1 Diagnosis of ILD. ILD cases were evaluated and determined according to the flowchart. The ILD review subcommittee assessed each ILD case report based on the clinical and radiographic findings. ILD interstitial lung disease

d safety measures. The committee was convened 10 times from November 2010 to December 2012.Fig. 1 Diagnosis of ILD. ILD cases were evaluated and determined according to the flowchart. The ILD review subcommittee assessed each ILD case report based on the clinical and radiographic findings. ILD interstitial lung disease If a reported term was contained within the Standard MedDRA Query (SMQ) “ILD” (narrow and broad), the event was required to be reviewed by the committee. If potential ILD events including pulmonary toxicities such as pneumonia, dyspnea, acute respiratory failure, and pulmonary edema that were not included in the SMQ “ILD” were indicated, these patients were assessed by the committee after a medical review. If ILD or potential ILD was reported, detailed clinical information, including laboratory data, and treatment and medical histories were additionally collected and the reporting physicians were requested to submit the radiographic and computed tomography (CT) images.

If a reported term was contained within the Standard MedDRA Query (SMQ) “ILD” (narrow and broad), the event was required to be reviewed by the committee. If potential ILD events including pulmonary toxicities such as pneumonia, dyspnea, acute respiratory failure, and pulmonary edema that were not included in the SMQ “ILD” were indicated, these patients were assessed by the committee after a medical review. If ILD or potential ILD was reported, detailed clinical information, including laboratory data, and treatment and medical histories were additionally collected and the reporting physicians were requested to submit the radiographic and computed tomography (CT) images. Statistical analysis Exploratory multivariate analysis was performed to investigate risk factors for ILD. Two ILD events considered by the committee not to be related to panitumumab were also included in the analyses to avoid a bias. Any patient who developed ILD more than 10 months after the first administration was excluded. The potential variables were first selected based on the stratified frequency of ILD. The medical interests of ILD and dependency variables were taken into account, and the major variables were selected and multivariate analysis in a stepwise manner was performed using Cox’s proportional hazards model (Table 1). The following variables were contained as explanatory variables: sex, age, Eastern Cooperative Oncology Group Performance Status (ECOG PS), previous or concurrent ILD, previous drug treatment for colorectal cancer, a concomitant chemotherapy with FOLFOX, and smoking status. The statistical analysis was performed with SAS version 9.1 software.Table 1 Method of multivariate analysis

: sex, age, Eastern Cooperative Oncology Group Performance Status (ECOG PS), previous or concurrent ILD, previous drug treatment for colorectal cancer, a concomitant chemotherapy with FOLFOX, and smoking status. The statistical analysis was performed with SAS version 9.1 software.Table 1 Method of multivariate analysis Statistical method Multivariate analysis using Cox’s proportional hazards model, stepwise processa, level of significance 5 % Analysis population 2311 patients who had the full set of explanatory variables among 3085 patients who participated in the safety analysis Response variable ILD (diagnosis by the ILD review subcommittee, adverse events) Yes/no Explanatory variable Sex Male/female Age <65 years/≥65 years ECOG PS PS: 0, 1/PS: 2–4 Previous or concurrent ILD No/yes Previous drug treatment for colorectal cancer No/yes Concomitant chemotherapy FOLFOX No/yes Smoking status No/yes (smoking and smoked in the past) ILD interstitial lung disease, ECOG PS Eastern Cooperative Oncology Group Performance Status aGender, age, ECOG PS and smoking status were set as variables when a model was selected

Statistical method Multivariate analysis using Cox’s proportional hazards model, stepwise processa, level of significance 5 % Analysis population 2311 patients who had the full set of explanatory variables among 3085 patients who participated in the safety analysis Response variable ILD (diagnosis by the ILD review subcommittee, adverse events) Yes/no Explanatory variable Sex Male/female Age <65 years/≥65 years ECOG PS PS: 0, 1/PS: 2–4 Previous or concurrent ILD No/yes Previous drug treatment for colorectal cancer No/yes Concomitant chemotherapy FOLFOX No/yes Smoking status No/yes (smoking and smoked in the past) ILD interstitial lung disease, ECOG PS Eastern Cooperative Oncology Group Performance Status aGender, age, ECOG PS and smoking status were set as variables when a model was selected Results Patient characteristics Forty-eight possible ILD case reports were evaluated and 39 events were determined to be panitumumab-induced ILD (Fig. 1). Patient demographics are shown in Table 2. Five of the 9 excluded patients were reported by primary physicians to have either a history of or concurrent ILD. In addition, 4 patients were excluded as they were considered by the committee, based on the evaluation of images, to have either a history or a complication of ILD, although the primary physicians reported that they had neither a history nor a complication of ILD.Table 2 Patient demographics Baseline characteristic Safety analysis set (N = 3085) ILD patientsa (N = 39)

Results Patient characteristics Forty-eight possible ILD case reports were evaluated and 39 events were determined to be panitumumab-induced ILD (Fig. 1). Patient demographics are shown in Table 2. Five of the 9 excluded patients were reported by primary physicians to have either a history of or concurrent ILD. In addition, 4 patients were excluded as they were considered by the committee, based on the evaluation of images, to have either a history or a complication of ILD, although the primary physicians reported that they had neither a history nor a complication of ILD.Table 2 Patient demographics Baseline characteristic Safety analysis set (N = 3085) ILD patientsa (N = 39) n % n % Gender Male 1965 63.7 32 1.6 Female 1120 36.3 7 0.6 Age <65 years 1524 49.4 13 0.9 65–74 years 1058 34.3 16 1.5 ≥75 years 503 16.3 10 2.0 Median (range) 65.0 (18–90) – 69.0 (40–90)

Results Patient characteristics Forty-eight possible ILD case reports were evaluated and 39 events were determined to be panitumumab-induced ILD (Fig. 1). Patient demographics are shown in Table 2. Five of the 9 excluded patients were reported by primary physicians to have either a history of or concurrent ILD. In addition, 4 patients were excluded as they were considered by the committee, based on the evaluation of images, to have either a history or a complication of ILD, although the primary physicians reported that they had neither a history nor a complication of ILD.Table 2 Patient demographics Baseline characteristic Safety analysis set (N = 3085) ILD patientsa (N = 39) n % n % Gender Male 1965 63.7 32 1.6 Female 1120 36.3 7 0.6 Age <65 years 1524 49.4 13 0.9 65–74 years 1058 34.3 16 1.5 ≥75 years 503 16.3 10 2.0 Median (range) 65.0 (18–90) – 69.0 (40–90) KRAS status Wild 3003 97.3 37 1.2 Mutant 3 0.1 1 33.3 Not determinable 79 2.6 1 1.3 ECOG PS 0 1877 60.8 23 1.2 1 942 30.5 10 1.1 2 241 7.8 5 2.1 3 22 0.7 1 4.5 4 3 0.1 0 0.0 Treatment lines First line 310 10.1 11 3.5 Second line 543 17.6 8 1.5 Third line or later 2232 72.4 20 0.9 Past treatment regimens No 173 5.6 8 4.6 Yes (duplicate counting) 2911 94.4 31 1.1 FOLFOX 2439 79.1 25 1.0 FOLFIRI 1907 61.8 20 1.0 Bevacizumab 2113 68.5 23 1.1 Cetuximab 917 29.7 9 1.0 Others 2067 67.0 23 1.1 Unknown 1 0.0 0 0 Treatment regimen Monotherapy 1254 40.7 16 1.3 Chemotherapy (duplicate counting) 1831 59.4 23 1.3 FOLFOX 573 18.6 13 2.3 FOLFIRI 1045 33.9 13 1.2 CPT-11 277 9.0 0 0.0 Others 191 6.2 1 0.5 Smoking history No 1365 44.3 14 1.0 Yes 947 30.7 19 2.0 Current smoking 234 7.6 5 2.1 Smoked in the past 713 23.1 14 2.0 Unknown 773 25.1 6 0.8 Previous or concurrent ILD No 3051 98.9 34 1.1 Yes (reported by physicians) 34 1.1 5 14.7

FOX 573 18.6 13 2.3 FOLFIRI 1045 33.9 13 1.2 CPT-11 277 9.0 0 0.0 Others 191 6.2 1 0.5 Smoking history No 1365 44.3 14 1.0 Yes 947 30.7 19 2.0 Current smoking 234 7.6 5 2.1 Smoked in the past 713 23.1 14 2.0 Unknown 773 25.1 6 0.8 Previous or concurrent ILD No 3051 98.9 34 1.1 Yes (reported by physicians) 34 1.1 5 14.7 ECOG PS Eastern Cooperative Oncology Group Performance Status, ILD interstitial lung disease aDetected by the ILD review subcommittee The total ILD frequency was 1.3 % (39/3085) [monotherapy group: 1.3 % (16/1254); combination therapy group: 1.3 % (23/1831); combination therapy with FOLFOX group: 2.3 % (13/573); combination therapy with FOLFIRI group: 1.2 % (13/1045); others: 0.5 % (1/191)]. The committee concluded that the mortality due to ILD was 51.3 % (20/39). Although the reporting physicians regarded that 3 patients among those who had ILD onset died due to their primary disease, it was considered that a causal relationship between the ILD and the death could not be ruled out by the committee. Therefore, they were included as fatal ILD. Imaging pattern A diffuse alveolar damage (DAD) pattern was identified in 18 patients, including 5 patients who developed ILD with a non-DAD pattern at an earlier time point of panitumumab administration and progressed to ILD with DAD. Hypersensitivity (HP) and organizing pneumonia (OP) were also found in 9 and 8 patients, respectively. Two ILD patients whose imaging data were not provided were assessed based on the clinical course and laboratory data. The imaging patterns were categorized as “unknown.”

administration and progressed to ILD with DAD. Hypersensitivity (HP) and organizing pneumonia (OP) were also found in 9 and 8 patients, respectively. Two ILD patients whose imaging data were not provided were assessed based on the clinical course and laboratory data. The imaging patterns were categorized as “unknown.” The mortality rate of patients with DAD was 83.3 % (15/18), while that of patients with non-DAD patterns, i.e., HP and OP, was 11.8 % (2/17) (Table 3).Table 3 Number of ILD patients and length of time to the onset of ILD for each image pattern Duration (months) Total ≤1 ≤2 ≤3 ≤4 ≤5 ≤6 ≤7 ≤8 ≤9 ≤10 >10 Number of patients 3085 2874 2570 2157 1774 1454 1210 1010 867 720 597 451 Number of ILD patients 39 (20) 8 (6) 4 (2) 11 (4) 1 (0) 4 (2) 5 (2) 0 (0) 3 (1) 2 (2) 0 (0) 1 (1) Image pattern DAD 18 (15) 4 (4) 1 (1) 6 (4) 1 (0) 2 (2) 2 (2) 0 (0) 1 (1) 0 (0) 0 (0) 1 (1) HP 9 (1) 1 (0) 1 (1) 3 (0) 0 (0) 1 (0) 1 (0) 0 (0) 2 (0) 0 (0) 0 (0) 0 (0) OP 8 (1) 1 (0) 2 (0) 2 (0) 0 (0) 1 (0) 1 (0) 0 (0) 0 (0) 1 (1) 0 (0) 0 (0) Unknown 4 (3) 2 (2) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0) 0 (0) 0 (0) 1 (1) 0 (0) 0 (0) Numbers of fatal cases are given in parentheses ILD interstitial lung disease, DAD diffuse alveolar damage, HP hypersensitivity pneumonia, OP organizing pneumonia

Duration (months) Total ≤1 ≤2 ≤3 ≤4 ≤5 ≤6 ≤7 ≤8 ≤9 ≤10 >10 Number of patients 3085 2874 2570 2157 1774 1454 1210 1010 867 720 597 451 Number of ILD patients 39 (20) 8 (6) 4 (2) 11 (4) 1 (0) 4 (2) 5 (2) 0 (0) 3 (1) 2 (2) 0 (0) 1 (1) Image pattern DAD 18 (15) 4 (4) 1 (1) 6 (4) 1 (0) 2 (2) 2 (2) 0 (0) 1 (1) 0 (0) 0 (0) 1 (1) HP 9 (1) 1 (0) 1 (1) 3 (0) 0 (0) 1 (0) 1 (0) 0 (0) 2 (0) 0 (0) 0 (0) 0 (0) OP 8 (1) 1 (0) 2 (0) 2 (0) 0 (0) 1 (0) 1 (0) 0 (0) 0 (0) 1 (1) 0 (0) 0 (0) Unknown 4 (3) 2 (2) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0) 0 (0) 0 (0) 1 (1) 0 (0) 0 (0) Numbers of fatal cases are given in parentheses ILD interstitial lung disease, DAD diffuse alveolar damage, HP hypersensitivity pneumonia, OP organizing pneumonia Timing of ILD onset Figure 2 and Table 3 show the number of ILD patients and the length of time to the onset of ILD. No significant correlations were found between the timing of ILD onset and the imaging patterns (time to onset from start of administration: 2–313 days; 1–19 courses). In 11 patients, ILD occurred 6 months or longer after the first administration of panitumumab.Fig. 2 Time to onset of ILD. The numbers of ILD cases assessed by the ILD review subcommittee were classified by the length of time to the onset of ILD. Each outcome consists of non-fatal (grey) and fatal (black) cases. Note that there were no specific trends of ILD occurrence observed during the study. ILD interstitial lung disease

onset of ILD. The numbers of ILD cases assessed by the ILD review subcommittee were classified by the length of time to the onset of ILD. Each outcome consists of non-fatal (grey) and fatal (black) cases. Note that there were no specific trends of ILD occurrence observed during the study. ILD interstitial lung disease Risk factors Of the 3085 patients, 774 (25.1 %), including several who developed ILD, were excluded from the multivariate analysis because of unknown background information (information about a smoking history in 773 patients and a prior treatment history in 1 patient was not available). Therefore, only 2311 patients were used for the multivariate analysis. Of these patients, 34 were assessed to have ILD. Among the 34 patients, the risk of ILD occurrence was significantly higher in those with a history/complication of ILD, no history of previous drug treatment, and male sex (Table 4; Fig. 3). The adjusted hazard ratio (HR) was greater than 2.0 in the groups of 65 years or older and ECOG PS 2–4.Table 4 Results of multivariate analysis

atients, the risk of ILD occurrence was significantly higher in those with a history/complication of ILD, no history of previous drug treatment, and male sex (Table 4; Fig. 3). The adjusted hazard ratio (HR) was greater than 2.0 in the groups of 65 years or older and ECOG PS 2–4.Table 4 Results of multivariate analysis Background factor No. of patients Follow-up (person-months) Patients with ILD Incidence (%) Incidence rate (/100 person-months) Criterion variable Explanatory variable HR (unadjusted) HR (adjusted) 95 % CI p value Lower Upper History/complication of ILD No 2283 15422 30 1.31 0.19 No Yes 11.01 7.99 2.75 23.28 <0.001 Yes 28 188 4 14.29 2.13 Sex Male 1399 9446 29 2.07 0.31 Female Male 3.78 3.15 1.11 8.90 0.031 Female 912 6164 5 0.55 0.08 ECOG PS PS: 0, 1 2104 14713 30 1.43 0.20 PS: 0, 1 PS: 2–4 2.27 2.74 0.95 7.91 0.062 PS: 2–4 207 897 4 1.93 0.45 Age <65 years 1143 7740 10 0.87 0.13 <65 ≥65 2.36 2.02 0.96 4.28 0.065 ≥65 years 1168 7870 24 2.05 0.30 Smoking history No 1364 9189 15 1.10 0.16 No Yes 1.81 1.20 0.58 2.52 0.625 Yes 947 6421 19 2.01 0.30 History of previous drug treatment of colorectal cancer No 131 948 7 5.34 0.74 Yes No 3.99 3.83 1.66 8.84 0.002 Yes 2180 14662 27 1.24 0.18 ILD interstitial lung disease, HR hazard ratio, CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status

Background factor No. of patients Follow-up (person-months) Patients with ILD Incidence (%) Incidence rate (/100 person-months) Criterion variable Explanatory variable HR (unadjusted) HR (adjusted) 95 % CI p value Lower Upper History/complication of ILD No 2283 15422 30 1.31 0.19 No Yes 11.01 7.99 2.75 23.28 <0.001 Yes 28 188 4 14.29 2.13 Sex Male 1399 9446 29 2.07 0.31 Female Male 3.78 3.15 1.11 8.90 0.031 Female 912 6164 5 0.55 0.08 ECOG PS PS: 0, 1 2104 14713 30 1.43 0.20 PS: 0, 1 PS: 2–4 2.27 2.74 0.95 7.91 0.062 PS: 2–4 207 897 4 1.93 0.45 Age <65 years 1143 7740 10 0.87 0.13 <65 ≥65 2.36 2.02 0.96 4.28 0.065 ≥65 years 1168 7870 24 2.05 0.30 Smoking history No 1364 9189 15 1.10 0.16 No Yes 1.81 1.20 0.58 2.52 0.625 Yes 947 6421 19 2.01 0.30 History of previous drug treatment of colorectal cancer No 131 948 7 5.34 0.74 Yes No 3.99 3.83 1.66 8.84 0.002 Yes 2180 14662 27 1.24 0.18 ILD interstitial lung disease, HR hazard ratio, CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status Fig. 3 Forest plot of risk factors. By employing multivariate analysis using Cox’s proportional hazard model, higher risk factors of ILD occurrence were identified, and the adjusted HR is shown. ILD interstitial lung disease, ECOG PS Eastern Cooperative Oncology Group Performance Status, HR hazard ratio

ILD interstitial lung disease, HR hazard ratio, CI confidence interval, ECOG PS Eastern Cooperative Oncology Group Performance Status Fig. 3 Forest plot of risk factors. By employing multivariate analysis using Cox’s proportional hazard model, higher risk factors of ILD occurrence were identified, and the adjusted HR is shown. ILD interstitial lung disease, ECOG PS Eastern Cooperative Oncology Group Performance Status, HR hazard ratio Discussion In order to manage and prevent fatal outcomes due to ILD, the current study focused on the clinical features and risk factors of panitumumab-induced ILD. Several postmarketing surveillance studies have suggested that the frequency of drug-induced ILD was higher in Japan than in other countries [13, 15, 16, 20, 21]. This difference might be due to many reasons: e.g., genetic susceptibility based on the different genetic background, underlying comorbidity, previous environmental exposures, clinical practices, frequency of diagnosis device, detection bias, and preference of reporting terms [15]. In the clinical trials of panitumumab monotherapy in metastatic colorectal cancer involving 1052 patients, there were no reports of adverse drug reactions in patients considered to have panitumumab-induced ILD, while in the clinical trials of the combination therapy with FOLFIRI [8] and with FOLFOX4 [7], adverse drug reactions considered to be panitumumab-induced ILD were reported at the rate of 0.7 % (2/302 patients; fatal, 0/2) and 0.6 % (2/322 patients; fatal due to ILD, 2/2), respectively. The incidence rate of ILD in this postmarketing all-case surveillance study in Japan was 1.3 % (39/3085 patients; fatal due to ILD, 20/39), which appeared slightly higher than that observed in premarketing clinical trials. Of the 20 patients who died due to ILD, 15 patients exhibited a DAD pattern and most of the patients were treated with steroid administration, but in 1 patient the treatment was considered to have been initiated late. Among the 20 deaths due to ILD, 3 out of 5 patients (including 3 non-evaluable patients) with a non-DAD pattern were treated with steroid administration, while the information regarding steroid treatment in the remaining 2 patients was missing. The ILD incidence and mortality rates were similar to those of the other anti-EGFR antibody [17, 20]. Data on the patients who died and those who did not were reviewed, but no particular differences in background factors or time to ILD onset, excluding a higher mortality rate in patients with a DAD pattern, were found between the 2 groups.

mortality rates were similar to those of the other anti-EGFR antibody [17, 20]. Data on the patients who died and those who did not were reviewed, but no particular differences in background factors or time to ILD onset, excluding a higher mortality rate in patients with a DAD pattern, were found between the 2 groups. It is beneficial for clinicians to understand the clinical features of drug-induced ILD in daily medical practice. For instance, capillary leak syndrome by bortezomib administration [22] and high frequency of grade 1–2 ILD by mammalian target of rapamycin (mTOR) inhibitor [23] are examples of specific ILD features related to molecularly targeted drugs [16]. Panitumumab as well as cetuximab and EGFR-TKIs such as gefitinib and erlotinib can induce ILD with a DAD pattern, which can sometimes lead to a fatal outcome [24–26]. However, in this study, no findings specific to panitumumab were identified based on CT images or clinical practice. It has been reported that an early onset of ILD occurrence was observed during EGFR-TKI administration [13]. Since no specific time-onset of ILD occurrence was found when panitumumab, as well as cetuximab, was administered, close and regular pulmonary monitoring is important for panitumumab administration.

It is beneficial for clinicians to understand the clinical features of drug-induced ILD in daily medical practice. For instance, capillary leak syndrome by bortezomib administration [22] and high frequency of grade 1–2 ILD by mammalian target of rapamycin (mTOR) inhibitor [23] are examples of specific ILD features related to molecularly targeted drugs [16]. Panitumumab as well as cetuximab and EGFR-TKIs such as gefitinib and erlotinib can induce ILD with a DAD pattern, which can sometimes lead to a fatal outcome [24–26]. However, in this study, no findings specific to panitumumab were identified based on CT images or clinical practice. It has been reported that an early onset of ILD occurrence was observed during EGFR-TKI administration [13]. Since no specific time-onset of ILD occurrence was found when panitumumab, as well as cetuximab, was administered, close and regular pulmonary monitoring is important for panitumumab administration. From the results of the multivariate analysis, a history/complication of ILD, no history of previous drug treatment, and male sex were considered to be significant risk factors for panitumumab-induced ILDs. ECOG PS 2–4 and age of 65 years or older were also indicated as potential risk factors. These risk factors for ILD associated with panitumumab use are almost the same as those reported for the EGFR-TKIs [13, 14, 18, 19] and the other anti-EGFR monoclonal antibody [20]. No history of previous drug treatment could be an apparent risk factor because of patient selection bias. The reasons are as follows: 666 patients previously treated with the other anti-EGFR monoclonal antibody, cetuximab, were included in the 2180 patients with previous treatment. These patients were considered to be less likely to experience ILD even when treated with panitumumab (the incidence of ILD was 0.66 % [6/914] in patients who received panitumumab among patients who had received the other anti-EGFR monoclonal antibody and had not experienced ILD). In addition to the patient selection bias, it was unlikely that the patients undergoing first-line treatment had a higher risk of ILD than those undergoing second-line or later treatment, because, generally speaking, a patient undergoing second-line or later treatment had disease progression and their general condition was worse. Similarly, the incidence of ILD was significantly higher in elderly patients and those who had prior ILD, according to the results of postmarketing all-case surveillance of cetuximab in Japan [20]. In this study, cetuximab was used as second-line or later therapy after some chemotherapy [17]. The risk factors for ILD occurrence during therapy with an anti-EGFR monoclonal antibody need further investigation.

had prior ILD, according to the results of postmarketing all-case surveillance of cetuximab in Japan [20]. In this study, cetuximab was used as second-line or later therapy after some chemotherapy [17]. The risk factors for ILD occurrence during therapy with an anti-EGFR monoclonal antibody need further investigation. The following 3 factors are important for detecting ILD at an early stage: (1) examining whether patients have signs or symptoms such as dry cough, dyspnoea, and pyrexia which suggest the patients have ILD, and consulting with pulmonologists at an early stage if the signs or symptoms are found; (2) informing patients or their family of signs or symptoms of ILD in advance and counseling the patients to see a physician and report the signs or symptoms immediately after onset; and (3) examining images of patients at the following times: (a) when physicians evaluate whether patients have lung metastasis before panitumumab is administered; (b) when the efficacy of panitumumab is evaluated; and (c) when signs or findings that show patients are suspected of having ILD are obtained.

after onset; and (3) examining images of patients at the following times: (a) when physicians evaluate whether patients have lung metastasis before panitumumab is administered; (b) when the efficacy of panitumumab is evaluated; and (c) when signs or findings that show patients are suspected of having ILD are obtained. The following are considered to be limitations in the current analysis: (A) the all-case surveillance was not designed to identify ILD risk factors, and therefore only selected risk factors (listed in Table 4) were taken into consideration; (B) adverse events of both the monotherapy and combination chemotherapy were analyzed together, and thus impacts of the combination chemotherapy could not be excluded; and (C) since 773 patients without information on smoking, a risk factor of ILD, were excluded from the analysis, the impact of smoking could not be fully considered. A benefit–risk balance must be thoroughly considered before giving the drug to patients with interstitial pneumonia or lung fibrosis or those who have histories of them, and careful decisions must be made when deciding to initiate panitumumab.

uded from the analysis, the impact of smoking could not be fully considered. A benefit–risk balance must be thoroughly considered before giving the drug to patients with interstitial pneumonia or lung fibrosis or those who have histories of them, and careful decisions must be made when deciding to initiate panitumumab. In conclusion, the ILD incidence mortality rates and risk factors of panitumumab were similar to those of the other anti-EGFR monoclonal antibody [17, 20]. Panitumumab-specific ILD findings were not observed in CT images or clinical practice. Panitumumab likely induces DAD just as the other anti-EGFR monoclonal antibody and EGFR-TKIs do, which may lead to death. A history/complication of ILD, male sex, poor general condition, and age of 65 years or older were indicated to be ILD risk factors in the multivariate analysis, and these are similarly observed in the reports with the EGFR-TKIs and anti-EGFR monoclonal antibody [13, 14, 20]. In addition, no history of previous drug treatment was considered to be an apparent risk factor. ILD can occur at any time after initiating panitumumab, and therefore close and regular monitoring is needed. Although several papers show that the frequency of EGFR-TKI-induced ILD was higher in Japan than in Western countries [12, 18, 19], the reason was unclear. Some studies suggest that genetic differences make a contribution [27, 28]. Anti-EGFR monoclonal antibodies might have similar mechanisms to EGFR-TKIs, and therefore ILD incidence is higher in Japan. Future studies are clearly warranted to investigate the benefit–risk balance in patients with the risk factors identified in this study.

gest that genetic differences make a contribution [27, 28]. Anti-EGFR monoclonal antibodies might have similar mechanisms to EGFR-TKIs, and therefore ILD incidence is higher in Japan. Future studies are clearly warranted to investigate the benefit–risk balance in patients with the risk factors identified in this study. The authors would like to thank all the patients and their families, the investigators, the medical staff, and the Vectibix Appropriate Use/Vectibix Safety Evaluation Committee members (Atsushi Ohtsu, Kenichi Sugihara, Narikazu Boku, Yuko Kitagawa, Hiroya Takiuchi, Kiyohiko Hatake, Naoya Yamazaki, Kei Muro, and Takayuki Yoshino). The authors would like to acknowledge Hiroshi Ueno, Yoshiyuki Takasu, Masanori Kitagawa, Kimitake Okazaki, Yoji Takesaki, Hidenori Mio, Masanori Nishimura, and the study team for their important contributions to the study. This work was funded by Takeda Pharmaceutical Co., Ltd. WysiWyg Co., Ltd. provided editorial assistance in the preparation of the manuscript.

i Ueno, Yoshiyuki Takasu, Masanori Kitagawa, Kimitake Okazaki, Yoji Takesaki, Hidenori Mio, Masanori Nishimura, and the study team for their important contributions to the study. This work was funded by Takeda Pharmaceutical Co., Ltd. WysiWyg Co., Ltd. provided editorial assistance in the preparation of the manuscript. Conflict of interest Akihiko Gemma, Shoji Kudoh, Fumikazu Sakai, Masahiro Endo, and Tetsuya Hamaguchi received consultancy fees from Takeda Pharmaceutical Co., Ltd. as members of the Vectibix ILD review subcommittee. Masahiro Osawa was an employee of Bayer Healthcare. Masahiro Osawa, Yumiko Ogino, Miyo Yoneoka, Motonobu Sakaguchi, and Hiroyuki Nishimoto are employees of Takeda Pharmaceutical Co., Ltd. The study was conducted by Takeda Pharmaceutical Co., Ltd. in conjunction with the Vectibix Appropriate Use/Vectibix Safety Evaluation Committees. Takeda Pharmaceutical Co., Ltd. funded, collected, and analyzed the data, and contributed to the interpretation of the study. Bell Medical Solutions, Inc. conducted statistical analysis.

Introduction Overexpression or gene amplification of human epidermal growth factor receptor 2 (HER2) occurs in approximately 20 % of breast cancer cases. It is known to be an independent prognostic factor that shortens survival of patients, and is associated with high rates of cell proliferation and lymph node metastases [1, 2]. At present, the standard regimens used worldwide are the trastuzumab-containing regimens, which have significantly improved the prognosis for HER2-positive breast cancer [3, 4]. One of the key combination regimens containing trastuzumab is with taxane and is recommended by clinical practice guidelines globally [3–5]. However, the majority of patients experience tumor recurrences and/or metastases and, hence, further treatment options for primary care as well as in the metastatic setting are required. Another important issue is the occurrence of brain metastases due to the inability of trastuzumab to cross the intact blood–brain barrier. Cardiotoxicity has been recognized as one of the important side-effects [6–11]. Lapatinib tosylate hydrate is a small molecule that reversibly inhibits the activity of epidermal growth factor receptor (EGFR) and HER2 tyrosine kinases. Lapatinib was first approved in the USA in 2006 in combination with capecitabine for HER2-positive metastatic breast cancer (MBC) which persisted or recurred after anthracycline, taxane and trastuzumab treatment. The approved condition is the same worldwide, including in Japan. Lapatinib has been evaluated in other combinations, such as trastuzumab, aromatase inhibitor and paclitaxel. Its combination with paclitaxel is approved in some countries/regions as first-line treatment for HER2-positive MBC patients in whom trastuzumab is not appropriate.

ion is the same worldwide, including in Japan. Lapatinib has been evaluated in other combinations, such as trastuzumab, aromatase inhibitor and paclitaxel. Its combination with paclitaxel is approved in some countries/regions as first-line treatment for HER2-positive MBC patients in whom trastuzumab is not appropriate. Lapatinib is a small molecule that passes through the compromised blood–brain barrier and is suggested to exert anti-tumor activity in metastatic brain lesions [12, 13]. This characteristic approach of lapatinib led to the development of lapatinib in combination with paclitaxel as first-line therapy of HER2-positive MBC, as a replacement for the trastuzumab regimens.

the compromised blood–brain barrier and is suggested to exert anti-tumor activity in metastatic brain lesions [12, 13]. This characteristic approach of lapatinib led to the development of lapatinib in combination with paclitaxel as first-line therapy of HER2-positive MBC, as a replacement for the trastuzumab regimens. The first Phase I study of a lapatinib and paclitaxel combination (L+P) in HER2-positive MBC patients was conducted outside Japan. This study demonstrated the tolerability of lapatinib (1500 mg/day) in combination with both weekly paclitaxel 80 mg/m2 and with tri-weekly paclitaxel 135, 175, 200 and 225 mg/m2 [14]. Following this, the combination of lapatinib (1500 mg/day) and tri-weekly paclitaxel (175 mg/m2) as first-line therapy was evaluated in a Phase III study, in which 579 HER2-negative or unknown, advanced or recurrent breast cancer patients were enrolled [15]. Of 579 patients enrolled, 83 had HER2-positive MBC, and the combination regimen showed significant improvement in progression-free survival (PFS) compared to paclitaxel monotherapy [hazard ratio (HR) = 0.49; 95 % confidence interval (CI) 0.3–0.8; p = 0.008]. In another Phase III study that was mainly conducted in China and South America, the combination of lapatinib (1500 mg/day) with weekly paclitaxel (80 mg/m2) as first-line therapy was evaluated in 444 patients with HER2-positive MBC [16]. The overall survival (OS) of the combination was prolonged compared to paclitaxel monotherapy (HR = 0.74; 95 % CI 0.58–0.94; p = 0.0124). The combinations of lapatinib (1250 mg/day) or trastuzumab either with paclitaxel or docetaxel as first-line therapy for HER2-positive MBC were evaluated in a global Phase III study including Japan, which was conducted mainly in the USA/EU [17]. The study enrolled 636 patients; the results, reported in 2012, showed that the PFS of the lapatinib combination was not significantly different compared with the trastuzumab regimen [HR = 1.33 (95 % CI 1.06–1.67); p = 0.01].

ed in a global Phase III study including Japan, which was conducted mainly in the USA/EU [17]. The study enrolled 636 patients; the results, reported in 2012, showed that the PFS of the lapatinib combination was not significantly different compared with the trastuzumab regimen [HR = 1.33 (95 % CI 1.06–1.67); p = 0.01]. In order to extrapolate the data from the above studies conducted outside Japan to clinical practice in Japan, we have conducted this study to investigate the tolerability, safety and pharmacokinetics (PK) of a lapatinib 1500 mg and weekly paclitaxel combination. Patients and methods Patients Eligible patients were Japanese women older than 18 years with histologically confirmed invasive HER2-positive MBC, which was defined as immunohistochemistry (IHC) 3+ or fluorescence in situ hybridization (FISH)-positive (an amplification ratio ≥2.2) evaluated by a local laboratory. Patients were required to have at least one measurable lesion according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0 [18]. Patients must have received no prior therapy for metastatic diseases. Additional inclusion criteria were: Eastern Cooperative Oncology Group performance status of 0 or 1; left ventricular ejection fraction (LVEF) within the institutional normal range (or ≥50 %, if unavailable); adequate renal, hepatic and hematologic functions.

must have received no prior therapy for metastatic diseases. Additional inclusion criteria were: Eastern Cooperative Oncology Group performance status of 0 or 1; left ventricular ejection fraction (LVEF) within the institutional normal range (or ≥50 %, if unavailable); adequate renal, hepatic and hematologic functions. Major exclusion criteria were: prior therapy with an EGFR and/or HER2 inhibitor other than trastuzumab; unresolved/unstable, serious toxicity from prior therapy with investigational drug and/or anticancer treatment; uncontrolled infection; ≥Grade 2 peripheral neuropathy according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0; malabsorption syndrome or other conditions that would prevent the efficacy and safety evaluation of the study regimen. All patients provided written informed consent. The study was conducted in accordance with good clinical practice and all applicable regulatory requirements, including the Declaration of Helsinki.

3.0; malabsorption syndrome or other conditions that would prevent the efficacy and safety evaluation of the study regimen. All patients provided written informed consent. The study was conducted in accordance with good clinical practice and all applicable regulatory requirements, including the Declaration of Helsinki. Study design This was a single-arm, two-part, open-label Phase I/II study (ClinicalTrials.gov Identifier: NCT01138046). The objectives were to evaluate the tolerability, safety, efficacy and PK of L+P in Japanese patients with HER2-positive MBC who have not received prior chemotherapy or trastuzumab for metastatic diseases. Six patients were enrolled in the first part (Part 1) to evaluate the tolerability of this regimen, and once the tolerability was confirmed, a further 6 patients were enrolled in the second part (Part 2). Sample size was not decided by statistical considerations.

ior chemotherapy or trastuzumab for metastatic diseases. Six patients were enrolled in the first part (Part 1) to evaluate the tolerability of this regimen, and once the tolerability was confirmed, a further 6 patients were enrolled in the second part (Part 2). Sample size was not decided by statistical considerations. Patients were given the standard treatment consisting of 6 cycles of weekly paclitaxel 80 mg/m2 (for 3 weeks in a 4-week cycle) combined with lapatinib 1500 mg daily; the latter was given until disease progression or withdrawal from study treatment due to unacceptable toxicity or consent withdrawal. The investigators could chose to continue concurrent paclitaxel administration for more cycles. For patients enrolled in Part 1, lapatinib was not given on day 1, but started on the following day, and paclitaxel was not given on day 15, but was given within 2 days after that date during the first cycle for PK evaluation purposes. If disease progression, unacceptable paclitaxel-related toxicities, or termination of lapatinib occurred, then paclitaxel was terminated at any time during the study, even before completing 6 cycles. Study endpoints The objective in Part 1 was tolerability. If one or no patient out of the 6 experienced any events included in the tolerability criteria, the study treatment was concluded to be tolerable. PK parameters were also evaluated.

Patients were given the standard treatment consisting of 6 cycles of weekly paclitaxel 80 mg/m2 (for 3 weeks in a 4-week cycle) combined with lapatinib 1500 mg daily; the latter was given until disease progression or withdrawal from study treatment due to unacceptable toxicity or consent withdrawal. The investigators could chose to continue concurrent paclitaxel administration for more cycles. For patients enrolled in Part 1, lapatinib was not given on day 1, but started on the following day, and paclitaxel was not given on day 15, but was given within 2 days after that date during the first cycle for PK evaluation purposes. If disease progression, unacceptable paclitaxel-related toxicities, or termination of lapatinib occurred, then paclitaxel was terminated at any time during the study, even before completing 6 cycles. Study endpoints The objective in Part 1 was tolerability. If one or no patient out of the 6 experienced any events included in the tolerability criteria, the study treatment was concluded to be tolerable. PK parameters were also evaluated. The efficacy and safety of the treatment in twelve patients were the objectives of Part 2. The study was not designed based on statistical hypotheses, as the study targeted a small population and was designed as single-arm stud;, however, in order to compare with the OS of the previous study, our efficacy evaluation was primarily focused on OS. The other endpoints of efficacy were PFS, time to response, duration of response, objective tumor response rate (ORR) and clinical benefit rate [complete response (CR) or partial response (PR) or stable disease (SD) ≥24 weeks]. Safety and biomarkers were also evaluated. All these endpoints were evaluated in the patients enrolled in both parts.

ficacy were PFS, time to response, duration of response, objective tumor response rate (ORR) and clinical benefit rate [complete response (CR) or partial response (PR) or stable disease (SD) ≥24 weeks]. Safety and biomarkers were also evaluated. All these endpoints were evaluated in the patients enrolled in both parts. Safety and efficacy assessments Safety assessment including laboratory tests were performed every week during the combination treatment and at the discontinuation of paclitaxel if it was decided to continue longer than 6 cycles. If paclitaxel was discontinued at the sixth cycle, then safety assessment was conducted every 8 weeks until the end of treatment. LVEF assessment by echocardiogram was performed at the end of even-numbered cycles during combination treatment and every 8 weeks while on lapatinib monotherapy. Adverse events (AEs) were graded according to NCI-CTCAE version 3.0. AE terms were coded by MedDRA Ver13.1. The protocol defined serious AEs as all Grade 4 laboratory abnormalities, Grade 3 or 4 decrease in LVEF, ≥20 % decrease in LVEF relative to baseline and also below the institution’s lower limit of normal (if the lower limit of normal was unavailable, decrease to less than 50 %), Grade 3 pneumonitis, alanine aminotransferase (ALT) >3 × upper limit of normal (ULN), and total bilirubin >2.0 × ULN (>35 % direct; bilirubin fractionation required). The tolerability criteria were defined as the toxicities related to study treatment and applicable to any of the following: Grade 4 neutropenia sustained for ≥7 days, Grade 4 thrombocytopenia, ≥Grade 3 or clinically significant non-hematologic toxicities (other than nausea), or unable to start cycle 2 within 2 weeks of scheduled dosing due to unresolved toxicity. Patients withdrawn from the study without disease progression were assessed every 12 weeks until progression, start of post-anticancer therapy or death. Efficacy assessment was performed at baseline and at the ends of every even-numbered cycle until withdrawal from the study. Tumor response was assessed by the investigators, using images or photographic data, in accordance with the RECIST [18]. Biomarker analysis for HER2 status was conducted based on the results determined by both IHC and FISH at the central laboratory.

the ends of every even-numbered cycle until withdrawal from the study. Tumor response was assessed by the investigators, using images or photographic data, in accordance with the RECIST [18]. Biomarker analysis for HER2 status was conducted based on the results determined by both IHC and FISH at the central laboratory. Pharmacokinetics evaluation Pharmacokinetics of lapatinib and/or paclitaxel were evaluated in all patients enrolled in Part 1 on days 1, 8 and 14: day 14 for PK of lapatinib monotherapy, day 1 for paclitaxel monotherapy and day 8 for combination therapy. Plasma samples were taken pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h post-dose. The PK parameters calculated by non-compartmental analysis were maximum plasma concentration (Cmax), time to Cmax (tmax) and area under the plasma concentration curve (AUC) from 0 to 24 h (AUC0–24) of lapatinib as well as Cmax, tmax, half-life, AUC extrapolated to infinity (AUC0–inf) and AUC0–24 of paclitaxel. Statistical analysis The sample size was determined based on study feasibility. This study did not assert or test any statistical hypotheses. The dose-limiting toxicity (DLT) incidences were evaluated in 6 patients. The intent-to-treat (ITT) population was analyzed for safety and efficacy analyses. PFS and OS were summarized using the Kaplan–Meier method. All the patients who had provided ample plasma samples for the PK parameter evaluation were treated as the PK population. For PK parameters, the items evaluated are shown in Table 5.

intent-to-treat (ITT) population was analyzed for safety and efficacy analyses. PFS and OS were summarized using the Kaplan–Meier method. All the patients who had provided ample plasma samples for the PK parameter evaluation were treated as the PK population. For PK parameters, the items evaluated are shown in Table 5. Version 9.1.3 Unix SAS® system (a registered trademark of the SAS Institute, Inc., Cary, NC, USA) was used for analysis. Results Patient characteristics A total of 12 patients were enrolled from 9 centers between April 2010 and June 2011, and were treated with the study regimen. As of 31 January 2014 (the final data cut-off date), 6 patients had completed the study and 6 patients were followed up for survival. Out of 12 patients enrolled, 8 patients had both visceral and non-visceral metastatic lesions, 2 patients had visceral lesions only, while the other 2 patients had non-visceral lesions only (Table 1). The median time since diagnosis was 12.9 months; 4 patients had received prior chemotherapy, of whom one had received prior trastuzumab. Six patients had estrogen receptor (ER)-positive breast cancer as assessed by a local laboratory, of whom 4 patients were positive for both ER and progesterone receptor.Table 1 Baseline characteristics of intent-to-treat population

4 patients had received prior chemotherapy, of whom one had received prior trastuzumab. Six patients had estrogen receptor (ER)-positive breast cancer as assessed by a local laboratory, of whom 4 patients were positive for both ER and progesterone receptor.Table 1 Baseline characteristics of intent-to-treat population Age, years Median (range) 59.0 (45–70) Time since diagnosis (months) Median (min–max) 12.9 (0–115) 1st Quartile 1.2 3rd Quartile 76.4 Prior anti-cancer therapy, n (%) Chemotherapy 4 (33) Anthracyclines 1 (8) Taxanes 3 (25) Trastuzumab 1 (8) Surgery 6 (50) Radiotherapy 2 (17) Endocrine therapy 4 (33) Immunotherapy 0 Metastatic sites, n (%) Visceral 2 (17) Non-visceral 2 (17) Visceral and non-visceral 8 (67) Hormone receptor status, n (%) ER+ and/or PgR+ 6 (50) ER+ and PgR+ 4 (33) ER+ and PgR− 2 (17) ER− and PgR− 6 (50) Unknown 0 Based on diagnosis made by investigators ER estrogen receptor, PgR progesterone receptor

Age, years Median (range) 59.0 (45–70) Time since diagnosis (months) Median (min–max) 12.9 (0–115) 1st Quartile 1.2 3rd Quartile 76.4 Prior anti-cancer therapy, n (%) Chemotherapy 4 (33) Anthracyclines 1 (8) Taxanes 3 (25) Trastuzumab 1 (8) Surgery 6 (50) Radiotherapy 2 (17) Endocrine therapy 4 (33) Immunotherapy 0 Metastatic sites, n (%) Visceral 2 (17) Non-visceral 2 (17) Visceral and non-visceral 8 (67) Hormone receptor status, n (%) ER+ and/or PgR+ 6 (50) ER+ and PgR+ 4 (33) ER+ and PgR− 2 (17) ER− and PgR− 6 (50) Unknown 0 Based on diagnosis made by investigators ER estrogen receptor, PgR progesterone receptor Tolerability and safety The median duration of lapatinib treatment was 50.9 weeks (range 4–117 weeks). Toxicities other than hematologic or neurologic toxicities leading to dose reduction occurred in 4 patients; however, DLT was not observed. The numbers of dose reductions observed were once (1250 mg) in 2 patients, twice (1000 mg) in 1 patient and three times (750 mg) in 1 patient. The primary reasons for dose reduction were rash, acne, diarrhea, increased ALT and increased aspartate aminotransferase (AST). Dose interruptions of lapatinib were reported in 10 patients, 73 times in total, mainly due to hematologic or neurologic toxicities. The median duration of interruption was 7 days (range 1–21 days).

imary reasons for dose reduction were rash, acne, diarrhea, increased ALT and increased aspartate aminotransferase (AST). Dose interruptions of lapatinib were reported in 10 patients, 73 times in total, mainly due to hematologic or neurologic toxicities. The median duration of interruption was 7 days (range 1–21 days). For paclitaxel, the median number of cycles was 10 cycles (range 2–36 cycles), in which eight patients received more than 6 cycles. Neurologic toxicity was the cause of the dose reduction in one patient and of the dose interruptions of paclitaxel in 10 patients. All 12 patients were withdrawn from L+P, mostly due to disease progression. In Part 1, the tolerability and safety of the study treatment in Japanese patients were confirmed. All patients experienced at least one AE regardless of the relationship with the study treatments, and most of them were at Grades 1 or 2. The most common AEs reported were alopecia, neutropenia, diarrhea, decreased hemoglobin and rash (Table 2). Grade 3 treatment-related AEs found in more than 2 patients were: neutropenia (n = 7), leukopenia (n = 5), diarrhea (n = 3), increased ALT (n = 3) and increased AST (n = 2). A Grade 4 treatment-related event, neutropenia, occurred in 2 patients.Table 2 Summary of adverse events with at least 50 % occurrence

(Table 2). Grade 3 treatment-related AEs found in more than 2 patients were: neutropenia (n = 7), leukopenia (n = 5), diarrhea (n = 3), increased ALT (n = 3) and increased AST (n = 2). A Grade 4 treatment-related event, neutropenia, occurred in 2 patients.Table 2 Summary of adverse events with at least 50 % occurrence Adverse event, n (%) Grade 1 Grade 2 Grade 3 Grade 4 Total Alopecia 6 (50) 6 (50) 0 0 12 (100) Diarrhea 4 (33) 4 (33) 3 (25) 0 11 (92) Neutropenia 0 2 (17) 7 (58) 2 (17) 11 (92) Decreased hemoglobin 3 (25) 6 (50) 1 (8) 0 10 (83) Rash 6 (50) 3 (25) 0 0 9 (75) Stomatitis 8 (67) 0 0 0 8 (67) Fatigue 8 (67) 0 0 0 8 (67) Peripheral sensory neuropathy 6 (50) 2 (17) 0 0 8 (67) Leukopenia 0 3 (25) 5 (42) 0 8 (67) Decreased apatite 4 (33) 3 (25) 0 0 7 (58) Paronychia 5 (42) 2 (17) 0 0 7 (58) ALT Increased 0 4 (33) 3 (25) 0 7 (58) AST Increased 1 (8) 3 (25) 2 (17) 0 6 (50) Lymphopenia 1 (8) 4 (33) 1 (8) 0 6 (50) Decreased hematocrit 5 (42) 1 (8) 0 0 6 (50) Vomiting 4 (33) 2 (17) 0 0 6 (50) Nasopharyngitis 5 (42) 1 (8) 0 0 6 (50) Nail disorder 4 (33) 2 (17) 0 0 6 (50) ALT alanine aminotransferase, AST aspartate aminotransferase Rash and diarrhea were the special interest AEs for lapatinib. No ≥Grade 3 or serious rash was reported. One Grade 2 rash event led to withdrawal from study treatment in one patient who had concurrently experienced Grade 2 erythema of the eyelid and on the back of both hands. Although Grade 3 diarrhea events occurred in 3 patients, no diarrhea was reported as ≥Grade 4 or serious, and there was no withdrawal from study treatment due to diarrhea.

led to withdrawal from study treatment in one patient who had concurrently experienced Grade 2 erythema of the eyelid and on the back of both hands. Although Grade 3 diarrhea events occurred in 3 patients, no diarrhea was reported as ≥Grade 4 or serious, and there was no withdrawal from study treatment due to diarrhea. No fatal serious AE was reported. Four protocol-defined serious AEs were reported in 3 patients; these were decreased neutrophil count in 2 patients, left ventricular dysfunction in a patient with a history of prior anthracycline treatment for other past malignancy, and pneumonia in a patient who was diagnosed by X-ray imaging. All these were considered by investigators to be treatment-related. Although the follow-up of left ventricular dysfunction was discontinued due to the start of another treatment, other serious AEs resolved within 2 weeks. Efficacy As of the end of the study, 6 patients had died. The remaining 6 patients were censored at the last visit. The median OS as primary endpoint was 35.6 months (95 % CI 23.9, not reached; Fig. 1). PFS was analyzed using the results evaluated by the investigators and the median was 13.9 months (95 % CI 7.6, 27.9; Fig. 2).Fig. 1 Kaplan–Meier estimates for overall survival Fig. 2 Kaplan–Meier estimates for progression-free survival assessed by investigators

Efficacy As of the end of the study, 6 patients had died. The remaining 6 patients were censored at the last visit. The median OS as primary endpoint was 35.6 months (95 % CI 23.9, not reached; Fig. 1). PFS was analyzed using the results evaluated by the investigators and the median was 13.9 months (95 % CI 7.6, 27.9; Fig. 2).Fig. 1 Kaplan–Meier estimates for overall survival Fig. 2 Kaplan–Meier estimates for progression-free survival assessed by investigators Ten out of 12 patients (83 %) achieved clinical benefit (95 % CI 51.6, 97.9) based on the investigators’ assessment (Table 3). The ORR in the ITT population was 83 % (95 % CI 51.6, 97.9) with 10 PRs, while one patient had SD for less than 24 weeks and progressive disease was observed in one patient.Table 3 Summary of tumor response in intent-to-treat population Best response, n (%) CR 0 PR 10 (83) SD, ≥24 weeks 0 SD, <24 weeks 1 (8) PD 1 (8) NE 0 ORR 83 (95 % CI 51.6, 97.9) CBR 83 (95 % CI 51.6, 97.9) CBR clinical benefit rate (CR; PR; SD ≥24 weeks), CR complete response, NE not evaluable, ORR overall tumor response rate, PD progressive disease, PR partial response, SD stable disease

Best response, n (%) CR 0 PR 10 (83) SD, ≥24 weeks 0 SD, <24 weeks 1 (8) PD 1 (8) NE 0 ORR 83 (95 % CI 51.6, 97.9) CBR 83 (95 % CI 51.6, 97.9) CBR clinical benefit rate (CR; PR; SD ≥24 weeks), CR complete response, NE not evaluable, ORR overall tumor response rate, PD progressive disease, PR partial response, SD stable disease Pharmacokinetics The plasma concentration–time profile of lapatinib after repeated oral dosing of lapatinib 1500 mg with or without concomitant administration of paclitaxel is shown in Fig. 3, and the plasma concentration–time profile of paclitaxel after 1 h intravenous infusion of paclitaxel 80 mg/m2 with or without concomitant administration of lapatinib is shown in Fig. 4.Fig. 3 Plasma concentration–time profile of lapatinib after dosing of lapatinib 1500 mg with or without concomitant administration of paclitaxel 80 mg/m2 Fig. 4 Plasma concentration–time profile of paclitaxel after dosing of paclitaxel 80 mg/m2 with or without concomitant administration of lapatinib 1500 mg The geometric means of Cmax and AUC(0–24) of lapatinib were increased by 59 and 42 % under the paclitaxel 80 mg/m2 combination, in comparison to lapatinib alone. No change was observed in tmax (Table 4). The geometric means of Cmax and tmax of paclitaxel were not changed by combination with lapatinib; however, that of AUC(0–inf) was increased by 23 % (Table 5).Table 4 Pharmacokinetic parameters of lapatinib after repeat dosing with or without concomitant administration of paclitaxel Parameter Lapatinib 1500 mg alone (n = 6) Lapatinib 1500 mg + paclitaxel 80 mg/m2 (n = 6) Ratio (90 % CI)a

The geometric means of Cmax and AUC(0–24) of lapatinib were increased by 59 and 42 % under the paclitaxel 80 mg/m2 combination, in comparison to lapatinib alone. No change was observed in tmax (Table 4). The geometric means of Cmax and tmax of paclitaxel were not changed by combination with lapatinib; however, that of AUC(0–inf) was increased by 23 % (Table 5).Table 4 Pharmacokinetic parameters of lapatinib after repeat dosing with or without concomitant administration of paclitaxel Parameter Lapatinib 1500 mg alone (n = 6) Lapatinib 1500 mg + paclitaxel 80 mg/m2 (n = 6) Ratio (90 % CI)a C max (ng/mL) 5945.0 (3077.9, 11483.0) 9470.4 (7157.9, 12530.0) 1.59 (0.96, 2.64) t max (h) 4.992b (1.50, 6.08)c 4.975b (1.95, 6.00)c 0.00d (−2.06, 2.21) AUC(0–24) (ng.h/mL) 79518.0 (34664.6, 182408.5) 113078.3 (74630.1, 171334.3) 1.42 (0.82, 2.46) Geometric mean (95 % CI) AUC (0–24) area under the curve from 0 to 24 h, CI confidence interval, C max maximum plasma concentration, t max time to reach maximum concentration aRatio = (lapatinib + paclitaxel)/lapatinib alone bMedian cMin and max dMedian difference Table 5 Pharmacokinetic parameters of paclitaxel after dosing with or without concomitant administration of lapatinib Parameter Paclitaxel 80 mg/m2 alone (n = 6) Paclitaxel 80 mg/m2 + lapatinib 1500 mg (n = 6) Ratioa (90 % CI) C max (ng/mL) 3485.2 (2693.0, 4510.5) 3412.3 (2753.1, 4229.4) 0.98 (0.85, 1.13)

cMin and max dMedian difference Table 5 Pharmacokinetic parameters of paclitaxel after dosing with or without concomitant administration of lapatinib Parameter Paclitaxel 80 mg/m2 alone (n = 6) Paclitaxel 80 mg/m2 + lapatinib 1500 mg (n = 6) Ratioa (90 % CI) C max (ng/mL) 3485.2 (2693.0, 4510.5) 3412.3 (2753.1, 4229.4) 0.98 (0.85, 1.13) t max (h) 0.992b (0.95, 1.08)c 0.975b (0.50, 1.02)c −0.05d (−0.27, 0.00) AUC(0–24) (ng.h/mL) 4657.2 (3942.6, 5501.3) 5786.1 (4667.5, 7172.8) 1.24 (1.13, 1.36) AUC(0–inf) (ng.h/mL) 5125.9 (4371.3, 6010.8) 6280.0 (5005.6, 7878.8) 1.23 (1.10, 1.37) t 1/2 (h) 12.19 (10.06, 14.77) 9.86 (8.48, 11.46) 0.81 (0.71, 0.92) Geometric mean (95 % CI) AUC (0–24) area under the curve, AUC (0–inf) area under the curve extrapolated to infinity, C max maximum plasma concentration, CI confidence interval, t max time to reach maximum plasma concentration, t 1/2 half-life aRatio = (paclitaxel + lapatinib)/paclitaxel alone bMedian cMin and max dMedian difference Discussion The tolerability of the lapatinib (1500 mg/day) and weekly paclitaxel (80 mg/m2) combination was confirmed in Japanese patients with HER2-positive MBC. Reported results from the pilot part of the Asian Phase III study targeting gastric cancer patients showed a similar tolerability of this regimen [19].

dMedian difference Discussion The tolerability of the lapatinib (1500 mg/day) and weekly paclitaxel (80 mg/m2) combination was confirmed in Japanese patients with HER2-positive MBC. Reported results from the pilot part of the Asian Phase III study targeting gastric cancer patients showed a similar tolerability of this regimen [19]. Drug–drug interaction of lapatinib and paclitaxel was found, as the AUC and Cmax of Japanese breast cancer patients given the combination were affected. A similar trend of interaction was observed in a Phase I study targeting solid tumor patients conducted outside of Japan [14]. The extent of drug interaction of L+P was also similar to those of the pilot part of the Asian Phase III study which included patients with a history of gastrectomy [19]. The interaction found in PK profiles of lapatinib and paclitaxel is consistent regardless of the cancer type, and such interaction was considered to be the result of lapatinib being a weak metabolism-dependent inhibitor of CYP3A4. The AEs reported in this study were generally consistent with those reported in overseas clinical studies evaluating L+P and with known safety profiles of lapatinib and paclitaxel given as monotherapy [14–17]. The grade of diarrhea was slightly worsened compared with lapatinib monotherapy, which means that L+P would require more careful management in clinical practice.

stent with those reported in overseas clinical studies evaluating L+P and with known safety profiles of lapatinib and paclitaxel given as monotherapy [14–17]. The grade of diarrhea was slightly worsened compared with lapatinib monotherapy, which means that L+P would require more careful management in clinical practice. Our study demonstrated that the combination therapy of L+P was efficacious for the treatment of HER2-positive MBC, which was consistent with the results of a Phase III study [16]. Meanwhile, results became available from another global Phase III study which showed a significant PFS of a trastuzumab-containing regimen compared with a lapatinib and taxane regimen [17]. Moreover, the results of a trastuzumab, docetaxel and pertuzumab tri-regimen became available after the results of that global study were reported, and, up to 2014, this tri-regimen became the standard in first-line therapy of MBC worldwide [20]. As the new treatment of trastuzumab, pertuzumab and docetaxel tri-regimen became available for MBC and the results of direct comparison between lapatinib and trastuzumab were confirmed, it is now proven to be difficult to recommend L+P as the first-line therapy, which we originally expected.

rapy of MBC worldwide [20]. As the new treatment of trastuzumab, pertuzumab and docetaxel tri-regimen became available for MBC and the results of direct comparison between lapatinib and trastuzumab were confirmed, it is now proven to be difficult to recommend L+P as the first-line therapy, which we originally expected. Overall, our study provides valuable results that show the drug–drug interaction and PK interaction between lapatinib and paclitaxel in Japanese patients with MBC. Although our study does not impact upon the clinical positions or the treatment strategy, it confirms that the combination of lapatinib with paclitaxel is tolerable in Japanese patients with MBC. Nevertheless, paclitaxel remains a key drug in breast cancer therapies and many multiple-agent regimens with paclitaxel have been evaluated worldwide. For this, it is considered that our findings of PK and safety data of L+P may be beneficial to those who seek the appropriate dose and safety management of the regimen. Currently, studies targeting the adjuvant/neoadjuvant setting in which to evaluate combinations such as lapatinib, paclitaxel and trastuzumab are ongoing. There is no evidence to recommend the use of lapatinib in comparison to trastuzumab; however, our data can be utilized for considering the best practice of HER2 targeting therapies. In conclusion, L+P was tolerable in Japanese patients with MBC, with manageable safety profiles, and a similar trend of the interaction of L+P to that previously reported in other ethnicities, as well as in different cancer types, was found.

Overall, our study provides valuable results that show the drug–drug interaction and PK interaction between lapatinib and paclitaxel in Japanese patients with MBC. Although our study does not impact upon the clinical positions or the treatment strategy, it confirms that the combination of lapatinib with paclitaxel is tolerable in Japanese patients with MBC. Nevertheless, paclitaxel remains a key drug in breast cancer therapies and many multiple-agent regimens with paclitaxel have been evaluated worldwide. For this, it is considered that our findings of PK and safety data of L+P may be beneficial to those who seek the appropriate dose and safety management of the regimen. Currently, studies targeting the adjuvant/neoadjuvant setting in which to evaluate combinations such as lapatinib, paclitaxel and trastuzumab are ongoing. There is no evidence to recommend the use of lapatinib in comparison to trastuzumab; however, our data can be utilized for considering the best practice of HER2 targeting therapies. In conclusion, L+P was tolerable in Japanese patients with MBC, with manageable safety profiles, and a similar trend of the interaction of L+P to that previously reported in other ethnicities, as well as in different cancer types, was found. We thank all the patients who participated in this study and their families; the investigators; medical nurses and research staffs at all the study centers. This study was fully funded and lapatinib was supplied by GlaxoSmithKline K.K.

In conclusion, L+P was tolerable in Japanese patients with MBC, with manageable safety profiles, and a similar trend of the interaction of L+P to that previously reported in other ethnicities, as well as in different cancer types, was found. We thank all the patients who participated in this study and their families; the investigators; medical nurses and research staffs at all the study centers. This study was fully funded and lapatinib was supplied by GlaxoSmithKline K.K. Conflict of interest Kenichi Inoue, Katsumasa Kuroi, Satoru Shimizu, Yoshiaki Rai, Kenjiro Aogi, Norikazu Masuda, Takahiro Nakayama, Hiroji Iwata, Yasutsuna Sasaki had no conflicts of interest; Alison Armour and Yuichiro Nishimura are employees of GlaxoSmithKline.

Introduction Irinotecan hydrochloride hydrate (CPT-11), a derivative of the antitumor alkaloid camptothecin, inhibits topoisomerase I, and is used to treat various types of tumor, including those arising as gastroenterological, lung, or gynecological cancers. As such, it forms part of one of the standard chemotherapy regimens for colorectal cancer, FOLFIRI, where it is used in combination with 5-fluorouracil (5-FU) and l-leucovorin (l-LV) [1, 2]. Irinotecan is hydrolyzed to its active metabolite, SN-38, by carboxylesterase, primarily in the human liver [3, 4]. SN-38 is then metabolized to a non-toxic glucuronide, SN-38G, by UDP-glucuronosyltransferase (UGT) 1A1, a molecular species of UGT in the liver. This metabolite is primarily excreted into the bile and transferred to the intestine [5, 6]. Genetic polymorphisms of UGT1A1 include UGT1A1*28 and UGT1A1*6, which are associated with a decrease in the formation of SN-38G, and thus delayed metabolism of SN-38 in the order wild-type, heterozygous, and homozygous. As a result, patients with UGT1A1 polymorphisms are more susceptible to toxicities such as neutropenia [7–9]. In 2005, the US Food and Drug Administration revised the Dosage and Administration section on the labeling of CPT-11 as follows: “When administered in combination with other agents, or as a single-agent, a reduction in the starting dose by at least one level of CAMPTOSAR (brand name of CPT-11) should be considered for patients known to be homozygous for the UGT1A1*28 allele. However, the precise dose reduction in this patient population is not known”.

administered in combination with other agents, or as a single-agent, a reduction in the starting dose by at least one level of CAMPTOSAR (brand name of CPT-11) should be considered for patients known to be homozygous for the UGT1A1*28 allele. However, the precise dose reduction in this patient population is not known”. The allele frequency of UGT1A1*28 is lower in Asians (8.6–13.0 % in Japanese) than in Caucasians (29.5–38.8 %). In contrast, although the frequency of the UGT1A1*6 allele is very low in Caucasians, it is relatively common in Asians (13.0–17.7 % in Japanese) [10], which partly contributes to an increased risk of CPT-11-induced toxicity in this group. Therefore, in Japan, some studies have investigated the relationship between UGT1A1 genetic polymorphisms and risk, focusing on the following three groups—*1/*1 (wild-type group); *28/*1 and *6/*1 (heterozygous group); and *28/*28, *6/*6, and *28/*6 (homozygous group). Double heterozygosity (*28/*6) was classified as homozygous based on the results of other earlier studies [7, 11, 12]. The results of one of these earlier clinical studies were inconclusive as to the optimum dose of CPT-11 for a homozygous group [11]. Approval for testing for UGT1A1*6 and *28 genetic polymorphisms using the Invader UGT1A1 Molecular Assay (Sekisui Medical Co., Ltd, Tokyo, Japan) under national insurance was finally given in November 2008 in Japan. The test kit then became available in March 2009, making this an easy and viable part of therapy in a clinical setting.

sting for UGT1A1*6 and *28 genetic polymorphisms using the Invader UGT1A1 Molecular Assay (Sekisui Medical Co., Ltd, Tokyo, Japan) under national insurance was finally given in November 2008 in Japan. The test kit then became available in March 2009, making this an easy and viable part of therapy in a clinical setting. The purpose of this prospective study was to investigate whether UGT1A1*6 and *28 polymorphisms could be used to determine the initial dose level of CPT-11 to improve safety in patients receiving FOLFIRI for colorectal cancer. Patients and methods Patients Patients were eligible for inclusion in the present study if they met all of the following criteria—CPT-11-naive; no evidence of myelosuppression, infectious disease, diarrhea (watery stool), intestinal paralysis or obstruction, ascites, or jaundice; a diagnosis of colorectal cancer; scheduled to receive FOLFIRI; and provided informed consent to participate in this study. This study was conducted in accordance with Good Post-marketing Study Practice (GPSP) of the Ministry of Health, Labour and Welfare, Japan. Approval by the Institutional Review Board of each institution was not mandatory, as the GPSP does not require such approval for post-marketing surveillance.

icipate in this study. This study was conducted in accordance with Good Post-marketing Study Practice (GPSP) of the Ministry of Health, Labour and Welfare, Japan. Approval by the Institutional Review Board of each institution was not mandatory, as the GPSP does not require such approval for post-marketing surveillance. Classification of UGT1A1 polymorphisms Three groups were established based on the results of testing for UGT1A1*6 and *28 genetic polymorphisms—wild-type group (*1/*1); heterozygous group (*28/*1 and *6/*1); and homozygous group (*28/*28, *6/*6, and *28/*6). Double heterozygosity (*28/*6) was classified as homozygous based on the results of previous studies [7, 11, 12]. Treatment FOLFIRI was administered according to a previously reported standard schedule in Japan—CPT-11, 5-FU (bolus), 5-FU (infusion), and l-LV at 150, 400, 2,400, and 200 mg/m2, respectively [2, 13]. Because this was a non-interventional study, the dose level and dosing interval were modified at the discretion of the primary physician. In this study, a dose reduction was defined as 5 % lower (142.5 mg/m2) than the standard dose in Japan (150 mg/m2). Information on treatment days and dosing levels was collected for 1 year after the start of treatment. Time-to-treatment failure (TTF) was defined as the period of time from the start of treatment until the end of treatment due to ‘progressive disease or tumor death’, ‘adverse event-related (side-effect) or treatment-related death’, or ‘withdrawal from FOLFIRI’.

els was collected for 1 year after the start of treatment. Time-to-treatment failure (TTF) was defined as the period of time from the start of treatment until the end of treatment due to ‘progressive disease or tumor death’, ‘adverse event-related (side-effect) or treatment-related death’, or ‘withdrawal from FOLFIRI’. Safety evaluation Adverse events were evaluated according to the Common Terminology Criteria for Adverse Events v3.0. The follow-up period was defined as 1 year after the start of treatment. Statistical analyses All analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA). TTF according to UGT1A1 genetic polymorphisms was assessed using Kaplan–Meier curves, and significance was determined using the log-rank test. Using the first cycle as the target, the incidence of grade ≥3 neutropenia was analyzed in a multivariate model using a step-down procedure to select variables; significance level was set at p = 0.1. The variables selected were genetic polymorphism, age, sex, Eastern Cooperative Oncology Group performance status (PS), prior chemotherapy, new onset/recurrence, coelomic fluid, complications, and starting dose reduction. Differences were considered statistically significant when the two-tailed p value was <0.05.

0.1. The variables selected were genetic polymorphism, age, sex, Eastern Cooperative Oncology Group performance status (PS), prior chemotherapy, new onset/recurrence, coelomic fluid, complications, and starting dose reduction. Differences were considered statistically significant when the two-tailed p value was <0.05. Results Baseline characteristics of patients A total of 823 patients from 210 institutions were initially enrolled in the study between April 2009 and March 2011. Of these, 795 patients were included in the safety evaluation after excluding two patients not treated with CPT-11, 24 for whom no data were available, and two who did not meet the selection criteria (Fig. 1). Of the 795 patients evaluated, 398 (50.1 %) were wild-type, 327 (41.1 %) were heterozygous, and 70 (8.8 %) were homozygous according to UGT1A1*6 and *28 polymorphisms (Table 1). The median ages of the patients in the wild-type, heterozygous, and homozygous groups were 67, 66, and 65 years, respectively; 151 (37.9 %), 112 (34.3 %), and 20 (28.6 %) patients were aged ≥70 years in each group, respectively. No marked imbalance was noted in any other baseline characteristics (Table 2).Fig. 1 Flow chart. A total of 823 patients tested for UGT1A1 genetic polymorphisms were enrolled in the study. Overall, 795 patients were included in safety evaluation after excluding 28 patients who did not meet the inclusion criteria Table 1 Frequencies of UGT1A1 genotype in this study

Results Baseline characteristics of patients A total of 823 patients from 210 institutions were initially enrolled in the study between April 2009 and March 2011. Of these, 795 patients were included in the safety evaluation after excluding two patients not treated with CPT-11, 24 for whom no data were available, and two who did not meet the selection criteria (Fig. 1). Of the 795 patients evaluated, 398 (50.1 %) were wild-type, 327 (41.1 %) were heterozygous, and 70 (8.8 %) were homozygous according to UGT1A1*6 and *28 polymorphisms (Table 1). The median ages of the patients in the wild-type, heterozygous, and homozygous groups were 67, 66, and 65 years, respectively; 151 (37.9 %), 112 (34.3 %), and 20 (28.6 %) patients were aged ≥70 years in each group, respectively. No marked imbalance was noted in any other baseline characteristics (Table 2).Fig. 1 Flow chart. A total of 823 patients tested for UGT1A1 genetic polymorphisms were enrolled in the study. Overall, 795 patients were included in safety evaluation after excluding 28 patients who did not meet the inclusion criteria Table 1 Frequencies of UGT1A1 genotype in this study UGT1A1 genotype n (%) All patients 795 Wild-type group 398 (50.1) *1/*1 398 (50.1) Heterozygous group 327 (41.1) *6/*1 195 (24.6) *28/*1 132 (16.6) Homozygous group 70 (8.8) *6/*6 14 (1.8) *28/*28 12 (1.5) *28/*6 44 (5.5) UGT UDP-glucuronosyltransferase Table 2 Baseline characteristics of patients

Table 1 Frequencies of UGT1A1 genotype in this study UGT1A1 genotype n (%) All patients 795 Wild-type group 398 (50.1) *1/*1 398 (50.1) Heterozygous group 327 (41.1) *6/*1 195 (24.6) *28/*1 132 (16.6) Homozygous group 70 (8.8) *6/*6 14 (1.8) *28/*28 12 (1.5) *28/*6 44 (5.5) UGT UDP-glucuronosyltransferase Table 2 Baseline characteristics of patients Characteristics Wild-type (n = 398), n (%) Heterozygous (n = 327), n (%) Homozygous (n = 70), n (%) Age (years) Median 67.0 66.0 65.0 Range 29–86 35–86 36–81 <70 247 (62.1) 215 (65.7) 50 (71.4) ≥70 151 (37.9) 112 (34.3) 20 (28.6) Sex Male 234 (58.8) 205 (62.7) 41 (58.6) Female 164 (41.2) 122 (37.3) 29 (41.4) Performance status 0 298 (74.9) 242 (74.0) 48 (68.6) 1 88 (22.1) 72 (22.0) 22 (31.4) ≥2 12 (3.0) 13 (4.0) 0 (−) Prior chemotherapy Absent 73 (18.3) 57 (17.4) 12 (17.1) Present 325 (81.7) 270 (82.6) 58 (82.9) New onset/recurrent New onset 201 (50.5) 174 (53.2) 33 (47.1) Recurrent 197 (49.5) 153 (46.8) 37 (52.9) Coelomic fluid Absent 374 (94.0) 295 (90.2) 65 (92.9) Present 24 (6.0) 32 (9.8) 5 (7.1) Complications Absent 250 (62.8) 193 (59.0) 48 (68.6) Present 148 (37.2) 134 (41.0) 22 (31.4)

ent 325 (81.7) 270 (82.6) 58 (82.9) New onset/recurrent New onset 201 (50.5) 174 (53.2) 33 (47.1) Recurrent 197 (49.5) 153 (46.8) 37 (52.9) Coelomic fluid Absent 374 (94.0) 295 (90.2) 65 (92.9) Present 24 (6.0) 32 (9.8) 5 (7.1) Complications Absent 250 (62.8) 193 (59.0) 48 (68.6) Present 148 (37.2) 134 (41.0) 22 (31.4) CPT-11 administration Table 3 shows the starting doses of each drug administered as part of FOLFIRI. The dose level of 5-FU did not differ among the three groups, but the median starting doses of CPT-11 were 143.0, 143.0, and 115.0 mg/m2 in the wild-type, heterozygous, and homozygous groups, respectively (Table 3). A total of 204 patients (50.1 %) in the wild-type group, 164 (51.3 %) in the heterozygous group, and 23 (32.9 %) in the homozygous group received a starting dose of CPT-11 of ≥142.5 mg/m2. A bimodal distribution was observed in the homozygous group, but not in the wild-type or heterozygous groups (Fig. 2).Table 3 State of clinical use Wild-type (n = 398) Heterozygous (n = 327) Homozygous (n = 70) Starting dose (mg/m2) CPT-11, median (range) 143.0 (56–185) 143.0 (23–181) 115.0 (41–180) Bolus 5-FU, median (range) 384.0 (0–796) 380.0 (0–800) 383.5 (0–517) Infusional 5-FU, median (range) 2327.0 (538–2542) 2312.0 (178–2830) 2299.0 (0–2469) Distribution of starting CPT-11 dose <142.5 mg/m2 (%) 194 (48.7) 163 (49.8) 47 (67.1) ≥142.5 mg/m2 (%) 204 (51.3) 164 (50.2) 23 (32.9) CPT-11 irinotecan, 5-FU 5-fluorouracil Fig. 2 Distribution of starting dose of CPT-11 in a wild-type (n = 398), b heterozygous (n = 327), and c homozygous groups (n = 70)

Wild-type (n = 398) Heterozygous (n = 327) Homozygous (n = 70) Starting dose (mg/m2) CPT-11, median (range) 143.0 (56–185) 143.0 (23–181) 115.0 (41–180) Bolus 5-FU, median (range) 384.0 (0–796) 380.0 (0–800) 383.5 (0–517) Infusional 5-FU, median (range) 2327.0 (538–2542) 2312.0 (178–2830) 2299.0 (0–2469) Distribution of starting CPT-11 dose <142.5 mg/m2 (%) 194 (48.7) 163 (49.8) 47 (67.1) ≥142.5 mg/m2 (%) 204 (51.3) 164 (50.2) 23 (32.9) CPT-11 irinotecan, 5-FU 5-fluorouracil Fig. 2 Distribution of starting dose of CPT-11 in a wild-type (n = 398), b heterozygous (n = 327), and c homozygous groups (n = 70) Safety The incidence of grade ≥3 neutropenia in the first cycle tended to increase in the order wild-type < heterozygous < homozygous (17.3, 25.4, 28.6 %, respectively), and this tendency persisted throughout the treatment (44.7, 54.1, and 57.1 %, respectively) (Table 4). The percentages of patients withdrawn from treatment were similar for all UGT1A1 genotypes (Table 5).Table 4 Median TTF and reasons for treatment discontinuation Wild-type (n = 398) Heterozygous (n = 327) Homozygous (n = 70) TTF Median 161.5 165.0 136.0 95 % CI 142.0–183.0 148.0–177.0 106.0–177.0 Reasons for discontinuing FOLFIRI Progressive disease (%) 183 (46.0) 169 (51.7) 33 (47.1) Adverse events (%) 59 (14.8) 52 (15.9) 10 (14.3) Withdrawal of FOLFIRI (%) 75 (18.8) 53 (16.2) 14 (20.0) TTF time-to-treatment failure, CI confidence interval, FOLFIRI l-leucovorin, 5-fluorouracil, and irinotecan Table 5 Association between UGT1A1 genotype and irinotecan toxicities

Wild-type (n = 398) Heterozygous (n = 327) Homozygous (n = 70) TTF Median 161.5 165.0 136.0 95 % CI 142.0–183.0 148.0–177.0 106.0–177.0 Reasons for discontinuing FOLFIRI Progressive disease (%) 183 (46.0) 169 (51.7) 33 (47.1) Adverse events (%) 59 (14.8) 52 (15.9) 10 (14.3) Withdrawal of FOLFIRI (%) 75 (18.8) 53 (16.2) 14 (20.0) TTF time-to-treatment failure, CI confidence interval, FOLFIRI l-leucovorin, 5-fluorouracil, and irinotecan Table 5 Association between UGT1A1 genotype and irinotecan toxicities Toxicities Wild-type (n = 398), n (%) Heterozygous (n = 327), n (%) Homozygous (n = 70), n (%) First-cycle neutropenia Grade ≥1 209 (52.5) 199 (60.9) 42 (60.0) Grade ≥3 69 (17.3) 83 (25.4) 20 (28.6) Neutropenia Grade ≥1 311 (78.1) 266 (81.3) 55 (78.6) Grade ≥3 178 (44.7) 177 (54.1) 40 (57.1) All adverse events Grade ≥1 383 (96.2) 321 (98.2) 68 (97.1) Grade ≥3 229 (57.5) 223 (68.2) 48 (68.6) Multivariate analysis was performed to identify risk factors for first-cycle grade ≥3 neutropenia. Heterozygosity and homozygosity were identified as significant risk factors compared with wild-type [heterozygous group: p = 0.0060; odds ratio (OR) 1.67; 95 % confidence interval (CI) 1.16–2.42, and homozygous group: p = 0.0088; OR 2.22; 95 % CI 1.22–4.02]. Age ≥70 years (p = 0.0017; OR 1.77; 95 % CI 1.24–2.53), coelomic fluid (p = 0.0343; OR 1.84; 95 % CI 1.05–3.25), and non-reduction in starting dose (p = 0.0176; OR 1.53; 95 % CI 1.08–2.18) were also identified as independent significant risk factors (Table 6).Table 6 Multivariate predictors of treatment-related grade ≥3 neutropenia

(p = 0.0017; OR 1.77; 95 % CI 1.24–2.53), coelomic fluid (p = 0.0343; OR 1.84; 95 % CI 1.05–3.25), and non-reduction in starting dose (p = 0.0176; OR 1.53; 95 % CI 1.08–2.18) were also identified as independent significant risk factors (Table 6).Table 6 Multivariate predictors of treatment-related grade ≥3 neutropenia Neutropenia OR (95 % CI) p value* UGT1A1 genotype (wild-type vs heterozygous) 1.67 1.16–2.42 0.0060 UGT1A1 genotype (wild-type vs homozygous) 2.22 1.22–4.02 0.0088 Age (years) (<70 vs ≥70) 1.77 1.24–2.53 0.0017 Sex (male vs female) 1.38 0.97–1.95 0.0726 Coelomic fluid (absent vs present) 1.84 1.05–3.25 0.0343 Starting dose reduction (reduction vs non-reduction) 1.53 1.08–2.18 0.0176 UGT UDP-glucuronosyltransferase, OR odds ratio, CI confidence interval * Chi-squared test

UGT1A1 genotype (wild-type vs homozygous) 2.22 1.22–4.02 0.0088 Age (years) (<70 vs ≥70) 1.77 1.24–2.53 0.0017 Sex (male vs female) 1.38 0.97–1.95 0.0726 Coelomic fluid (absent vs present) 1.84 1.05–3.25 0.0343 Starting dose reduction (reduction vs non-reduction) 1.53 1.08–2.18 0.0176 UGT UDP-glucuronosyltransferase, OR odds ratio, CI confidence interval * Chi-squared test Efficacy Figure 3 shows the Kaplan–Meier curves obtained for TTF according to UGT1A1 genetic polymorphism. No significant difference was observed among the UGT1A1 genetic polymorphisms [wild-type group vs heterozygous or homozygous group: p = 0.7390; hazard ratio (HR) 1.025; 95 % CI 0.888–1.183, and wild-type or heterozygous group vs homozygous group: p = 0.1582; HR 1.197; 95 % CI 0.931–1.540]. The median TTF was 161.5 (95 % CI 142.0–183.0), 165.0 (95 % CI 148.0–177.0), and 136.0 (95 % CI 106.0–177.0) days in the wild-type, heterozygous, and homozygous groups, respectively (Table 4).Fig. 3 Kaplan–Meier curves for time from start of treatment to discontinuation of treatment according to UGT1A1 genetic polymorphisms. Patients not withdrawn from treatment during the follow-up period (1 year after start of treatment) were censored on the last dosing day of the final treatment cycle The present results indicated little difference in effect of each polymorphism on TTF, regardless of starting dose (data not shown).

Efficacy Figure 3 shows the Kaplan–Meier curves obtained for TTF according to UGT1A1 genetic polymorphism. No significant difference was observed among the UGT1A1 genetic polymorphisms [wild-type group vs heterozygous or homozygous group: p = 0.7390; hazard ratio (HR) 1.025; 95 % CI 0.888–1.183, and wild-type or heterozygous group vs homozygous group: p = 0.1582; HR 1.197; 95 % CI 0.931–1.540]. The median TTF was 161.5 (95 % CI 142.0–183.0), 165.0 (95 % CI 148.0–177.0), and 136.0 (95 % CI 106.0–177.0) days in the wild-type, heterozygous, and homozygous groups, respectively (Table 4).Fig. 3 Kaplan–Meier curves for time from start of treatment to discontinuation of treatment according to UGT1A1 genetic polymorphisms. Patients not withdrawn from treatment during the follow-up period (1 year after start of treatment) were censored on the last dosing day of the final treatment cycle The present results indicated little difference in effect of each polymorphism on TTF, regardless of starting dose (data not shown). Discussion The goal of the present study was to investigate whether UGT1A1*6 and *28 polymorphisms could be used to determine the initial dose level of CPT-11 to improve safety in patients receiving FOLFIRI for colorectal cancer. These results comprise the largest published prospective analysis to date.

The present results indicated little difference in effect of each polymorphism on TTF, regardless of starting dose (data not shown). Discussion The goal of the present study was to investigate whether UGT1A1*6 and *28 polymorphisms could be used to determine the initial dose level of CPT-11 to improve safety in patients receiving FOLFIRI for colorectal cancer. These results comprise the largest published prospective analysis to date. The multivariate analysis revealed that the incidence of neutropenia increased significantly in those groups harboring one or more alleles compared with the wild-type group—the heterozygous and homozygous groups showed a 1.67-fold and 2.22-fold increase in risk of grade ≥3 neutropenia, respectively. Minami et al. reported that neutropenia was associated with homozygosity for UGT1A1*6 or *28 [9]. However, Hoskins et al. [14] and Hu et al. [15] reported inconsistent results from meta-analyses of the relationship between homozygosity for UGT1A1*28 and neutropenia in patients treated with CPT-11 at a starting dose of <150 mg/m2, and no definitive relationship was therefore demonstrated. The results of the present study, albeit non-interventional, support the findings of Minami et al. and Hu et al. Moreover, the present study was based on an analysis of data from a single set of 795 patients, and therefore comprises a larger sample size than those in the above-mentioned studies. Satho et al. [11] and Hazama et al. [16] found that the pharmacokinetics of SN 38 were higher in patients harboring one variant allele than in those who were not. Moreover, in the Satho study, there was a significant correlation between an increase in the rate of severe neutropenia and an increase in the pharmacokinetics of SN38. Taken together, these results suggest that the initial dose of CPT-11 should be considered with care in patients homozygous or heterozygous for UGT1A1 in clinical practice in Japan. However, previous studies reported that the risk for those who were heterozygous was small, indicating the need for further evaluation of clinical information on UGT1A1*6 and *28 heterozygous groups in future studies.

considered with care in patients homozygous or heterozygous for UGT1A1 in clinical practice in Japan. However, previous studies reported that the risk for those who were heterozygous was small, indicating the need for further evaluation of clinical information on UGT1A1*6 and *28 heterozygous groups in future studies. The observed OR for the risk of neutropenia in the homozygous group compared with the wild-type group (2.22) was lower than (5.21–8.63) reported in previous studies [11, 17]. Hoskins et al. [14] reported that the risk of neutropenia abated with a decrease in the dose of CPT-11. In their study, the OR was 1.80 at lower doses, which represented no significant difference between homozygous and wild-type or heterozygous individuals, similar to the findings in the present study.

tudies [11, 17]. Hoskins et al. [14] reported that the risk of neutropenia abated with a decrease in the dose of CPT-11. In their study, the OR was 1.80 at lower doses, which represented no significant difference between homozygous and wild-type or heterozygous individuals, similar to the findings in the present study. In addition to UGT1A1 polymorphisms, the present study also identified age ≥70 years, coelomic fluid, and non-reduction in starting dose as risk factors for grade ≥3 neutropenia. The relationship between age and risk of neutropenia remains unexplained, although older patients are generally more likely to experience adverse events, probably because of age-related changes in pharmacokinetics or pharmacodynamics and an increased prevalence of chronic diseases. The trend observed in the present study is in line with the results of an earlier study showing that CPT-11 monotherapy was associated with a higher risk of grade ≥3 neutropenia in the elderly [18]. The relationship between coelomic fluid and risk of neutropenia also remains poorly understood, although an increased risk of CPT-11-related leukopenia in pleural effusion and massive ascites was reported [19]. We believe that the retention of coelomic fluid decreases intestinal peristalsis, thereby delaying excretion of CPT-11 and increasing toxicity. In addition, there appears to be an association between coelomic fluid and peritoneal dissemination. This suggests that the disease is likely to be at a more advanced stage by the time treatment is initiated in patients with coelomic fluid. Taken together with the results from earlier studies, the present results suggest that patient age and presence of coelomic fluid should also be taken into consideration when planning treatment in a clinical setting.

kely to be at a more advanced stage by the time treatment is initiated in patients with coelomic fluid. Taken together with the results from earlier studies, the present results suggest that patient age and presence of coelomic fluid should also be taken into consideration when planning treatment in a clinical setting. In the present study, the starting dose of CPT-11 was reduced in approximately 67 % of patients in the homozygous group, but not in the wild-type or heterozygous groups, resulting in a median starting dose that was approximately 20 % lower in the homozygous group. A previous study [11] investigating dose levels in Japanese patients according to UGT1A1*6 and *28 polymorphisms demonstrated the safety of 150 mg/m2 in wild-type and heterozygous groups; however, no recommended dose was identified for homozygous patients because of a large individual variation in pharmacokinetics. The results of the present study indicate that testing for UGT1A1*6 and *28 genetic polymorphisms could be useful in determining the appropriate starting dose of CPT-11 in clinical practice.

In the present study, the starting dose of CPT-11 was reduced in approximately 67 % of patients in the homozygous group, but not in the wild-type or heterozygous groups, resulting in a median starting dose that was approximately 20 % lower in the homozygous group. A previous study [11] investigating dose levels in Japanese patients according to UGT1A1*6 and *28 polymorphisms demonstrated the safety of 150 mg/m2 in wild-type and heterozygous groups; however, no recommended dose was identified for homozygous patients because of a large individual variation in pharmacokinetics. The results of the present study indicate that testing for UGT1A1*6 and *28 genetic polymorphisms could be useful in determining the appropriate starting dose of CPT-11 in clinical practice. An analysis of treatment duration in relation to UGT1A1 genetic polymorphisms revealed that TTF was similar between the wild-type, heterozygous, and homozygous groups, as were the reasons for treatment discontinuation. Blood drug concentrations were not measured in the current study, but the metabolism of SN-38 is delayed in individuals homozygous for UGT1A1 polymorphisms [9, 20]. In the present study, the dose level in the homozygous group was reduced, allowing the pharmacokinetics of SN-38 to be maintained without compromising its antitumor efficacy and avoiding serious adverse events requiring treatment discontinuation; therefore, there was no effect on the treatment duration. However, caution should be taken in reducing the dose, as it may increase the risk of ineffective treatment.

cokinetics of SN-38 to be maintained without compromising its antitumor efficacy and avoiding serious adverse events requiring treatment discontinuation; therefore, there was no effect on the treatment duration. However, caution should be taken in reducing the dose, as it may increase the risk of ineffective treatment. In conclusion, the present results revealed that patients harboring one or more alleles had a higher risk of neutropenia at initiation of treatment, indicating the importance of testing for UGT1A1 genetic polymorphisms before commencing therapy. These results also suggest that when a reduction in dose is required in patients harboring two variant alleles, the decrease should be approximately 20 %. We would like to thank all the patients and healthcare professionals who cooperated in this study, as well as Yakult Honsha Co., Ltd. Compliance with ethical standards Conflict of interest Mitsuaki Manabe is a Yakult Honsha employee. Yoshinori Miyata, Tetsuo Touyama, Takaya Kusumi, Yoshitaka Morita, Nobuyuki Mizunuma, and Fumihiro Taniguchi have no conflict of interest to declare.

Introduction As prostate cancer growth is dependent on androgens, androgen deprivation therapy (ADT), which includes surgical castration or medical therapy with gonadotropin-releasing hormone (GnRH) agonists or GnRH antagonists, is standard therapy for patients with metastatic prostate cancer recurrence after definitive therapy, or inoperable prostate cancer. In Japan, it is common practice in primary ADT to use androgen blockade combined with bicalutamide, a non-steroidal anti-androgen [1–4]. Progression of the disease despite castrate levels of testosterone under primary ADT is considered castration-resistant prostate cancer (CRPC) [5] and it generally represents a transition to the lethal state of the disease. CRPC is frequently treated with hormone therapy alternating with anti-androgens, low dose steroids or estrogenic compounds [6]. However, prolonged survival of patients with CRPC by these secondary hormonal treatments is not confirmed [7]. Until early 2014, docetaxel plus prednisone were the only approved drugs for patients with advanced CRPC in Japan [8, 9]. Recent treatment options that have demonstrated a survival improvement in patients with metastatic CRPC include cabazitaxel plus prednisone [10] and abiraterone plus prednisone [11, 12]. Enzalutamide [13, 14], sipuleucel-T [15] and radium Ra 223 dichloride [16] have also been approved for use in several countries.

Introduction As prostate cancer growth is dependent on androgens, androgen deprivation therapy (ADT), which includes surgical castration or medical therapy with gonadotropin-releasing hormone (GnRH) agonists or GnRH antagonists, is standard therapy for patients with metastatic prostate cancer recurrence after definitive therapy, or inoperable prostate cancer. In Japan, it is common practice in primary ADT to use androgen blockade combined with bicalutamide, a non-steroidal anti-androgen [1–4]. Progression of the disease despite castrate levels of testosterone under primary ADT is considered castration-resistant prostate cancer (CRPC) [5] and it generally represents a transition to the lethal state of the disease. CRPC is frequently treated with hormone therapy alternating with anti-androgens, low dose steroids or estrogenic compounds [6]. However, prolonged survival of patients with CRPC by these secondary hormonal treatments is not confirmed [7]. Until early 2014, docetaxel plus prednisone were the only approved drugs for patients with advanced CRPC in Japan [8, 9]. Recent treatment options that have demonstrated a survival improvement in patients with metastatic CRPC include cabazitaxel plus prednisone [10] and abiraterone plus prednisone [11, 12]. Enzalutamide [13, 14], sipuleucel-T [15] and radium Ra 223 dichloride [16] have also been approved for use in several countries. Enzalutamide is a novel androgen receptor inhibitor that significantly prolongs survival of men with CRPC regardless of prior docetaxel therapy [13]. Enzalutamide inhibits multiple steps in the androgen receptor signaling pathway and is devoid of agonist activity in preclinical models [17]. Preclinical pharmacology studies have demonstrated that enzalutamide competitively inhibits androgen-induced receptor activation, nuclear translocation of activated androgen receptors, and the association of the activated androgen receptor with chromatin, even in the setting of androgen receptor over-expression and in prostate cancer cells resistant to anti-androgens [17].

f progression, death, or last follow-up, whichever occurred first. Impact of surgery result on survival was assessed by constructing Kaplan–Meier curves with a log-rank test. Cox regression analyses were performed to assess the prognostic factors on survival. All reported significance was two tailed at a level of 0.05. Results Clinical characteristics Among 43 patients enrolled, 1 was diagnosed as having glassy cell carcinoma based on the postoperative histopathological examination. Therefore, we analyzed the other 42 patients. The median age was 45 years (range, 25–63). The performance status was 0 in 37 patients and 1 in 5, and clinical progression was stage IB2 in 9 patients, stage IIA2 in 2, stage IIB2 in 27, and stage IIIB in 4. Histological subtypes were keratinizing type in 11 patients and non-keratinizing type in 31. Computed tomography before NAC showed lymph node metastasis in 16 patients and no lymph node metastasis in 26. The tumor size was less than 5 cm in 18 patients and 5 cm or more in 24 by MRI before NAC. Thirteen patients were positive and 29 patients were negative for pathological lymph node metastasis. Postoperative treatments were radiation therapy in 13 patients, chemotherapy in 15, and chemoradiotherapy in 3; no postoperative treatment was given to 9 patients (Table 1).Table 1 Patient characteristics

enzalutamide competitively inhibits androgen-induced receptor activation, nuclear translocation of activated androgen receptors, and the association of the activated androgen receptor with chromatin, even in the setting of androgen receptor over-expression and in prostate cancer cells resistant to anti-androgens [17]. The efficacy of enzalutamide was evaluated in two multinational phase III studies in men with metastatic CRPC; AFFIRM and PREVAIL. The AFFIRM trial showed overall survival (OS) benefit of enzalutamide in post-docetaxel patients with metastatic CRPC versus placebo. Median survival was 18.4 months with enzalutamide and 13.6 months with placebo [hazard ratio 0.63; 95 % confidence interval (CI) 0.53–0.75; p < 0.001] [14]. The PREVAIL trial confirmed clinical benefit of enzalutamide in chemotherapy-naïve patients with metastatic CRPC. The hazard ratio of OS and radiographic progression-free survival (rPFS) were 0.71 (95 % CI 0.60–0.84; p < 0.001) and 0.19 (95 % CI 0.15–0.23; p < 0.001), respectively [13]. Median OS was 32.4 months with enzalutamide and 30.2 months with placebo. Median rPFS was not reached with enzalutamide and was 3.9 months with placebo [13]. The present phase I/II clinical study evaluated the safety, tolerability and pharmacokinetics (PK) of enzalutamide in patients with CRPC and the anti-tumor activity and safety of enzalutamide in Japanese post-docetaxel patients with CRPC to provide supporting data for the regulatory approval of enzalutamide in Japan.

The efficacy of enzalutamide was evaluated in two multinational phase III studies in men with metastatic CRPC; AFFIRM and PREVAIL. The AFFIRM trial showed overall survival (OS) benefit of enzalutamide in post-docetaxel patients with metastatic CRPC versus placebo. Median survival was 18.4 months with enzalutamide and 13.6 months with placebo [hazard ratio 0.63; 95 % confidence interval (CI) 0.53–0.75; p < 0.001] [14]. The PREVAIL trial confirmed clinical benefit of enzalutamide in chemotherapy-naïve patients with metastatic CRPC. The hazard ratio of OS and radiographic progression-free survival (rPFS) were 0.71 (95 % CI 0.60–0.84; p < 0.001) and 0.19 (95 % CI 0.15–0.23; p < 0.001), respectively [13]. Median OS was 32.4 months with enzalutamide and 30.2 months with placebo. Median rPFS was not reached with enzalutamide and was 3.9 months with placebo [13]. The present phase I/II clinical study evaluated the safety, tolerability and pharmacokinetics (PK) of enzalutamide in patients with CRPC and the anti-tumor activity and safety of enzalutamide in Japanese post-docetaxel patients with CRPC to provide supporting data for the regulatory approval of enzalutamide in Japan. Patients and methods Study design This was a multicenter, open-label, uncontrolled study of orally administered enzalutamide, involving two phases (http://ClinicalTrials.gov NCT01284920). Phase I involved dose escalation in patients with CRPC. Phase II involved dose expansion in post-docetaxel patients with CRPC.

The present phase I/II clinical study evaluated the safety, tolerability and pharmacokinetics (PK) of enzalutamide in patients with CRPC and the anti-tumor activity and safety of enzalutamide in Japanese post-docetaxel patients with CRPC to provide supporting data for the regulatory approval of enzalutamide in Japan. Patients and methods Study design This was a multicenter, open-label, uncontrolled study of orally administered enzalutamide, involving two phases (http://ClinicalTrials.gov NCT01284920). Phase I involved dose escalation in patients with CRPC. Phase II involved dose expansion in post-docetaxel patients with CRPC. All participating sites obtained approval for conducting the study by their institutional review boards. The study was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice Guidelines and the Pharmaceutical Affairs Law in Japan. All patients provided written informed consent to participate in the study.

pating sites obtained approval for conducting the study by their institutional review boards. The study was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice Guidelines and the Pharmaceutical Affairs Law in Japan. All patients provided written informed consent to participate in the study. In phase I, patients received a single dose of enzalutamide (80, 160 or 240 mg/day) and blood samples for PK analysis were collected over 7 days. Subsequently, patients received multiple doses of enzalutamide at the same dosage levels as in the single-dose period. Tolerability was evaluated 29 days after initiation of repeat dosing by an independent data monitoring committee. Patients who received 240 mg in the single-dose period subsequently received multiple doses of 160 mg/day (the recommended dose in the AFFIRM study [12]). Following the evaluation of enzalutamide tolerability and PK parameters after single and multiple doses at 160 mg in phase I, an open-label, uncontrolled phase II study was initiated to evaluate the efficacy, safety and PK in patients receiving enzalutamide 160 mg/day. The study design was discussed with the Pharmaceuticals and Medicine Devices Agency from a perspective of regulatory approval of enzalutamide in Japan. Consequently, overall response rate was selected to be the primary outcome variable in this study, thereby requiring enrolment of patients with measureable disease into the study.

ash (the most common skin toxicity) receiving steroids within 4 days of diagnosis. Of the patients experiencing xeroderma, more than 75 % received steroids within 5 days of onset or diagnosis, and many patients with paronychia were already on steroids, for an average of 10 days, before diagnosis or onset of paronychia. Regardless of rash grade, earlier initiation of topical steroids resulted in quicker recovery (Fig. 1). Although the early initiation groups (before onset, 0–1, 2–6, and 7–13 days) had similar median recovery times (30–39 days depending on initiation group for grade 1 rash, 48–51 days depending on initiation group for grade 2 rash), there was a noticeable increase in recovery time from the 14–20 days group (93 days for grade 1 rash, 71 days for grade 2 rash). In the group initiated more than 21 days after onset, recovery appeared to be considerably longer than the other groups, resulting in a recovery time of more than 100 days regardless of rash grade, compared with the before onset groups who had median time to recovery of 35–51 days depending on rash grade.Fig. 1 Time to recovery according to time to treatment initiation: grade 1 rash (a), grade 2 rash (b), grade ≥3 rash (c)

esign was discussed with the Pharmaceuticals and Medicine Devices Agency from a perspective of regulatory approval of enzalutamide in Japan. Consequently, overall response rate was selected to be the primary outcome variable in this study, thereby requiring enrolment of patients with measureable disease into the study. Patients Patients with metastatic CRPC who had disease progression while on castration therapy were eligible for participation. Patients had to have received ADT with a GnRH analogue or a bilateral orchiectomy with serum testosterone level maintained within castration level (≤50 ng/dL). The criteria used to define disease progression for trial entry are available in the Online Resource. Eligible patients had Eastern Cooperative Oncology Group performance status of 0 or 1 (or 2 if only due to metastatic bone pain at the screening). Post-chemotherapy patients had to have received prior chemotherapy with docetaxel and no more than two prior chemotherapy regimens. In particular for phase II, patients had to have measurable lesions as determined by response evaluation criteria for solid tumors (RECIST) guidelines. Exclusion criteria were history of seizure (including any febrile seizure, loss of consciousness or transient ischemic attack within 12 months prior to initiation of study drug) or any condition that may predispose to seizure. The complete list of exclusion criteria is available in the Online Resource.

The criteria used to define disease progression for trial entry are available in the Online Resource. Eligible patients had Eastern Cooperative Oncology Group performance status of 0 or 1 (or 2 if only due to metastatic bone pain at the screening). Post-chemotherapy patients had to have received prior chemotherapy with docetaxel and no more than two prior chemotherapy regimens. In particular for phase II, patients had to have measurable lesions as determined by response evaluation criteria for solid tumors (RECIST) guidelines. Exclusion criteria were history of seizure (including any febrile seizure, loss of consciousness or transient ischemic attack within 12 months prior to initiation of study drug) or any condition that may predispose to seizure. The complete list of exclusion criteria is available in the Online Resource. Assessments The primary outcome in anti-tumor activity in phase II was best overall response by 12 weeks; defined by RECIST guidelines as complete response (CR) or partial response (PR) and assessed by an investigator. Confirmation of CR or PR was required by a subsequent scan at least 4 weeks later. When the investigator confirmed CR or PR, the assessment was finally evaluated by an independent RECIST assessment committee. Measurements had to meet the stable disease criteria by day 85 for determination of stable disease. Radiographic imaging for the target region was conducted at the screening visit, on day 29, day 57 and day 85, and at each subsequent visit every 84 days. Bone scans were examined at the screening visit and at each 84-day visit.

ents had to meet the stable disease criteria by day 85 for determination of stable disease. Radiographic imaging for the target region was conducted at the screening visit, on day 29, day 57 and day 85, and at each subsequent visit every 84 days. Bone scans were examined at the screening visit and at each 84-day visit. The secondary endpoint was prostate-specific antigen (PSA) response rate (proportion of subjects with ≥50 % decline in serum PSA from baseline). Serum PSA measurements were conducted at the screening visit and at each subsequent visit every 28 days. Safety was evaluated from the start of study treatment to 30 days after completion of the study treatment. All adverse events (AEs) were recorded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 4.0, and Medical Dictionary for Regulatory Activities (MedDRA), version 14.1. Laboratory values, vital signs, body weight and 12-lead echocardiograms were assessed at predefined time points.

vents (AEs) were recorded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 4.0, and Medical Dictionary for Regulatory Activities (MedDRA), version 14.1. Laboratory values, vital signs, body weight and 12-lead echocardiograms were assessed at predefined time points. Blood samples were collected at predefined time points in phase I and phase II. Plasma concentrations of enzalutamide and its active metabolite, N-desmethyl enzalutamide, were determined by a validated bioanalytical method based on liquid chromatography combined with mass spectrometry [18]. PK parameters were estimated by non-compartmental methods in WinNonlin® (Pharsight Corp., Palo Alto, CA, USA) and included maximum plasma concentration (Cmax) and area under the plasma concentration–time curve from time 0 to infinity (AUC∞). To investigate potential PK differences between Japanese and non-Japanese patients, the PK data were compared with PK data from AFFIRM. Statistical analysis The number and percentage of patients with a best overall response by day 85 and two-sided 90 and 95 % Clopper–Pearson CIs were used in the primary analysis. A waterfall plot of maximum percent change from baseline of serum PSA was created. The number and percentage of patients with best PSA response at time of nadir were summarized. All data processing, summarization and analyses were performed using SAS Drug Development, version 3.4, and PC-SAS, version 9.1.3. All analyses were performed by the sponsor using data obtained by the cut-off date of 12 July 2012.

umber and percentage of patients with best PSA response at time of nadir were summarized. All data processing, summarization and analyses were performed using SAS Drug Development, version 3.4, and PC-SAS, version 9.1.3. All analyses were performed by the sponsor using data obtained by the cut-off date of 12 July 2012. Post hoc analysis An additional post hoc exploratory analysis was conducted to further compare enzalutamide anti-tumor activity in Japanese patients with non-Japanese patients with measurable disease from the AFFIRM study. The best overall response by number of prior hormonal therapy lines, and defined by RECIST and PSA response rate, was calculated.

post hoc exploratory analysis was conducted to further compare enzalutamide anti-tumor activity in Japanese patients with non-Japanese patients with measurable disease from the AFFIRM study. The best overall response by number of prior hormonal therapy lines, and defined by RECIST and PSA response rate, was calculated. Results Patients In phase I, three patients were assigned to each of the 80, 160 and 240 mg groups. The median duration of exposure in each group was 584.0, 171.0 and 252.0 days, respectively. Thirty-eight post-docetaxel patients with CRPC and measurable disease as defined by RECIST were enrolled into phase II at a dose of 160 mg/day. Median duration of exposure was 121 days. No remarkable differences were observed in the demographic and clinical baseline characteristics between the phase I and phase II study populations (Table 1). Patients in phase II were heavily pretreated, with >90 % having had ≥4 prior hormonal treatments (Table 2). Eight of 38 patients (21.1 %) in phase II discontinued due to AEs. Of these, five patients withdrew due to disease progression (Table 3). All patients had received complete androgen blockade (CAB) therapy with bicalutamide soon after their initial diagnosis of prostate cancer. Overall, 42.1 % had >10 bone metastases and all patients had measurable disease by RECIST (Table 1).Table 1 Summary of demographics and other baseline characteristics

on (Table 3). All patients had received complete androgen blockade (CAB) therapy with bicalutamide soon after their initial diagnosis of prostate cancer. Overall, 42.1 % had >10 bone metastases and all patients had measurable disease by RECIST (Table 1).Table 1 Summary of demographics and other baseline characteristics Demographic/characteristic Phase I (N = 9) Phase II (N = 38) Age (years) Median 73.0 71.5 Min–max 62–86 50–85 Height (cm) Median 166.0 165.7 Min–max 156.2–174.4 153.4–181.0 Weight (kg) Median 71.2 65.7 Min–max 49.2–88.9 49.2–93.0 ECOG PSa Grade 0 8 (88.9) 25 (65.8) Grade 1 1 (11.1) 13 (34.2) Total Gleason scoreb at initial diagnosisa Low, 2–4 0 0 Medium, 5–7 0 8 (21.1) High, 8–10 9 (100.0) 29 (76.3) Unknown 0 1 (2.6) Clinical tumor stage (T)c at initial diagnosisa TX 1 (11.1) 1 (2.6) T0 0 0 T1 0 0 T2 1 (11.1) 10 (26.3) T3 6 (66.7) 16 (42.1) T4 1 (11.1) 10 (26.3) Unknown 0 1 (2.6) Clinical lymph node stage at initial diagnosisa NX 2 (22.2) 1 (2.6) N0 3 (33.3) 14 (36.8) N1 4 (44.4) 22 (57.9) Unknown 0 1 (2.6) Distant metastasis (M)c at initial diagnosisb MX 0 1 (2.6) M0 3 (33.3) 17 (44.7) M1 6 (66.7) 19 (50.0) Unknown 0 1 (2.6) Number of bone metastases 0 3 (33.3) 8 (21.1) 1 1 (11.1) 1 (2.6) 2–4 1 (11.1) 6 (15.8) 5–9 0 7 (18.4) ≥10 4 (44.4) 16 (42.1) Anti-androgen withdrawal syndromea Yes 0 4 (10.5) Stage of prostate cancera,b

NX 2 (22.2) 1 (2.6) N0 3 (33.3) 14 (36.8) N1 4 (44.4) 22 (57.9) Unknown 0 1 (2.6) Distant metastasis (M)c at initial diagnosisb MX 0 1 (2.6) M0 3 (33.3) 17 (44.7) M1 6 (66.7) 19 (50.0) Unknown 0 1 (2.6) Number of bone metastases 0 3 (33.3) 8 (21.1) 1 1 (11.1) 1 (2.6) 2–4 1 (11.1) 6 (15.8) 5–9 0 7 (18.4) ≥10 4 (44.4) 16 (42.1) Anti-androgen withdrawal syndromea Yes 0 4 (10.5) Stage of prostate cancera,b Localized 1 (11.1) 6 (15.8) Locally advanced 2 (22.2) 11 (28.9) Metastatic 6 (66.7) 19 (50.0) Not classifiable 0 2 (5.3) PSA at baseline (ng/mL) Mean (SD) 634.82 (1403.52) 174.94 (307.97) Median 21.60 65.80 Duration of disease at screening (months) Mean (SD) 47.36 (22.23) 63.11 (38.15) Median 39.93 52.83 ECOG PS Eastern Cooperative Oncology Group performance status, PSA prostate-specific antigen, SD standard deviation aNumber (%) of patients bGleason [27] cClassified using the TNM classification [28] as follows: localized, T1/2 and (NX or N0) and M0; locally advanced, T3/4 and (NX or N0) and M0 or N1 and M0; metastatic, M1; Not classifiable, others Table 2 Prior treatments for prostate cancer in phase II

ECOG PS Eastern Cooperative Oncology Group performance status, PSA prostate-specific antigen, SD standard deviation aNumber (%) of patients bGleason [27] cClassified using the TNM classification [28] as follows: localized, T1/2 and (NX or N0) and M0; locally advanced, T3/4 and (NX or N0) and M0 or N1 and M0; metastatic, M1; Not classifiable, others Table 2 Prior treatments for prostate cancer in phase II Parameter Category/statistic Phase II (N = 38) Cancer treatment history, radiation Yes 19 (50.0 %) Cancer treatment history, procedure Yes 6 (15.8 %) Quantity of prior hormone therapy linesa 3 3 (7.9 %) 4 5 (13.2 %) 5 13 (34.2 %) 6 11 (28.9 %) ≥7 6 (15.8 %) Typical prior hormone therapy, other than GnRH analogue Bicalutamide 38 (100.0 %) Flutamide 29 (76.3 %) Estramustine 30 (78.9 %) Docetaxel 38 (100 %) Number of prior chemotherapy regimens 1 8 (21.1 %) 2 30 (78.9 %) Duration of prior docetaxel (days) Median 198 Min–max 1–1012 GnRH gonadotropin-releasing hormone aSum of prior hormonal treatment agents including castration therapy Table 3 Primary reasons for discontinuation Category and reason Phase I (N = 9) Phase II (N = 38) Discontinuation in multiple-dose period (early termination), n (%) Adverse event 0 5 (13.2) Worsening of disease 1 (11.1) 5 (13.2) Withdrawal by subject 0 2 (5.3) Discontinuation in overall study, n (%) Adverse event 0 8 (21.1) Worsening of disease 5 (55.6) 18 (47.4) Withdrawal by subject 0 2 (5.3)

I before NAC. Thirteen patients were positive and 29 patients were negative for pathological lymph node metastasis. Postoperative treatments were radiation therapy in 13 patients, chemotherapy in 15, and chemoradiotherapy in 3; no postoperative treatment was given to 9 patients (Table 1).Table 1 Patient characteristics Characteristic n = 42 Age (years) Median (range) 45 (25–63) PS 0/1 37/5 FIGO stage 1/11/III 9/29/4 Histological type Keratinizing type/non-keratinizing type 11/31 Preoperative lymph node metastasis Positive/negative 16/26 Tumor diameter >5 cm/<5 cm 24/18 Pathological lymph node metastasis Positive/negative 13/29 Postoperative treatment None 9 Radiation 13 Chemotherapy 15 Chemoradiotherapy 3 Response The antitumor effect was complete response (CR) in 7 patients, partial response (PR) in 28, stable disease (SD) in 6, and progressive disease (PD) in 1, and the response rate was 83.3 % [95 % confidence interval (CI), 68.6–93.0]. Response rates by progression were 100 % for stage I, 82.8 % for stage II, and 50.0 % for stage III (Table 2).Table 2 Response (n = 42) CR PR SD PD Objective response (CR + PR) Total (n = 42) 7 28 6 1 35 (83.3 %) Stage l (n = 9) 2 7 0 0 9 (100 %) Stage ll (n = 29) 2 20 4 1 24 (82.8 %) Stage lll (n = 4) 1 1 2 0 2 (50.0 %) CR complete response, PR partial response, SD stable disease, PD progressive disease

9) Phase II (N = 38) Discontinuation in multiple-dose period (early termination), n (%) Adverse event 0 5 (13.2) Worsening of disease 1 (11.1) 5 (13.2) Withdrawal by subject 0 2 (5.3) Discontinuation in overall study, n (%) Adverse event 0 8 (21.1) Worsening of disease 5 (55.6) 18 (47.4) Withdrawal by subject 0 2 (5.3) Anti-tumor activity Response The best overall response rate (CR and PR) by day 85, as evaluated by the RECIST assessment committee and investigators, was seen in 5.3 % of patients (two out of 38; 95 % CI 0.6–17.7 %; 90 % CI 0.9–15.7 %). The best overall disease control rate (CR plus PR plus stable disease) by day 85 was 47.4 % of patients (18 out of 38; 95 % CI 31.0–64.2 %; 90 % CI 33.3–61.8 %) (Table 4). The rate with which the sum of diameters of target lesions was reduced by ≥30 % was 18.4 % (7 out of 38 patients).Table 4 Best overall responses by day 85 Best overall response Evaluation by RECIST assessment committee and investigatora (N = 38) CR, n 0 PR, n (%) 2 (5.3) Stable disease, n (%) 16 (42.1) PD, n (%) 16 (42.1) Not evaluated 4 (10.5) CR or PR, n (%) (response rate) 2 (5.3) 95 % CIb 0.6–17.7 % 90 % CIb 0.9–15.7 % CR or PR or stable disease, n (%) (disease control rate) 18 (47.4) 95 % CIb 31.0–64.2 % 90 % CIb 33.3–61.8 % Tumor response (overall response) for each patient was assessed by the investigator and subsequently evaluated by an independent RECIST assessment committee (when the investigator assessed that a patient had been accomplished CR or PR) CR complete response, PD progressive disease, PR partial response

Best overall response Evaluation by RECIST assessment committee and investigatora (N = 38) CR, n 0 PR, n (%) 2 (5.3) Stable disease, n (%) 16 (42.1) PD, n (%) 16 (42.1) Not evaluated 4 (10.5) CR or PR, n (%) (response rate) 2 (5.3) 95 % CIb 0.6–17.7 % 90 % CIb 0.9–15.7 % CR or PR or stable disease, n (%) (disease control rate) 18 (47.4) 95 % CIb 31.0–64.2 % 90 % CIb 33.3–61.8 % Tumor response (overall response) for each patient was assessed by the investigator and subsequently evaluated by an independent RECIST assessment committee (when the investigator assessed that a patient had been accomplished CR or PR) CR complete response, PD progressive disease, PR partial response aWhen there were evaluation data from both the RECIST committee and investigator, RECIST assessment committee data were adopted bBased on exact binomial confidence interval (Clopper–Pearson) PSA Eleven out of 38 patients in phase II (28.9 %; 95 % CI 15.4–45.9 %) had a ≥50 % decrease in PSA levels at the time of nadir, as compared with baseline (Fig. 1; Table 5).Fig. 1 Waterfall plot of maximum percent change from baseline of serum PSA in phase II. PSA, prostate-specific antigen Table 5 Best PSA response at time of nadir Response 160 mg/day (N = 38) Decline from baseline, patients, n (%) ≥ 30 % 15 (39.5) 95 % CI 24.0–56.6 % ≥50 % 11 (28.9) 95 % CI 15.4–45.9 % ≥90 % 4 (10.5) 95 % CI 2.9–24.8 % CI confidence interval, PSA prostate-specific antigen

PSA Eleven out of 38 patients in phase II (28.9 %; 95 % CI 15.4–45.9 %) had a ≥50 % decrease in PSA levels at the time of nadir, as compared with baseline (Fig. 1; Table 5).Fig. 1 Waterfall plot of maximum percent change from baseline of serum PSA in phase II. PSA, prostate-specific antigen Table 5 Best PSA response at time of nadir Response 160 mg/day (N = 38) Decline from baseline, patients, n (%) ≥ 30 % 15 (39.5) 95 % CI 24.0–56.6 % ≥50 % 11 (28.9) 95 % CI 15.4–45.9 % ≥90 % 4 (10.5) 95 % CI 2.9–24.8 % CI confidence interval, PSA prostate-specific antigen Safety The most frequent treatment-emergent AEs (TEAEs) with an incidence of ≥20 % across both phases were weight decrease (36.2 %), decreased appetite (27.7 %) and constipation (25.5 %) (Table 6). Of the adverse drug reactions reported in ≥10 % of patients, those considered to be related to the study drug were hypertension (14.9 %), constipation (14.9 %), fatigue (12.8 %), decreased appetite (12.8 %), weight decrease (10.6 %) and electrocardiogram QT prolonged (10.6 %). None of the TEAEs resulted in death and no seizures were reported. The most common serious TEAE was cancer pain (N = 3) (Table 7).Table 6 Common adverse events (reported in at least 10 % of patients in total)

ue (12.8 %), decreased appetite (12.8 %), weight decrease (10.6 %) and electrocardiogram QT prolonged (10.6 %). None of the TEAEs resulted in death and no seizures were reported. The most common serious TEAE was cancer pain (N = 3) (Table 7).Table 6 Common adverse events (reported in at least 10 % of patients in total) MedDRA, version 14.1, preferred term All adverse events Adverse events considered to be related to study drug Phase I (N = 9) Phase II (N = 38) Total (N = 47) Phase I (N = 9) Phase II (N = 38) Total (N = 47) Overall 9 (100.0) 36 (94.7) 45 (95.7) 7 (77.8) 24 (63.2) 31 (66.0) Weight decreased 1 (11.1) 16 (42.1) 17 (36.2) 0 5 (13.2) 5 (10.6) Decreased appetite 3 (33.3) 10 (26.3) 13 (27.7) 2 (22.2) 4 (10.5) 6 (12.8) Constipation 2 (22.2) 10 (26.3) 12 (25.5) 1 (11.1) 6 (15.8) 7 (14.9) Hypertension 3 (33.3) 6 (15.8) 9 (19.1) 3 (33.3) 4 (10.5) 7 (14.9) Cancer pain 1 (11.1) 8 (21.1) 9 (19.1) 0 1 (2.6) 1 (2.1) Nausea 4 (44.4) 5 (13.2) 9 (19.1) 1 (11.1) 2 (5.3) 3 (6.4) Electrocardiogram QT prolonged 0 6 (15.8) 6 (12.8) 0 5 (13.2) 5 (10.6) Fatigue 2 (22.2) 4 (10.5) 6 (12.8) 2 (22.2) 4 (10.5) 6 (12.8) Nasopharyngitis 1 (11.1) 5 (13.2) 6 (12.8) 0 0 0 Pyrexia 1 (11.1) 4 (10.5) 5 (10.6) 1 (11.1) 0 1 (2.1) Somnolence 0 5 (13.2) 5 (10.6) 0 1 (2.6) 1 (2.1) Rash 0 5 (13.2) 5 (10.6) 0 1 (2.6) 1 (2.1) Number of patients (%) MedDRA Medical Dictionary for Regulatory Activities Table 7 Serious treatment-emergent adverse events (with an incidence of ≥2 events in the study)

MedDRA, version 14.1, preferred term All adverse events Adverse events considered to be related to study drug Phase I (N = 9) Phase II (N = 38) Total (N = 47) Phase I (N = 9) Phase II (N = 38) Total (N = 47) Overall 9 (100.0) 36 (94.7) 45 (95.7) 7 (77.8) 24 (63.2) 31 (66.0) Weight decreased 1 (11.1) 16 (42.1) 17 (36.2) 0 5 (13.2) 5 (10.6) Decreased appetite 3 (33.3) 10 (26.3) 13 (27.7) 2 (22.2) 4 (10.5) 6 (12.8) Constipation 2 (22.2) 10 (26.3) 12 (25.5) 1 (11.1) 6 (15.8) 7 (14.9) Hypertension 3 (33.3) 6 (15.8) 9 (19.1) 3 (33.3) 4 (10.5) 7 (14.9) Cancer pain 1 (11.1) 8 (21.1) 9 (19.1) 0 1 (2.6) 1 (2.1) Nausea 4 (44.4) 5 (13.2) 9 (19.1) 1 (11.1) 2 (5.3) 3 (6.4) Electrocardiogram QT prolonged 0 6 (15.8) 6 (12.8) 0 5 (13.2) 5 (10.6) Fatigue 2 (22.2) 4 (10.5) 6 (12.8) 2 (22.2) 4 (10.5) 6 (12.8) Nasopharyngitis 1 (11.1) 5 (13.2) 6 (12.8) 0 0 0 Pyrexia 1 (11.1) 4 (10.5) 5 (10.6) 1 (11.1) 0 1 (2.1) Somnolence 0 5 (13.2) 5 (10.6) 0 1 (2.6) 1 (2.1) Rash 0 5 (13.2) 5 (10.6) 0 1 (2.6) 1 (2.1) Number of patients (%) MedDRA Medical Dictionary for Regulatory Activities Table 7 Serious treatment-emergent adverse events (with an incidence of ≥2 events in the study) MedDRA, version 14.1, preferred term Phase I totala (N = 9) Phase II 160 mg (N = 38) Overall 2 (22.2) 13 (34.2) Cancer pain 1 (11.1) 2 (5.3) Anemia 0 2 (5.3) Disseminated intravascular coagulation 0 2 (5.3) General physical health deterioration 0 2 (5.3) Cellulitis 0 2 (5.3) Tumor pain 0 2 (5.3) Bladder tamponade 0 2 (5.3) Number of patients (%) MedDRA Medical Dictionary for Regulatory Activities

MedDRA, version 14.1, preferred term Phase I totala (N = 9) Phase II 160 mg (N = 38) Overall 2 (22.2) 13 (34.2) Cancer pain 1 (11.1) 2 (5.3) Anemia 0 2 (5.3) Disseminated intravascular coagulation 0 2 (5.3) General physical health deterioration 0 2 (5.3) Cellulitis 0 2 (5.3) Tumor pain 0 2 (5.3) Bladder tamponade 0 2 (5.3) Number of patients (%) MedDRA Medical Dictionary for Regulatory Activities aIn phase I, safety data from single doses (80, 160 and 240 mg) and multiple doses (80 and 160 mg) are included. All patients in the 240 mg group received enzalutamide at a dose of 160 mg after single dosing Pharmacokinetics Enzalutamide was absorbed rapidly after oral administration in Japanese patients and the PK was dose-proportional after a single dose ranging from 80 to 240 mg (Fig. 2). The PK profile of a single dose of enzalutamide in Japanese patients was similar to that of non-Japanese patient data from the first enzalutamide study in humans (phase I/II study; http://ClinicalTrials.gov NCT00510718) (Fig. 2) [19]. PK profiles of the sum of enzalutamide plus its active metabolite in plasma were similar between Japanese patients from the current study and non-Japanese populations from AFFIRM (Fig. 3).Fig. 2 Comparison of individual enzalutamide Cmax and AUC∞ during single-dosing. aPhase I, open-label, dose-escalation safety and pharmacokinetic study of enzalutamide in patients with CRPC conducted overseas [19]

similar between Japanese patients from the current study and non-Japanese populations from AFFIRM (Fig. 3).Fig. 2 Comparison of individual enzalutamide Cmax and AUC∞ during single-dosing. aPhase I, open-label, dose-escalation safety and pharmacokinetic study of enzalutamide in patients with CRPC conducted overseas [19] Fig. 3 Individual trough plasma sum of enzalutamide and active metabolite concentration versus time plot in the Japanese phase I/II and AFFIRM (International, phase III, randomized, double-blind, placebo-controlled study of enzalutamide in patients with prostate cancer who had previously been treated with one or two chemotherapy regimens, at least one of which contained docetaxel [14]) studies, up to day 169 Post hoc analysis An exploratory post hoc analysis compared the subgroup of non-Japanese patients with measurable disease from AFFIRM [N = 446 out of 800 enzalutamide-treated patients (cut-off date: 25 September 2011)] and Japanese patients from the current study (N = 38). The quantity of prior hormonal therapy lines used as prostate cancer treatment in the two studies, which excluded medical or surgical castration therapy, is available in the Online Resource in Table S1. While approximately 90 % of patients in the AFFIRM study had received ≤2 hormonal therapy lines, approximately 90 % of patients in the current study had received ≥3 hormonal therapy lines. Best overall response rate, by RECIST, and PSA response rate for each amount of prior hormonal therapy lines are available in the Online Resource in Table S2 and Table S3, respectively.

dy had received ≤2 hormonal therapy lines, approximately 90 % of patients in the current study had received ≥3 hormonal therapy lines. Best overall response rate, by RECIST, and PSA response rate for each amount of prior hormonal therapy lines are available in the Online Resource in Table S2 and Table S3, respectively. Discussion In patients who receive primary androgen deprivation therapy, the proportion of patients with high risk and/or advanced prostate cancer is higher in Japan than in the United States [2]. A randomized, controlled study of primary ADT by CAB with chemical castration by GnRH agonist and bicalutamide 80 mg in Japanese patients showed significant prolongation of OS compared with chemical castration alone [20]. Based on this result, CAB is used in Japan as a standard initial therapy for high-risk or progressive prostate cancer [2, 4]. Results from this first clinical study of enzalutamide in the Japanese post-docetaxel CRPC patient population showed that enzalutamide was well tolerated when orally administered at a dose of 160 mg once daily. PK of enzalutamide was dose-proportional in the doses ranging from 80 to 240 mg and similar to PK data from non-Japanese patients. Furthermore, enzalutamide administered orally at 160 mg once daily had anti-tumor activity in Japanese post-chemotherapy patients with CRPC, in terms of best overall response or tumor-shrinking tendency and PSA response.

portional in the doses ranging from 80 to 240 mg and similar to PK data from non-Japanese patients. Furthermore, enzalutamide administered orally at 160 mg once daily had anti-tumor activity in Japanese post-chemotherapy patients with CRPC, in terms of best overall response or tumor-shrinking tendency and PSA response. However, this study did not achieve radiographic and PSA response rates as high as those in AFFIRM. The radiographic response rate by day 85 was 5.3 % in the current study versus 28.9 % in AFFIRM. The PSA response rate (≥50 % reduction from baseline) was 54.0 % in AFFIRM, compared with 28.9 % in this study. Differences between the two studies (i.e., patient populations enrolled, study setting and design, patient samples in each trial) may account for the lower radiographic and PSA response rates. As prior docetaxel exposure received by patients in this study (median, 198 days; approximately 9–10 cycles) was similar to that reported by patients in the AFFIRM trial (median, 8.5 cycles) [14], the most important difference could be that patients in the current study had received more hormonal therapy lines prior to enzalutamide compared with those in AFFIRM. With the exclusion of castration therapies, approximately 90 % of patients in this study had received ≥3 prior hormonal therapy lines (i.e., CAB, anti-androgen alternative therapy, steroids, and estrogens), whereas patients typically received ≤2 lines in AFFIRM. This difference may be related to the recommended treatment strategy in Japan, which includes extensive exposure to CAB, with bicalutamide 80 mg as the primary ADT and further treatment with alternating hormone therapy. This observation is also supported by the results of a previous surveillance study by the Japan Study Group of Prostate Cancer (J-CaP) that considered the current status of endocrine therapy for prostate cancer [4]. Of the 3337 patients who initially received primary ADT, 2477 patients (74.2 %) were given CAB in the J-Cap surveillance [4]. The pattern of primary ADT usage was more common in Japan than in the United States and primary ADT by CAB was associated with better survival than other forms of primary ADT in Japanese high-risk patients [2]. Although extensive direct comparisons between Japan and the United States are not possible, there are some differences between the two countries in the initial prostate cancer treatment selection and outcome [2, 4].

was associated with better survival than other forms of primary ADT in Japanese high-risk patients [2]. Although extensive direct comparisons between Japan and the United States are not possible, there are some differences between the two countries in the initial prostate cancer treatment selection and outcome [2, 4]. Furthermore, although hormonal treatments have been the mainstay of treatment in advanced prostate cancer, recent data suggest potential development of cross-resistance after multiple lines of hormonal therapy [21, 22]. In the first-in-man enzalutamide phase I/II study in patients in the United States, the rate of PSA decline of ≥50 % was significantly lower in patients with previous ketoconazole treatment versus those without [37 % (95 % CI 25–50 %) versus 71 % (95 % CI 60–81 %; p = 0.0007)] [19]. Moreover, a study of abiraterone acetate plus prednisone showed that patients with prior exposure to ketoconazole had a lower percentage of PSA decline of ≥50 % compared with ketoconazole-naïve patients [23]. In the same study, time to PSA progression was shorter in patients with prior ketoconazole exposure compared with ketoconazole-naïve patients [23]. In addition, there have been several recent reports on cross-tolerance between abiraterone plus prednisone and enzalutamide. Reports from compassionate use programs involving heavily pretreated patients with metastatic CRPC suggest reduced efficacy for both enzalutamide and abiraterone in comparison to the efficacy reported in clinical trials [24, 25]. Furthermore, results of a recent study in 32 patients suggested a potential effect of androgen receptor splice variant-7 on primary treatment resistance, observed with abiraterone plus prednisone or enzalutamide [26].

both enzalutamide and abiraterone in comparison to the efficacy reported in clinical trials [24, 25]. Furthermore, results of a recent study in 32 patients suggested a potential effect of androgen receptor splice variant-7 on primary treatment resistance, observed with abiraterone plus prednisone or enzalutamide [26]. Enzalutamide showed good tolerability in Japanese patients, with PK and safety profiles similar to those in non-Japanese populations included in other enzalutamide studies. The differences in anti-tumor activity observed in this study versus the AFFIRM trial may be attributed to differences in the study design and patients’ backgrounds in each trial. In particular, they may be attributed to differences in treatment history prior to starting enzalutamide. This may require further investigation to define the optimal timing and treatment strategy of enzalutamide for patients with CRPC. Particularly in Japan, the influence of sequence for hormone treatments, including CAB therapy, should be considered. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 22 kb)

Enzalutamide showed good tolerability in Japanese patients, with PK and safety profiles similar to those in non-Japanese populations included in other enzalutamide studies. The differences in anti-tumor activity observed in this study versus the AFFIRM trial may be attributed to differences in the study design and patients’ backgrounds in each trial. In particular, they may be attributed to differences in treatment history prior to starting enzalutamide. This may require further investigation to define the optimal timing and treatment strategy of enzalutamide for patients with CRPC. Particularly in Japan, the influence of sequence for hormone treatments, including CAB therapy, should be considered. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 22 kb) We thank the patients in the study for their cooperation, along with the following investigators: Hiroshi Kitamura, Sapporo Medical University Hospital; Hideo Saito, Tohoku University Hospital; Senji Hoshi, Yamagata Prefectural Central Hospital; Kazuhiro Suzuki, Gunma University Hospital; Takeshi Ueda, Chiba Cancer Center; Tomohiko Ichikawa, Chiba University Hospital; Yataro Yamanaka, Nihon University Itabashi Hospital; Takashi Fukagai, Showa University Hospital; Hirohiko Nagata, Keio University Hospital; Hiroyuki Fujimoto, National Cancer Center Hospital; Shin Egawa, Jikei University Hospital; Takatsugu Okegawa, Kyorin University Hospital; Hiroji Uemura, Yokohama City University Hospital; Tsutomu Nishiyama, Niigata University Medical and Dental Hospital; Atsushi Mizokami, Kanazawa University Hospital; Tatsuya Takayama, Hamamatsu University Hospital; Takahiro Inoue, Kyoto University Hospital; Tatsuya Nakatani, Osaka City University Hospital; Kazuo Nishimura, Osaka Medical Center for Cancer and Cardiovascular Diseases; Norio Nonomura, Osaka University Hospital; Masahiro Nozawa, Kinki University Hospital; Sadanori Kamikawa, Osaka City General Hospital; Tomoharu Fukumori, Tokushima University Hospital; Shigeru Sakano, Yamaguchi University Hospital; Akito Yamaguchi, Harasanshin Hospital; Akira Yokomizo, Kyushu University Hospital; Tsukasa Igawa, Nagasaki University Hospital. We also thank the following independent data monitoring committee and independent RECIST assessment committee members: Shigeo Horie, Juntendo University Graduate School of Medicine; Shiro Hinotsu, Okayama University Hospital; Kojiro Yamamoto, Gunma University; Koichiro Akakura, Tokyo Shinjuku Medical Center; Yuichi Watanabe, National Cancer Center Hospital. Enzalutamide is being co-developed by Medivation, Inc, and Astellas Pharma, Inc. This study was funded by Astellas Pharma, Inc. The authors would like to thank Lauren Smith of Complete HealthVizion for copyediting the manuscript, which was funded by Astellas Pharma, Inc and Medivation, Inc.

onal Cancer Center Hospital. Enzalutamide is being co-developed by Medivation, Inc, and Astellas Pharma, Inc. This study was funded by Astellas Pharma, Inc. The authors would like to thank Lauren Smith of Complete HealthVizion for copyediting the manuscript, which was funded by Astellas Pharma, Inc and Medivation, Inc. Compliance with ethical standards Conflict of interest H. A. has received personal fees from Astellas Pharma, Inc. for the submitted work and personal fees from Takeda Pharmaceutical Company Limited and Janssen Pharmaceutical K.K. outside the submitted work. Hirotsugu Uemura has received consulting fees from Astellas Pharma, Inc. for the submitted work. T. T. has received consulting fees from Astellas Pharma, Inc. S. O. has received personal fees and research funding from Astellas Pharma Inc., AstraZeneca K.K., Janssen Pharmaceutical K.K., Takeda Pharmaceutical Company Limited, and Sanofi K.K. O. O. has received consulting fees from Astellas Pharma, Inc. H. S. has received personal fees from Astellas Pharma for the submitted work; personal fees from Astellas, AstraZeneca, Takeda, and Janssen outside the submitted work; and research funds from Astellas, AstraZeneca, and Takeda outside the submitted work. M. O. has received personal fees from Astellas Pharma, Inc., Sanofi K.K., AstraZeneca K.K., ASKA Pharmaceutical, Janssen Pharmaceutical, Asahi Kasei Pharma Corp., Nippon Shinyaku Co., Nippon Kayaku Co., Kyorin Pharmaceutical, Medicon, Kissei Pharmaceutical, Shiongi & Co., Pfizer Japan, Daiichi Sankyo Co., Boston Scientific Japan K.K., Taiho Pharmaceutical Co., Novartis K.K., GSK K.K., Teijin Pharma Limited, Nihon Medi-Physics Co., Otsuka Pharmaceutical Co., Japan BCG Laboratory, Takeda Pharmaceutical, Kyowa Hakko Kirin Co., Bayer Yakuhin, and Philips Electronics Japan during the conduct of the study; research funding from AstraZeneca K.K., ASKA Pharmaceutical Co., Asahi Kasei Pharma, Nippon Shinyaku Co., Ltd, Pfizer Japan, and Takeda Pharmaceutical; and encouraged donations from Astellas Pharma, Inc., Sanofi K.K., Nippon Kayaku Co., Ltd., Kyorin Pharmaceutical Col., Ltd, Medicon, Inc., Kissei Pharmaceutical Co., Ltd., Shionogi & Co., Ltd., Daiichi Sankyo Company, Limited, Boston Scientific Japan K.K., Taiho Pharmaceutical Co., Ltd., Novartis Pharma K.K., GlaxoSmithKline K.K., Teijin Pharma Limited, Otsuka Pharmaceutical Co., Ltd, Japan BCG Laboratory, and Kyowa Hakko Kirin Co., Ltd. M. N. has received personal fees and research funding from Astellas Pharma, Inc.

Sankyo Company, Limited, Boston Scientific Japan K.K., Taiho Pharmaceutical Co., Ltd., Novartis Pharma K.K., GlaxoSmithKline K.K., Teijin Pharma Limited, Otsuka Pharmaceutical Co., Ltd, Japan BCG Laboratory, and Kyowa Hakko Kirin Co., Ltd. M. N. has received personal fees and research funding from Astellas Pharma, Inc. S. F., A. Y., and Hiroji Uemura have no conflict of interest. Y. O. has received an executive salary from Statcom, personal fees from Astellas for the submitted work; lecture fees from Daiichi Sankyo, Chugai Pharmaceutical, Shionogi, and EPS outside the submitted work; manuscript fees from Dentsu Sudler & Hennessey and DNP Media Create outside the submitted work; and research funding from Astellas for the submitted work, and research funding from Takeda Pharmaceutical, Kyowa Hakko Kirin, and Kowa Pharmaceutical outside the submitted work. H.M. is an employee of Astellas Pharma, Inc. and owns stock from Astellas. A. S. and K.T are employees of Astellas Pharma, Inc. S. N. received personal fees from Astellas during the conduct of the study and personal fees from Takeda, AstraZeneca, Janssen and GreenPeptide outside the submitted work.

Introduction Recently, three nephrometry scoring systems, namely, the R. E. N. A. L. nephrometry score (RNS) [1], PADUA score [2], and C-index [3], all of which are based on the anatomical features of renal tumors, were developed to assess the risk of nephron-sparing surgery for a small renal mass including renal cell carcinoma (RCC). These metrics have been validated to predict perioperative complications for partial nephrectomy in many previous studies. RNS characterizes tumors on the basis of the anatomical features of renal masses on computed tomography (CT) or magnetic resonance imaging (MRI) [1]. Of these scoring systems, only the RNS was associated with histological features of tumor aggressiveness in some reports [4–7]. Accordingly, RNS is expected to be a useful predictor of postoperative recurrence in patients with localized RCC. However, to our knowledge, there has been only one report regarding the association between RNS and postoperative recurrence [8]. For these reasons, in the present study, we decided to use RNS to analyze the association between anatomical features and postoperative recurrence after radical nephrectomy in patients with localized RCC (T1b–T2b).

e, there has been only one report regarding the association between RNS and postoperative recurrence [8]. For these reasons, in the present study, we decided to use RNS to analyze the association between anatomical features and postoperative recurrence after radical nephrectomy in patients with localized RCC (T1b–T2b). Patients and methods Patients We retrospectively analyzed a database comprising 91 consecutive patients with localized RCC (pT1b–2b) treated by radical nephrectomy between January 2002 and March 2013 at the Osaka University Hospital. Staging of RCC was performed according to the 7th edition of TNM staging proposed by the International Union for Cancer Control and the American Joint Committee on Cancer [9]. RNS (www.nephrometry.com) consists of R, E, N, A, and L components based on the following anatomical features of RCC: size, endophytic degree, proximity to the collecting system or renal sinus, position (anterior or posterior), and location relative to the polar line, respectively. If the tumor is in contact with the main renal artery or vein, the suffix h is assigned. RNS was calculated on the basis of findings of preoperative CT or MRI. Recurrence-free survival (RFS) time was calculated from the date of surgery until the date of recurrence or the date of the patient’s last follow-up visit. The study was approved by the institutional review board of Osaka University hospital (approval number: 11397-2).

n the basis of findings of preoperative CT or MRI. Recurrence-free survival (RFS) time was calculated from the date of surgery until the date of recurrence or the date of the patient’s last follow-up visit. The study was approved by the institutional review board of Osaka University hospital (approval number: 11397-2). Statistical analysis RFS rate was calculated using the Kaplan–Meier method, and comparisons were made using the log-rank test. The median value of each RNS parameter was used for the cutoff value. For preoperative serum C-reactive protein (CRP) level, 0.2 mg/dl was used for cutoff value. Univariate and multivariate analyses were performed using the COX proportional hazards model to predict RFS and calculate hazard ratio. Variables entered into the model for RFS analysis included patient age, gender, preoperative serum CRP level, histological grade, histological type, microscopic vascular involvement, RNS sum, and each component of RNS. The variables that had a P value <0.1 in the univariate analysis were entered into the multivariate analysis. Two kinds of multivariate analysis, one including the RNS sum and the other including each score-constituent parameter of RNS, were performed. All statistical analyses were performed using JMP Pro version 11 (SAS Institute, Tokyo, Japan), with P < 0.05 considered as statistically significant.

the multivariate analysis. Two kinds of multivariate analysis, one including the RNS sum and the other including each score-constituent parameter of RNS, were performed. All statistical analyses were performed using JMP Pro version 11 (SAS Institute, Tokyo, Japan), with P < 0.05 considered as statistically significant. Results The clinical and pathological characteristics of the 91 patients with localized RCC and their treatment outcomes are shown in Table 1. The median age at operation was 63 years (range, 30–85 years). The histological type of RCC of 80 patients was clear cell carcinoma and the remaining 11 were non-clear cell carcinoma. Postoperative recurrence occurred in 19 patients (21 %), and the median time to cancer recurrence after radical nephrectomy was 27 months (range, 1–79 months). The recurrence sites were as follows: lung (10 patients, 53 %), contralateral kidney (2 patients, 11 %), bone (1 patient, 5 %), contralateral ureter (1 patient, 5 %), gallbladder (1 patient, 5 %), pancreas (1 patient, 5 %), small intestine (1 patient, 5 %), hilar lymph node (1 patient, 5 %), and mediastinal lymph node (1 patient, 5 %). In addition, 3 patients (16 %) with cancer recurrence died of RCC progression during the observation periods. The median follow-up period was 65 months (range, 1–156 months). Anatomic characteristics based on RNS are shown in Table 2.Table 1 Clinicopathological characteristics of the 91 patients

node (1 patient, 5 %). In addition, 3 patients (16 %) with cancer recurrence died of RCC progression during the observation periods. The median follow-up period was 65 months (range, 1–156 months). Anatomic characteristics based on RNS are shown in Table 2.Table 1 Clinicopathological characteristics of the 91 patients Characteristics Total (n = 91) Age, years (median) 30–85 (63) Gender Male 59 (65 %) Female 32 (35 %) R/L Right 44 (48 %) Left 47 (52 %) Preoperative serum CRP level ≤0.2 mg/dl 72 (79 %) >0.2 mg/dl 18 (20 %) Unknown 1 (1 %) pT stage 1b 65 (71 %) 2a 18 (20 %) 2b 8 (9 %) Histological type Clear cell carcinoma 80 (88 %) Non-clear cell carcinoma 11 (12 %) Histological grade 1 5 (5 %) 2 73 (80 %) 3 13 (14 %) Microscopic vascular involvement Absent 82 (90 %) Present 5 (5 %) Unknown 4 (4 %) Recurrence No 72 (79 %) Yes 19 (21 %) Follow up, months (median) 1–156 (65) Table 2 Anatomic characteristics as based on R.E.N.A.L. nephrometry score Characteristics Total (n = 91) R.E.N.A.L. nephrometry score sum 4–6 2 (2 %) 7–9 44 (48 %) 10–12 45 (49 %) R component 1 0 (0 %) 2 60 (66 %) 3 31 (34 %) E component 1 33 (36 %) 2 53 (58 %) 3 5 (5 %) N component 1 2 (2 %) 2 2 (2 %) 3 87 (96 %) A component a 22 (24 %) p 17 (19 %) x 52 (57 %) L component 1 15 (16 %) 2 29 (32 %) 3 47 (52 %) Presence of h h (−) 49 (54 %) h (+) 42 (46 %)

4–6 2 (2 %) 7–9 44 (48 %) 10–12 45 (49 %) R component 1 0 (0 %) 2 60 (66 %) 3 31 (34 %) E component 1 33 (36 %) 2 53 (58 %) 3 5 (5 %) N component 1 2 (2 %) 2 2 (2 %) 3 87 (96 %) A component a 22 (24 %) p 17 (19 %) x 52 (57 %) L component 1 15 (16 %) 2 29 (32 %) 3 47 (52 %) Presence of h h (−) 49 (54 %) h (+) 42 (46 %) Figure 1 shows the Kaplan–Meier curve of RFS rate stratified according to RNS sum (4–9 vs. 10–12). RFS rate in patients with a low RNS sum was much better than that in patients with a high RNS sum (P = 0.0012). The 5- and 10-year RFS rates in patients with high RNS sum (70 % and 55 %, respectively) were significantly worse than those in patients with low RNS sum (95 % and 95 %, respectively). Figure 2 shows the Kaplan–Meier curve of the RFS rate stratified according to each component of RNS. RFS rate was significantly different when stratified by the R component (P = 0.0009), the L component (P = 0.0001), and the presence of hilar tumor (P = 0.0396).Fig. 1 Probability estimates of recurrence-free survival (RFS) rate in 91 patients in two groups based on R.E.N.A.L. nephrometry score (RNS) sum (4–9 vs. 10–12) Fig. 2 Probability estimates of recurrence-free survival rate (RFS) in 91 patients stratified based on R component (score: 2 vs. 3) (a), E component (score: 1 vs. 2, 3) (b), N component (score: 1, 2 vs. 3) (c), A component (a vs. p vs. x) (d), L component (score: 1, 2 vs. 3) (e), and presence of h (absent vs. present) (f)

Fig. 2 Probability estimates of recurrence-free survival rate (RFS) in 91 patients stratified based on R component (score: 2 vs. 3) (a), E component (score: 1 vs. 2, 3) (b), N component (score: 1, 2 vs. 3) (c), A component (a vs. p vs. x) (d), L component (score: 1, 2 vs. 3) (e), and presence of h (absent vs. present) (f) The results of univariate analysis are shown in Table 3. Of several factors examined, RNS sum, R component, L component, and the presence of h were significant factors in predicting postoperative cancer recurrence. Age at operation, preoperative serum CRP level and A component (x vs. p) were weakly associated with RFS rate on univariate analysis. The multivariate analysis including age at operation, preoperative serum CRP level, RNS sum, A component, and presence of h showed that only the RNS sum (HR, 9.05; 95 % CI, 2.11–63.9; P = 0.0019) was an independent predictive factor for postoperative cancer recurrence (Table 3). A second multivariate analysis replacing RNS sum with the R and L components, which were significant score-constituent factors of RNS on univariate analysis, showed that the L component (HR, 15.0; 95 % CI, 2.68–285; P = 0.0006) was an independent predictor of postoperative cancer recurrence (Table 3). Of the components of RNS, only the L component was significantly associated with RFS rate.Table 3 Cox proportional hazards analysis of R.E.N.A.L. nephrometry score and clinicopathological factors to predict recurrence-free survival

; P = 0.0006) was an independent predictor of postoperative cancer recurrence (Table 3). Of the components of RNS, only the L component was significantly associated with RFS rate.Table 3 Cox proportional hazards analysis of R.E.N.A.L. nephrometry score and clinicopathological factors to predict recurrence-free survival Characteristics Univariate Multivariate 1 Multivariate 2 HR (95 % CI) P value HR (95 % CI) P value HR (95 % CI) P value Age (years) (continuous) 1.04 (1.00–1.08) 0.0753 1.04 (1.00–1.08) 0.0739 1.03 (0.99–1.08) 0.0619 Gender (male vs. female) 1.34 (0.53–3.84) 0.5404 Preoperative serum CRP level (>0.2 vs. ≤0.2 mg/dl) 2.39 (0.83–6.13) 0.0996 2.39 (0.80–6.51) 0.1114 2.82 (0.91–8.26) 0.0710 Histological grade (G1, G2 vs. G3) 1.24 (0.29–3.75) 0.7378 Histological type (clear cell vs. non-clear cell) 1.13 (0.32–7.11) 0.8707 Microscopic vascular involvement (+ vs. −) 2.78 (0.44–9.83) 0.2306 R.E.N.A.L. nephrometry score sum (10–12 vs. 4–9) 7.77 (2.22–49.0) 0.0005 9.05 (2.11–63.9) 0.0019 R.E.N.A.L. nephrometry score component R (3 vs. 2) 4.49 (1.77–12.8) 0.0014 2.53 (0.96–7.62) 0.0619 E (2, 3 vs. 1) 1.99 (0.72–7.00) 0.1982 N (3 vs. 1, 2) 4.69 (0.06–401) 0.496 L (3 vs. 1, 2) 17.49 (3.61–315) <0.0001 15.0 (2.68–285) 0.0006 A (a vs. p) 4.48 (0.72–86.4) 0.1165 3.19 (0.48–62.7) 0.2520 2.57 (0.39–50.3) 0.3536 A (x vs. p) 4.88 (0.97–88.8) 0.0562 2.69 (0.51–49.6) 0.2859 2.15 (0.39–40.2) 0.4314 Presence of h (+ vs. −) 2.66 (1.05–7.57) 0.039 0.85 (0.30–2.74) 0.7754 0.75 (0.26–2.32) 0.6043

vs. 1, 2) 17.49 (3.61–315) <0.0001 15.0 (2.68–285) 0.0006 A (a vs. p) 4.48 (0.72–86.4) 0.1165 3.19 (0.48–62.7) 0.2520 2.57 (0.39–50.3) 0.3536 A (x vs. p) 4.88 (0.97–88.8) 0.0562 2.69 (0.51–49.6) 0.2859 2.15 (0.39–40.2) 0.4314 Presence of h (+ vs. −) 2.66 (1.05–7.57) 0.039 0.85 (0.30–2.74) 0.7754 0.75 (0.26–2.32) 0.6043 Discussion RNS was developed by Kutikov and Uzzo to standardize the assessment of anatomical features of renal tumors [1]. RNS consists of (R)adius (tumor size at maximal diameter), (E)xophytic/endophytic properties of the tumor, (N)earness of the deepest portion of tumor to the collecting system or sinus, (A)nterior (a)/posterior (p) descriptor, and the (L)ocation relative to the polar line. The suffix h (hilar) is assigned to tumors that are close to the main renal artery or vein. RNS is scored based on CT or MRI findings at diagnosis.

tumor, (N)earness of the deepest portion of tumor to the collecting system or sinus, (A)nterior (a)/posterior (p) descriptor, and the (L)ocation relative to the polar line. The suffix h (hilar) is assigned to tumors that are close to the main renal artery or vein. RNS is scored based on CT or MRI findings at diagnosis. Because RNS was developed to quantitatively evaluate the complexity of renal tumors, many studies confirmed the usefulness of RNS for predicting perioperative outcomes in patients treated by partial nephrectomy [10–15]. Recently, some studies revealed a relationship between RNS and histological features of tumor aggressiveness [4–7]. Kutikov et al. compared the individual components of RNS with histology and grade of 525 resected tumors and constructed a novel nomogram for predicting high-grade histology. In their analyses, high R and L scores were strongly associated with high-grade histology [4]. Wang et al. validated this nomogram in 391 Chinese RCC patients [5]. Mullins et al. revealed a high RNS sum was associated with high-grade pathology in a study of 886 patients treated by robot-assisted partial nephrectomy [7].

n their analyses, high R and L scores were strongly associated with high-grade histology [4]. Wang et al. validated this nomogram in 391 Chinese RCC patients [5]. Mullins et al. revealed a high RNS sum was associated with high-grade pathology in a study of 886 patients treated by robot-assisted partial nephrectomy [7]. The present study evaluated the relationship between RNS and postoperative cancer recurrence of non-small localized RCC (pT1b–pT2b). A high RNS sum was significantly associated with low RFS rate (P = 0.0012), and the RNS sum was a significant, independent factor for predicting postoperative cancer recurrence by multivariate analysis. Of the RNS components, only the L component was strongly associated with RFS rate. Kopp et al. revealed high RNS sum (10–12) and transfusion status were associated with shorter progression-free survival in a study of 202 patients with localized cT2 renal masses treated by radical nephrectomy or partial nephrectomy [8]. They also revealed high RNS sum, high nuclear grade, and transfusion status were associated with shorter overall survival. To date, there has been only one report suggesting that RNS was a predictive factor for postoperative cancer recurrence. The present study is the first to our knowledge that demonstrates a relationship between RNS, especially the L component, and postoperative recurrence.

were associated with shorter overall survival. To date, there has been only one report suggesting that RNS was a predictive factor for postoperative cancer recurrence. The present study is the first to our knowledge that demonstrates a relationship between RNS, especially the L component, and postoperative recurrence. Many predictive factors of recurrence after surgery in patients with RCC have been reported in the literature, and of anatomical tumor characteristics, tumor size has been identified as predictive of recurrence in many studies [16–18]. In the present study, the R component, namely, tumor size, was significantly associated with RFS rate on univariate analysis. However, upon multivariate analysis using all components of RNS, only the L component was significantly associated with RFS, whereas all other components were not significant, including R. To our knowledge, no other study has reported a similar result, making our current study unique in its findings. A study by Matsumoto et al. revealed a correlation between RNS and annual growth rates of renal masses scheduled for active surveillance [19], in which only the L component was significantly correlated with annual growth rate by multivariate analysis. These findings suggest that biological aggressiveness may be influenced by tumor location, although the underlying mechanism for this concept has not yet been fully clarified.

duled for active surveillance [19], in which only the L component was significantly correlated with annual growth rate by multivariate analysis. These findings suggest that biological aggressiveness may be influenced by tumor location, although the underlying mechanism for this concept has not yet been fully clarified. Among several histological characteristics, nuclear grade, presence of microvascular invasion, and tumor necrosis have been reported as predictive factors for postoperative cancer recurrence [16, 17, 20]. Of clinical and biochemical features, age at diagnosis, performance status, and the serum level of CRP have been reported as predictors of postoperative cancer recurrence [18, 20–23]. Several interesting studies have reported on molecular predictors of postoperative recurrence. Shvarts et al. reported that p53 expression in the tumor was related to nuclear grade and significantly associated with RFS rate in patients with localized RCC treated by radical nephrectomy [22]. Recently, Fujita et al. revealed that the level of vascular endothelial growth factor in preoperative serum was an independent predictor of postoperative recurrence in localized clear cell RCC [24]. Hongo et al. also revealed that cyclin-dependent kinase-related parameters were strongly associated with recurrence after surgery [25].

ta et al. revealed that the level of vascular endothelial growth factor in preoperative serum was an independent predictor of postoperative recurrence in localized clear cell RCC [24]. Hongo et al. also revealed that cyclin-dependent kinase-related parameters were strongly associated with recurrence after surgery [25]. The present study had some limitations. First, the patient cohort of this study was small, and their T stage ranged from T1b to T2b. Considering the increasing incidence of T1a-stage tumors among RCC cases, the results of the present study may not perfectly represent localized RCC. A large study including T1a disease may produce more reliable results, but because cancer recurrence in patients with T1a RCC is rare, such a study may require a large number of patients to reach statistical significance. Second, as already mentioned, although many clinicopathological and novel molecular predictors of postoperative recurrence have been reported, our study utilized only a small number of clinicopathological parameters. Further studies would be needed to fully assess the relevance of other known predictive factors in evaluating the prognostic value of RNS on RFS rate.

nicopathological and novel molecular predictors of postoperative recurrence have been reported, our study utilized only a small number of clinicopathological parameters. Further studies would be needed to fully assess the relevance of other known predictive factors in evaluating the prognostic value of RNS on RFS rate. In conclusion, the present study showed that RNS sum was independent predictors of postoperative recurrence in patients with non-small localized RCC (pT1b–T2b) treated by radical nephrectomy. Of the RNS components, only the L component was significantly associated with RFS. Accurate prediction of recurrence after surgical resection would be highly valuable in designing adjuvant trials, for example, in immunotherapy or molecular target therapy trials, and also for effectively scheduling follow-up visits and imaging studies. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

Introduction Adherence to the dosing schedule is important for the effectiveness of anticancer chemotherapy and affects therapeutic outcomes [1]. To improve adherence, measures that reduce adverse events (AEs) have been considered in the area of dosage form design, and the administration method has been modified. Drugs that suppress various symptoms have also been developed and used as supportive therapies to prevent AEs. Two examples of supportive therapies include antiemetics, which mitigate nausea, and granulocyte colony-stimulating factor (G-CSF), which treats neutropenia [2–4]. However, AEs are still difficult to control, and further development of supportive therapies is needed.

ped and used as supportive therapies to prevent AEs. Two examples of supportive therapies include antiemetics, which mitigate nausea, and granulocyte colony-stimulating factor (G-CSF), which treats neutropenia [2–4]. However, AEs are still difficult to control, and further development of supportive therapies is needed. Cystine and theanine is a supplement that contains 700 mg cystine and 280 mg theanine; it is available in Japan and the United States. Cystine consists of two molecules of cysteine, which is a sulfur-containing amino acid, that are connected by a disulfide bond, and it is reduced and converted to cysteine in the cell. Theanine breaks down into glutamic acid and ethylamine after it is absorbed. In the cell, cysteine and glutamic acid are synthesized with glycine to form the tripeptide glutathione (GSH) [5]. GSH is reportedly the most potent antioxidant in the body, and its levels have been shown to decrease after exercise and surgery [6, 7]. The need for GSH is thought to increase under these conditions. We previously described that the intake of cystine and theanine for 10 days during the perioperative period led to the early resolution of high postoperative levels of interleukin (IL)-6 and C-reactive protein (CRP) and early recovery from changes in neutrophil and lymphocyte counts [8]. Cystine and theanine has also been shown to produce similar effects in mouse digestive tract surgery models and to prevent decreases in intestinal GSH; these effects were considered to be partly explained by the supply of GSH [9].

ve protein (CRP) and early recovery from changes in neutrophil and lymphocyte counts [8]. Cystine and theanine has also been shown to produce similar effects in mouse digestive tract surgery models and to prevent decreases in intestinal GSH; these effects were considered to be partly explained by the supply of GSH [9]. Justino et al. showed that the GSH concentrations in the epithelial cells of the gastrointestinal mucosa in mice decreased after the administration of 5-FU; they also found that administering the intestinal bacterial species Saccharomyces boulardii prevented the decrease in GSH and alleviated diarrhea, an AE associated with the administration of 5-FU [10]. Preliminary studies using cystine and theanine have shown that this treatment reduced the severity of stomatitis caused by various chemotherapies [11], suggesting that cystine and theanine is a promising supportive therapy. Therefore, we performed a prospective randomized trial in patients undergoing surgery for either colon cancer or gastric cancer with postoperative S-1 adjuvant chemotherapy to determine the preventive effects of cystine and theanine against AEs caused by chemotherapy and to evaluate the usefulness of cystine and theanine as a supportive therapy.

rospective randomized trial in patients undergoing surgery for either colon cancer or gastric cancer with postoperative S-1 adjuvant chemotherapy to determine the preventive effects of cystine and theanine against AEs caused by chemotherapy and to evaluate the usefulness of cystine and theanine as a supportive therapy. Patients and methods Patients This study was approved by the Institutional Review Board of Sendai City Medical Center (approval number: 2012-0010), and consent was obtained from each patient after the study was sufficiently explained. The study was performed in accordance with the Declaration of Helsinki. The subjects comprised patients who underwent R0 surgery for either colon or gastric cancer at the surgery department of the Sendai City Medical Center and who were expected to receive postoperative S-1 adjuvant chemotherapy for 4 weeks with a 2-week drug-free interval. Patients were enrolled in the study if they were in PS0 or PS1, were 20 years of age or above, submitted written consent, and fulfilled the following criteria on their pre-registration laboratory tests: white blood cell (WBC) count >3,000/mm3, neutrophil count >1,500/mm3, platelet count >100,000/mm3, hemoglobin level >9.0 g/dl, total bilirubin level <2.0 mg/dl, aspartate aminotransferase (AST) level <100 IU, alanine aminotransferase (ALT) level <100 IU, and estimated glomerular filtration rate (eGFR) >60 ml/min. The patient registration period lasted from July 2012 to June 2014, and the intended number of enrolled subjects was 70.

>9.0 g/dl, total bilirubin level <2.0 mg/dl, aspartate aminotransferase (AST) level <100 IU, alanine aminotransferase (ALT) level <100 IU, and estimated glomerular filtration rate (eGFR) >60 ml/min. The patient registration period lasted from July 2012 to June 2014, and the intended number of enrolled subjects was 70. Study design and data collection The study design was a prospective randomized trial, and the subjects were allocated using the envelope method. The administration of S-1 (Taiho Pharmaceutical, Tokyo, Japan) was scheduled to be initiated within 6 weeks after surgery. The subjects in the C/T group were orally administered an amino acid supplement, which contained 700 mg cystine and 280 mg theanine (total weight, 1.7 g; Ajinomoto, Tokyo, Japan), once a day starting 1 week before and ending at the same time as the administration of S-1 (a total of 35 days) (Fig. 1). The control group received S-1 without cystine and theanine. The subjects in both groups were examined at the outpatient clinic immediately before starting S-1 chemotherapy. They underwent blood sampling, and it was confirmed that they fulfilled the inclusion criteria. The subjects visited the hospital 14 days after starting S-1 chemotherapy and at the end of the first treatment course (28 days after starting S-1 chemotherapy); during the visits, they underwent blood sampling and answered questions about AEs. The subjects were observed until the end of the first treatment course. A physician in charge of the outpatient clinic graded the AEs according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. If AEs that were of a grade 2 severity or more severe occurred during the chemotherapy, the administration of S-1 was suspended or discontinued, and the period in which S-1 was administered at the prescribed dose (i.e., not including the period when the dose was reduced) was recorded. Consultations regarding AEs were given by telephone and during hospital visits, and the administration of S-1 was immediately discontinued if the AE was judged to be of a grade 2 severity or more severe. The evaluation items included the incidence of AEs, which was determined through blood tests and medical interviews, and the completion rate of the first course of chemotherapy at the prescribed dose.Fig. 1 Administration schedules of S-1 and cystine and theanine after R0 surgery

e of a grade 2 severity or more severe. The evaluation items included the incidence of AEs, which was determined through blood tests and medical interviews, and the completion rate of the first course of chemotherapy at the prescribed dose.Fig. 1 Administration schedules of S-1 and cystine and theanine after R0 surgery Statistical analyses The hypothesis was that the completion rate of the first course of chemotherapy would be increased from 45 % (less than half) to 80 % by administration of cystine and theanine. It was calculated that 66 samples would be required. Expecting the loss of several samples, 70 samples were included in this study. All data were expressed as a percentage or the mean ± standard deviation (SD). The incidence of AEs and completion rate of chemotherapy were evaluated using Fisher’s exact test. The number of days that the prescribed dose of S-1 was administered in each group was compared using the t test. All statistical procedures were performed at a significance level of p < 0.05 using the Prism software package (GraphPad Software, La Jolla, CA, USA).

MRI images before treatment as the baseline. Antitumor efficacy was determined using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, with response being the best evaluation. Adverse events were evaluated according to the National Cancer Institute Common Toxicity Criteria (NCI-CTCAE) version 4.0. Main treatment Stage Ib2–IIIb patients were subjected to a radical hysterectomy unless the antitumor response was progression of disease or up-stage progression. A radical hysterectomy was performed on stage IIIb patients with a down-stage progression. As a rule, lymph node dissection included the lymph nodes within the pelvis (external iliac nodes, internal iliac nodes, common iliac nodes, suprainguinal nodes, parametrial nodes, and obturator nodes). Concurrent chemoradiation therapy (CCRT) was carried out for patients whose conditions were inoperable. Postoperative adjuvant therapy Patients with a positive surgical margin, metastatic lymph nodes, infiltration to the parametrium, and/or vascular invasion, as demonstrated by pathological examination of the resected specimens, underwent postoperative irradiation, chemotherapy or CCRT. Before 2008, irradiation was performed as postoperative adjuvant therapy, whereas chemotherapy was performed from 2008 onward. However, CCRT was performed for patients who had multiple lymph node metastases and/or infiltration to the parametrium.

f chemotherapy were evaluated using Fisher’s exact test. The number of days that the prescribed dose of S-1 was administered in each group was compared using the t test. All statistical procedures were performed at a significance level of p < 0.05 using the Prism software package (GraphPad Software, La Jolla, CA, USA). Results Of the 70 subjects who participated, 35 subjects were allocated to each group. Four subjects in the control group were excluded (1 had difficulty visiting the hospital because of low back pain, 2 were underdosed with S-1, and 1 was administered cystine and theanine), and 3 in the C/T group were excluded (1 developed ileus, 1 could not be administered S-1, and 1 was not administered cystine and theanine). Thus, 31 subjects in the control group and 32 subjects in the C/T group were evaluated (Fig. 2).Fig. 2 Thirty-five subjects each were randomly allocated to the control and C/T groups. After exclusion of 4 control group subjects and 3 C/T group subjects, the data from 31 and 32 subjects in the respective groups were analyzed The subjects in the control and C/T groups had the following characteristics: their ages were 63.5 ± 8.9 and 63.2 ± 8.5 years, their male:female ratios were 17:14 and 21:11, and their diagnoses were colon cancer (n = 24 and n = 22) and gastric cancer (n = 7 and n = 9), respectively; 1 patient in the C/T group had gastric cancer and colon cancer (Table 1).Table 1 Characteristics of the subjects in the control and C/T groups

nd 63.2 ± 8.5 years, their male:female ratios were 17:14 and 21:11, and their diagnoses were colon cancer (n = 24 and n = 22) and gastric cancer (n = 7 and n = 9), respectively; 1 patient in the C/T group had gastric cancer and colon cancer (Table 1).Table 1 Characteristics of the subjects in the control and C/T groups Control C/T p value Age (years) 63.5 ± 8.9 63.2 ± 8.5 0.917 Sex, male:female (cases) 17:14 21:11 0.556 Colon cancer (cases) 24 22 0.517 Gastric cancer (cases) 7 9 Gastric/colon cancer (cases) 0 1 Total (cases) 31 32 Age was expressed as mean ± SD and compared using the t test. Numbers of subjects were compared using percentage. p values were calculated using Fisher’s exact test C/T cystine and theanine Operative procedures in each group are shown in Table 2. Open or laparoscopic procedure was not distinguished.Table 2 Operative procedures in the control and C/T groups Control (cases) C/T (cases) Total (cases) Colon cancer Rt. colectomy 5 8 13 Sigmoid colectomy 8 5 13 Resection of rectum 6 6 12 Miles’ operation 5 2 7 Partial resection 0 1 1 Gastric cancer Distal gastrectomy 5 3 8 Total gastrectomy 2 6 8 Gastric/colon cancer Distal gastrectomy + rt. colectomy 0 1 1 Total (cases) 31 32 63 C/T cystine and theanine

Control (cases) C/T (cases) Total (cases) Colon cancer Rt. colectomy 5 8 13 Sigmoid colectomy 8 5 13 Resection of rectum 6 6 12 Miles’ operation 5 2 7 Partial resection 0 1 1 Gastric cancer Distal gastrectomy 5 3 8 Total gastrectomy 2 6 8 Gastric/colon cancer Distal gastrectomy + rt. colectomy 0 1 1 Total (cases) 31 32 63 C/T cystine and theanine The following incidences for AEs (any grades) were observed in the control and C/T groups, respectively: 16.1 % and 9.4 % for neutropenia, 29.0 % and 18.8 % for stomatitis, 38.7 % and 18.8 % for appetite loss, 16.1 % and 18.8 % for nausea, 41.9 % and 9.4 % for diarrhea, 22.6 % and 9.4 % for fatigue, 32.3 % and 25.0 % for pigmentation, 6.5 % and 0 % for exanthema, 9.7 % and 0 % for fever (not accompanied by neutropenia), 9.7 % and 0 % for abdominal pain, and 3.2 % and 0 % for vertigo. The incidences for all AEs, except nausea, were lower in the C/T group than in the control group, and a significant difference (p < 0.01) was observed in the incidence of diarrhea (Table 3). The following incidences for AEs that were of a grade 2 or more severity (at which point S-1 administration was suspended or discontinued) were observed in the control and the C/T groups, respectively: 16.1 % and 9.4 % for neutropenia, 12.9 % and 3.1 % for stomatitis, 19.4 % and 6.3 % for appetite loss, 9.7 % and 3.1 % for nausea, 25.8 % and 3.1 % for diarrhea, 12.9 % and 0 % for fatigue, and 0 % and 0 % for pigmentation. The incidences for all these AEs were lower in the CT group than in the control group, and a significant difference (p < 0.05) was observed in the incidence of diarrhea. Exanthema (3.2 %), fever (9.7 %), abdominal pain (9.7 %), and vertigo (3.2 %) were noted in only the control group (Table 3). With regard to operative procedure, incidences of all AEs, except neutropenia and appetite loss in gastric cancer patients, were lower in the C/T group than in the control group in both colon cancer and gastric cancer patients. The incidence of appetite loss and diarrhea of any grade was significantly lower (p < 0.05 and p < 0.01, respectively) in the C/T group than in the control group in colon cancer patients.Table 3 Incidence of adverse events (AEs) during the first course (28 days) of S-1 therapy

colon cancer and gastric cancer patients. The incidence of appetite loss and diarrhea of any grade was significantly lower (p < 0.05 and p < 0.01, respectively) in the C/T group than in the control group in colon cancer patients.Table 3 Incidence of adverse events (AEs) during the first course (28 days) of S-1 therapy Group Colon cancer Gastric cancer Total Control (n = 24) C/T (n = 22) Control (n = 7) C/T (n = 9) Control (n = 31) C/T (n = 32) Adverse event (AE) (grade) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Neutropenia % 16.7 (16.7) 0 (0) 14.2 (14.2) 33.3 (33.3) 16.1 (16.1) 9.4 (9.4) Stomatitis 29.2 (8.3) 13.6 (0) 28.6 (28.6) 22.0 (0) 29.0 (12.9) 18.8 (3.1) Appetite loss 41.7 (20.8) 9.1* (0) 28.6 (14.2) 44.4 (22.2) 38.7 (19.4) 18.8 (6.3) Nausea 16.7 (12.5) 18.2 (4.5) 14.2 (0) 22.2 (0) 16.1 (9.7) 18.8 (3.1) Diarrhea 41.7 (25.0) 4.5** (4.5) 42.9 (28.6) 11.1 (0) 41.9 (25.8) 9.4** (3.1*) Fatigue 16.7 (12.5) 9.1 (0) 42.9 (14.2) 11.1 (0) 22.6 (12.9) 9.4 (0) Pigmentation 25.0 (0) 22.7 (0) 57.1 (0) 22.2 (0) 32.3 (0) 25.0 (0) Exantheme 8.3 (4.2) 0 (0) 0 (0) 0 (0) 6.5 (3.2) 0 (0) Fever 4.2 (4.2) 0 (0) 28.6 (28.6) 0 (0) 9.7 (9.7) 0 (0) Abdominal pain 8.3 (8.3) 0 (0) 14.3 (14.3) 0 (0) 9.7 (9.7) 0 (0) Vertigo 4.2 (4.2) 0 (0) 14.3 (14.3) 0 (0) 3.2 (3.2) 0 (0) AEs were evaluated using the CTCAE (ver. 4.0) C/T cystine and theanine * p < 0.05; ** p < 0.01 vs. control

Group Colon cancer Gastric cancer Total Control (n = 24) C/T (n = 22) Control (n = 7) C/T (n = 9) Control (n = 31) C/T (n = 32) Adverse event (AE) (grade) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Any (G ≥ 2) Neutropenia % 16.7 (16.7) 0 (0) 14.2 (14.2) 33.3 (33.3) 16.1 (16.1) 9.4 (9.4) Stomatitis 29.2 (8.3) 13.6 (0) 28.6 (28.6) 22.0 (0) 29.0 (12.9) 18.8 (3.1) Appetite loss 41.7 (20.8) 9.1* (0) 28.6 (14.2) 44.4 (22.2) 38.7 (19.4) 18.8 (6.3) Nausea 16.7 (12.5) 18.2 (4.5) 14.2 (0) 22.2 (0) 16.1 (9.7) 18.8 (3.1) Diarrhea 41.7 (25.0) 4.5** (4.5) 42.9 (28.6) 11.1 (0) 41.9 (25.8) 9.4** (3.1*) Fatigue 16.7 (12.5) 9.1 (0) 42.9 (14.2) 11.1 (0) 22.6 (12.9) 9.4 (0) Pigmentation 25.0 (0) 22.7 (0) 57.1 (0) 22.2 (0) 32.3 (0) 25.0 (0) Exantheme 8.3 (4.2) 0 (0) 0 (0) 0 (0) 6.5 (3.2) 0 (0) Fever 4.2 (4.2) 0 (0) 28.6 (28.6) 0 (0) 9.7 (9.7) 0 (0) Abdominal pain 8.3 (8.3) 0 (0) 14.3 (14.3) 0 (0) 9.7 (9.7) 0 (0) Vertigo 4.2 (4.2) 0 (0) 14.3 (14.3) 0 (0) 3.2 (3.2) 0 (0) AEs were evaluated using the CTCAE (ver. 4.0) C/T cystine and theanine * p < 0.05; ** p < 0.01 vs. control The percent of subjects who were able to complete the first course of chemotherapy at the prescribed dose was significantly higher (p < 0.01) in the C/T group (75.0 %) than in the the control group (35.5 %) (Table 4). According to sub-analyses, completion rate was significantly higher (p < 0.01) in C/T group (90.9 %) than control group (41.7 %) in colon cancer patients, and higher, but not significant, in C/T group (44.4 %) than control group (14.3 %) in gastric cancer patients (Table 4).Table 4 Completion rate and the duration of the administration period in which S-1 should be administered at the prescribed dose without suspension or discontinuation during first treatment course (28 days)

gher, but not significant, in C/T group (44.4 %) than control group (14.3 %) in gastric cancer patients (Table 4).Table 4 Completion rate and the duration of the administration period in which S-1 should be administered at the prescribed dose without suspension or discontinuation during first treatment course (28 days) Group Colon cancer Gastric cancer Total Control C/T Control C/T Control C/T Completion rate of first course of treatment (cases) 10/24 20/22 1/7 4/9 11/31 24/32 Percent (%) 41.7 90.9** 14.3 44.4 35.5 75.0** Duration (days) 21.0 ± 7.4 26.8 ± 3.8** 16.3 ± 8.0 21.1 ± 6.8 20.0 ± 7.7 24.8 ± 5.8** Duration was expressed as mean ± SD and compared using the t test C/T cystine and theanine ** p < 0.01 vs. control The duration of the administration period in which S-1 could be administered at the prescribed dose without suspension or discontinuation during the first treatment course (scheduled to be 28 days) was significantly longer (p < 0.01) in the C/T group (24.8 ± 5.8 days) than in the control group (20.0 ± 7.7 days). According to sub-analyses, the duration was significantly longer (p < 0.01) in the C/T group (26.8 ± 3.8 days) than in the control group (21.0 ± 7.4 days) in colon cancer patients, and longer, but not significant, in the C/T group (21.1 ± 6.8 days) than in the control group (16.3 ± 8.0 days) in gastric cancer patients (Table 4).

to sub-analyses, the duration was significantly longer (p < 0.01) in the C/T group (26.8 ± 3.8 days) than in the control group (21.0 ± 7.4 days) in colon cancer patients, and longer, but not significant, in the C/T group (21.1 ± 6.8 days) than in the control group (16.3 ± 8.0 days) in gastric cancer patients (Table 4). Discussion The ACTS-GC study reported that the use of S-1 adjuvant chemotherapy for treating stage II/III gastric cancer increased the 5-year survival rate after surgery alone from 61.7 % to 71.7 % and improved the hazard risk by 32 % [12]. A study in Japan that assessed the regimen of orally administering uracil, tegafur, and leucovorin (UFT/LV) as an adjuvant chemotherapy after colon cancer surgery found that it was not inferior to intravenous 5-FU and levofolinate (5-Fu/LV) therapy [13]. UFT/LV is recommended in the guidelines; however, the ACTS-CC study demonstrated that S-1 is not inferior to UFT/LV [14]. The results of this study demonstrated that S-1 adjuvant chemotherapy was effective following surgery for gastric or colon cancer.

ferior to intravenous 5-FU and levofolinate (5-Fu/LV) therapy [13]. UFT/LV is recommended in the guidelines; however, the ACTS-CC study demonstrated that S-1 is not inferior to UFT/LV [14]. The results of this study demonstrated that S-1 adjuvant chemotherapy was effective following surgery for gastric or colon cancer. AEs associated with the administration of S-1 have been shown to affect the duration of the administration period; S-1 administered was continued for 12 months with suspensions and dose reductions in 65.8 % (340/517) of the patients after gastrectomy in the ACTS-GC study [12] and for 6 months in 76.5 % of the patients after large bowel resection in the ACTS-CC study [14]. As dose reductions were necessary because of the occurrence of AEs in 46.5 % (158 patients) of the 340 patients who completed the therapy in the ACTS-GC study, the percent of patients who completed the therapy without dose reductions was 35.2 % (182/517 patients) [12]. In a study by Maekawa et al., S-1 was administered for 12 months in 65 % of the 40 patients after gastrectomy, and only 23 % of the patients did not require a change in the administration schedule or a reduction in dose [15]. According to sub-analyses of the ACTS-GC study results, the outcomes were more favorable among the patients who could continue receiving treatment for 12 months and the patients who received 70 % or more of the planned dose [16]. Therefore, how well AEs can be controlled and how much of the scheduled dose can be administered within the scheduled treatment period are considered to markedly affect the therapeutic outcomes of S-1 adjuvant chemotherapy.

ing treatment for 12 months and the patients who received 70 % or more of the planned dose [16]. Therefore, how well AEs can be controlled and how much of the scheduled dose can be administered within the scheduled treatment period are considered to markedly affect the therapeutic outcomes of S-1 adjuvant chemotherapy. In the present clinical trial, cystine and theanine was shown to alleviate the AEs associated with the use of S-1. Moreover, during the first course of S-1 therapy, cystine and theanine significantly increased the number of days on which S-1 could be administered and significantly improved the completion rate of the therapy at the prescribed dose from 35.5 % to 75.0 %. Especially, the completion rate of colon cancer patients improved from 41.7 % to 90.9 %. The findings also suggest that cystine and theanine alleviates all AEs, in contrast to drugs such as antiemetics, that target particular symptoms. No study has yet demonstrated improvements in the completion rate of anticancer regimens through the use of particular supportive therapies. With respect to the specific symptoms, significant suppression was observed only for diarrhea; however, the effects on the other symptoms may also be significant in large-scale studies. The AEs associated with S-1 most frequently occur during the first few treatment courses. If cystine and theanine can improve the completion rate of the first treatment course, its suppressive effects on AEs may be sustained in subsequent courses in patients who continue to take it. In a previous study, we were able to administer the S-1 regimen as scheduled without AEs by the continuous administration of cystine and theanine in a small number of patients.

rate of the first treatment course, its suppressive effects on AEs may be sustained in subsequent courses in patients who continue to take it. In a previous study, we were able to administer the S-1 regimen as scheduled without AEs by the continuous administration of cystine and theanine in a small number of patients. With respect to the dose, the final dosage form of the cystine and theanine amino acid supplement, which contained 700 mg cystine and 280 mg theanine, weighed 1.7 g, and it was readily ingested even by patients with gastrointestinal symptoms, which is a major advantage. The findings of all the clinical trials to date are based on the combination of 700 mg cystine and 280 mg theanine; future studies should evaluate the optimal dose. In an experiment using mice, its effects were not intensified at higher doses, suggesting that there is an optimal dose [9, 17].

hich is a major advantage. The findings of all the clinical trials to date are based on the combination of 700 mg cystine and 280 mg theanine; future studies should evaluate the optimal dose. In an experiment using mice, its effects were not intensified at higher doses, suggesting that there is an optimal dose [9, 17]. Although the action mechanism of cystine and theanine has not yet been elucidated in detail, it likely involves an increase in the cellular concentration of GSH in the organs that are involved in the AEs which occur. Cystine and theanine provides cysteine and glutamic acid to cells, and in combination with glycine, they increase intracellular GSH concentrations. The use of an anticancer agent is expected to increase oxidative stress in cells and to reduce the concentration of GSH, which is a potent antioxidant. Because the administration of cystine and theanine for 5 days before laparotomy suppressed decreases in GSH in the small bowel mucosa and Peyer’s patches of mice [9], this may be the mechanism responsible for alleviation of the AEs caused by anticancer chemotherapy. A previous study involving 14 patients who had colorectal cancer and underwent adjuvant chemotherapy with 5-FU and oxaliplatin reported that the incidence of sensory neuropathy was significantly lower in 5 patients who were orally administered N-acetylcysteine, a GSH precursor, at 1200 mg than in the 9 patients who were not administered the supplement [18]. Another study found that among 52 patients who had colorectal cancer and underwent oxaliplatin-based chemotherapy, a significantly stronger neuroprotective effect was observed in the group that received intravenous GSH at 1500 mg/m2 before the administration of oxaliplatin [19]. The regimens evaluated in these clinical studies also aimed to alleviate the AEs caused by chemotherapy by increasing the GSH concentration in normal cells.

significantly stronger neuroprotective effect was observed in the group that received intravenous GSH at 1500 mg/m2 before the administration of oxaliplatin [19]. The regimens evaluated in these clinical studies also aimed to alleviate the AEs caused by chemotherapy by increasing the GSH concentration in normal cells. In conclusion, the intake of the amino acids cystine and theanine at 700 and 280 mg, respectively, alleviated the AEs caused by the anticancer drug S-1, reduced the frequency of therapy suspension or discontinuation caused by AEs, and improved the completion rate of the first course of therapy. The authors thank Makiko Kashima, B.Sc., Katsushi Takahashi, B.Sc., and Atsushi Kudoh, Ph.D. (Department of Pharmacy, Sendai City Medical Center, Sendai, Japan) for their support in the AE consultations that were conducted by telephone. Funding This work was supported by Ajinomoto Co., Inc. Compliance with ethical standards Conflict of interest No author has any conflict of interest.

Introduction Therapeutic methods for stage IB2 to IIB cervical cancer with a bulky mass differ between Japan and Western countries. Based on the results of large-scale randomized studies and meta-analyses, concurrent chemoradiotherapy (CCRT) is recommended as the standard treatment in Western countries [1–5]. However, an approach using neoadjuvant chemotherapy (NAC) is widely applied clinically in Japan, South Korea, China, and Italy [6]. Irinotecan (CPT-11), a drug developed in Japan, is reportedly useful as monotherapy or in combination with cisplatin (CDDP) for recurrent cervical cancer [7, 8]. We previously reported the efficacy and safety of a treatment regimen involving a 28-day cycle of CDDP administered on day 1 and CPT-11 on days 1, 8, and 15 [9]. From the aspect of reducing the time to surgical therapy as the primary treatment, we conducted a phase II clinical study of NAC in combination with radical hysterectomy with a dosing schedule employing a 21-day cycle with a higher than usual CDDP dose intensity. Subjects and methods Subjects The subjects were 42 patients with stages IB2 to IIIB squamous cell carcinoma of the uterine cervix with a bulky mass. These patients gave informed consent and were scheduled to undergo radical hysterectomy during the period from June 2002 to March 2014.

From the aspect of reducing the time to surgical therapy as the primary treatment, we conducted a phase II clinical study of NAC in combination with radical hysterectomy with a dosing schedule employing a 21-day cycle with a higher than usual CDDP dose intensity. Subjects and methods Subjects The subjects were 42 patients with stages IB2 to IIIB squamous cell carcinoma of the uterine cervix with a bulky mass. These patients gave informed consent and were scheduled to undergo radical hysterectomy during the period from June 2002 to March 2014. Justification for the target sample size We previously reported that the response rate to the CPT-11/CDDP regimen [CPT-11 60 mg/m2 (days 1, 8, 15), CDDP 70 mg/m2 (day 1) q28 days] was 59 % [9]. Accordingly, the threshold response rate was set at 50 %, because it would not be worthwhile to use this strategy in a clinical setting if the response rate was significantly lower than 50 %. The response rates to platinum-based NAC in patients with cervical cancer reportedly range from 76 % to 95 % [6, 10, 11]. Accordingly, the expected response rate was set at 80 %, anticipating that the response rate to this regimen would be 80 %. The number of patients required was calculated to be 36 based on a presumed binominal distribution with a threshold value of 59 %, expected response rate of 80 %, and two-sided α-level of 0.05 and β-level of 0.2 (1 − β = 0.8). The number of planned subjects was thus set at 40 patients, taking into account the possibility of a few patients becoming ineligible or dropping out.

ased on a presumed binominal distribution with a threshold value of 59 %, expected response rate of 80 %, and two-sided α-level of 0.05 and β-level of 0.2 (1 − β = 0.8). The number of planned subjects was thus set at 40 patients, taking into account the possibility of a few patients becoming ineligible or dropping out. Inclusion criteria The following set of inclusion criteria was employed for selection of study subjects. (1) Histologically verified squamous cell carcinoma of the uterine cervix; (2) age more than 20 years and less than 70 years; (3) locally advanced stage IB2 to IIIB; (4) Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–2; (5) initially treated case; (6) presence of a magnetic resonance imaging (MRI)-measurable bulky mass in the uterine cervix; (7) hematological and blood biochemical findings meeting the following criteria (WBC count ≥4,000/mm3; neutrophil count ≥2,000/mm3; platelet count ≥100,000/mm3; hemoglobin ≥10.0 g/dl; AST and ALT levels ≤2 times the upper limit of normal reference range at study site; serum total bilirubin level ≤1.5 mg/dl; serum creatinine ≤1.5 mg/dl; and creatinine clearance ≥60 ml/min); (8) life expectancy ≥6 months; and (9) written informed consent personally given by the subject.

000/mm3; hemoglobin ≥10.0 g/dl; AST and ALT levels ≤2 times the upper limit of normal reference range at study site; serum total bilirubin level ≤1.5 mg/dl; serum creatinine ≤1.5 mg/dl; and creatinine clearance ≥60 ml/min); (8) life expectancy ≥6 months; and (9) written informed consent personally given by the subject. Exclusion criteria Exclusion criteria were prescribed as follows. (1) Patients with overt infection; (2) patients with a serious complication (e.g., cardiac disease, poorly controlled diabetes mellitus, malignant hypertension, bleeding tendency); (3) patients with active multiple cancer; (4) patients with interstitial pneumonia or pulmonary fibrosis; (5) patients with effusions; (6) patients with a history of unstable angina or myocardial infarction within 6 months after registration, or with a concurrent serious arrhythmia requiring treatment; (7) patients for whom treatment with cisplatin and irinotecan is contraindicated; (8) patients with (watery) diarrhea; (9) patients with intestinal paralysis or ileus; (10) pregnant women, nursing mothers, or women wishing to become pregnant; (11) patients with a history of serious drug hypersensitivity or drug allergy; and (12) patients who were inadequate for safe conduct of this study as judged by the attending physician. Neoadjuvant chemotherapy CDDP at 70 mg/m2 was intravenously administered on day 1 and CPT-11 at 70 mg/m2 was intravenously administered on days 1 and 8 of a 21-day cycle. In principle, two cycles were administered followed by radical hysterectomy. Dose modification criteria

Exclusion criteria Exclusion criteria were prescribed as follows. (1) Patients with overt infection; (2) patients with a serious complication (e.g., cardiac disease, poorly controlled diabetes mellitus, malignant hypertension, bleeding tendency); (3) patients with active multiple cancer; (4) patients with interstitial pneumonia or pulmonary fibrosis; (5) patients with effusions; (6) patients with a history of unstable angina or myocardial infarction within 6 months after registration, or with a concurrent serious arrhythmia requiring treatment; (7) patients for whom treatment with cisplatin and irinotecan is contraindicated; (8) patients with (watery) diarrhea; (9) patients with intestinal paralysis or ileus; (10) pregnant women, nursing mothers, or women wishing to become pregnant; (11) patients with a history of serious drug hypersensitivity or drug allergy; and (12) patients who were inadequate for safe conduct of this study as judged by the attending physician. Neoadjuvant chemotherapy CDDP at 70 mg/m2 was intravenously administered on day 1 and CPT-11 at 70 mg/m2 was intravenously administered on days 1 and 8 of a 21-day cycle. In principle, two cycles were administered followed by radical hysterectomy. Dose modification criteria Criteria for CPT-11 dose skip. The CPT-11 dose on day 8 will be skipped if hematological test values within 2 days before day 8 fail to fulfill the following criteria: (1) neutrophil count ≥1,000/mm3, and (2) platelet count ≥75,000/mm3.

Neoadjuvant chemotherapy CDDP at 70 mg/m2 was intravenously administered on day 1 and CPT-11 at 70 mg/m2 was intravenously administered on days 1 and 8 of a 21-day cycle. In principle, two cycles were administered followed by radical hysterectomy. Dose modification criteria Criteria for CPT-11 dose skip. The CPT-11 dose on day 8 will be skipped if hematological test values within 2 days before day 8 fail to fulfill the following criteria: (1) neutrophil count ≥1,000/mm3, and (2) platelet count ≥75,000/mm3. Criteria for initiation of the second cycle. Initiation of the second cycle will be postponed up to a maximum of 2 weeks if hematological test values within 2 days before the scheduled second cycle initiation day fail to fulfill the following criteria: (1) neutrophil count ≥1,500/mm3, (2) platelet count ≥75,000/mm3, and (3) serum creatinine ≤1.5 mg/dl. Dose reduction criteria The doses of cisplatin and irinotecan will be reduced to 70 and 60 mg/m2, respectively, in the second course for patients for whom any of the following signs of toxicity is noted in the first cycle: (1) grade 4 neutropenia persisting for ≥7 days, (2) febrile neutropenia persisting for ≥4 days, (3) grade 4 thrombocytopenia, (4) grade 3 thrombocytopenia with hemorrhage, and (5) grade ≥3 nonhematological toxicity excluding nausea, vomiting, appetite loss, fatigue, and hair loss.

toxicity is noted in the first cycle: (1) grade 4 neutropenia persisting for ≥7 days, (2) febrile neutropenia persisting for ≥4 days, (3) grade 4 thrombocytopenia, (4) grade 3 thrombocytopenia with hemorrhage, and (5) grade ≥3 nonhematological toxicity excluding nausea, vomiting, appetite loss, fatigue, and hair loss. Supportive therapy Therapeutic administration of granulocyte-colony-stimulating factor (G-CSF) preparations was undertaken when grade 4 neutropenia was noted in the first cycle. In the second cycle and thereafter, prophylactic use of the preparation in patients with grade 3 neutropenia was acceptable if grade 4 neutropenia had been noted in the first cycle. Antiemetics were used for preventive purposes. Endpoints/variables The primary endpoint was antitumor efficacy, and the secondary endpoints were adverse events, completion rate of radical hysterectomy, operative time, surgical blood loss, progression-free survival (PFS), and overall survival (OS). Antitumor efficacy, PFS, and OS were also calculated by stage. For the determination of antitumor efficacy, MRI was performed after the completion of course 1 and course 2, using MRI images before treatment as the baseline. Antitumor efficacy was determined using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, with response being the best evaluation. Adverse events were evaluated according to the National Cancer Institute Common Toxicity Criteria (NCI-CTCAE) version 4.0.

ted specimens, underwent postoperative irradiation, chemotherapy or CCRT. Before 2008, irradiation was performed as postoperative adjuvant therapy, whereas chemotherapy was performed from 2008 onward. However, CCRT was performed for patients who had multiple lymph node metastases and/or infiltration to the parametrium. Statistical analysis Progression-free survival and overall survival were calculated from the date of start of NAC, to the documented date of progression, death, or last follow-up, whichever occurred first. Impact of surgery result on survival was assessed by constructing Kaplan–Meier curves with a log-rank test. Cox regression analyses were performed to assess the prognostic factors on survival. All reported significance was two tailed at a level of 0.05.

Response The antitumor effect was complete response (CR) in 7 patients, partial response (PR) in 28, stable disease (SD) in 6, and progressive disease (PD) in 1, and the response rate was 83.3 % [95 % confidence interval (CI), 68.6–93.0]. Response rates by progression were 100 % for stage I, 82.8 % for stage II, and 50.0 % for stage III (Table 2).Table 2 Response (n = 42) CR PR SD PD Objective response (CR + PR) Total (n = 42) 7 28 6 1 35 (83.3 %) Stage l (n = 9) 2 7 0 0 9 (100 %) Stage ll (n = 29) 2 20 4 1 24 (82.8 %) Stage lll (n = 4) 1 1 2 0 2 (50.0 %) CR complete response, PR partial response, SD stable disease, PD progressive disease Adverse events Grades 3 and 4 neutropenia were observed in 11 (26.2 %) and 12 (28.6 %) patients, respectively. Grade 3 or more severe anemia was noted in 4 patients (9.5 %); among them, a patient with grade 4 anemia required a blood transfusion. Grade 3 or more severe decreases in platelet counts were observed in 1 patient (2.4 %). As grade 3 or more severe nonhematological toxicity, grade 3 nausea occurred in 3 patients (7.1 %), vomiting in 1 (2.4 %), and grade 3 febrile neutropenia in 2 (7.1 %) (Table 3).Table 3 Adverse events (n = 42) Grade 1 Grade 2 Grade 3 Grade 4 Grade >3 Hematological toxicity Leukopenia 8 20 8 3 11 (26.2) Neutropenia 4 14 11 12 23 (54.8) Anemia 17 20 3 1 4 (9.5) Thrombocytopenia 10 2 1 0 1 (2.4) Nonhematological toxicity Nausea 25 9 3 0 3 (7.1) Vomiting 21 7 1 0 1 (2.4) Diarrhea 4 2 0 0 0 (0) Creatinine 2 0 0 0 0 (0) Febrile neutropenia 0 0 2 0 2 (4.8)

rade 4 Grade >3 Hematological toxicity Leukopenia 8 20 8 3 11 (26.2) Neutropenia 4 14 11 12 23 (54.8) Anemia 17 20 3 1 4 (9.5) Thrombocytopenia 10 2 1 0 1 (2.4) Nonhematological toxicity Nausea 25 9 3 0 3 (7.1) Vomiting 21 7 1 0 1 (2.4) Diarrhea 4 2 0 0 0 (0) Creatinine 2 0 0 0 0 (0) Febrile neutropenia 0 0 2 0 2 (4.8) Among the total 42 patients, 40 (95.2 %) completed chemotherapy as scheduled. The administration of CPT-11 on day 8 of cycle 2 was skipped in the remaining 2 patients. Grade 3 nausea persisted in these 2 patients; although it did not satisfy the skip criteria, in both cases the primary physician decided to skip the administration. The administration in cycle 2 was postponed in 3 patients (10.7 %) because neutrophil counts did not satisfy the therapy initiation criteria; however, all these 3 patients were able to receive treatment within 7 days. In 2 patients (10.0 %) in whom febrile neutropenia continued for 4 days or more, the doses of CPT-11 and CDDP were reduced from 70 to 60 mg/m2.

ed in 3 patients (10.7 %) because neutrophil counts did not satisfy the therapy initiation criteria; however, all these 3 patients were able to receive treatment within 7 days. In 2 patients (10.0 %) in whom febrile neutropenia continued for 4 days or more, the doses of CPT-11 and CDDP were reduced from 70 to 60 mg/m2. Operative details Among the total 42 patients, 2 with stage IIIB had inoperable disease. Forty patients underwent surgery. Radical hysterectomy was performed for 38 patients, but in 1 of them surgery was incomplete because of difficulty in resecting the lymph node metastasis. Simple total hysterectomy and exploratory laparotomy were performed in 1 patient each. The operable rate of surgery was 95.2 %; completion rate of radical hysterectomy was 88.1 % (Fig. 1). Median number of lymph nodes dissected was 22 (range, 3–49); median operative time was 260 min (range, 210–334); median blood loss was 500 ml (range, 393–898). Blood transfusions were given to 6 of 40 patients (15.0 %). The median time from surgery to discharge was 21 days (range, 16–26) (Table 4).Fig. 1 Operation consort diagram. SCC squamous cell carcinoma, NAC neoadjuvant chemotherapy Table 4 Operative details Characteristic n = 42 Type of surgery 40 RH 38 TAH 1 Probe laparotomy 1 Inoperable 2 Operation time (min) Median (IQR) 260 (210–334) Blood loss (ml) Median (IQR) 500 (393–898) Blood transfusion Yes 6 No 34 Time from surgery to discharge (Days) Median (IQR) 21 (16–26) Pathological lymph node metastasis Positive 13 Negative 29

teristic n = 42 Type of surgery 40 RH 38 TAH 1 Probe laparotomy 1 Inoperable 2 Operation time (min) Median (IQR) 260 (210–334) Blood loss (ml) Median (IQR) 500 (393–898) Blood transfusion Yes 6 No 34 Time from surgery to discharge (Days) Median (IQR) 21 (16–26) Pathological lymph node metastasis Positive 13 Negative 29 RH radical hysterectomy, TAH total abdominal hysterectomy, IQR interquartile range PFS and OS analysis Of the 38 patients who underwent radical hysterectomy (excluding 1 patient who underwent probelaparotomy), 10 patients (26.3 %) developed tumor recurrence, that is, recurrence was noted in 1 (50.0 %) of the 2 patients who received postoperative CCRT, 2 (13.3 %) of the 15 patients who received postoperative chemotherapy, 5 (41.7 %) of the 12 patients who received postoperative radiotherapy, and 2 (22.2 %) of the 9 patients who did not receive postoperative adjuvant therapy (Table 5). Tumor recurrence in the lymph nodes was noted in 4 patients (affecting the paraaortic lymph nodes in all 4 cases and accompanied by metastasis to the subclavicular lymph nodes in 1 case). Of the 4 patients unable to undergo radical hysterectomy, 1 patient underwent CCRT after a simple total hysterectomy. CCRT was used for the patient who underwent probelaparotomy, and CCRT and radiation were used for the 2 patients judged unable to receive post-NAC surgery, but all 3 of these patients developed intrapelvic recurrence.Table 5 Details of recurrent patients after radical hysterectomy (n = 38)

a simple total hysterectomy. CCRT was used for the patient who underwent probelaparotomy, and CCRT and radiation were used for the 2 patients judged unable to receive post-NAC surgery, but all 3 of these patients developed intrapelvic recurrence.Table 5 Details of recurrent patients after radical hysterectomy (n = 38) Adjuvant therapy Number Rate (%) Recurrent sites Chemoradiotherapy (n = 2) 1 50.0 PAN (l) Chemotherapy (n = 15) 2 13.3 PAN (l), pelvic cavity (l) Radiation (n = 12) 5 41.7 PAN (2), subclavicular lymph node (l), pelvic cavity (3) No therapy (n = 9) 2 22.2 Vaginal stump (2) PAN paraaortic lymph node The median follow-up period for 42 patients was 45 months (range, 8–143), and the 5-year PFS rate and 5-year OS rate were 67.2 % and 68.0 %, respectively. As to progression, the 5-year PFS rates for patients at stages I, II, and III were 53.3 %, 78.4 %, and 25.0 %, respectively, and the 5-year OS rates were 53.3 %, 79.2 %, and 25.0 %, respectively (Figs. 2, 3). In multivariate analysis of OS, lymph node metastasis before NAC [hazard radio (HR), 34.88; p = 0.0031] and response of NAC (HR, 30.58; p = 0.0014) were extracted as significant prognosis factors (Table 6).Fig. 2 Kaplan–Meier plot of progression-free survival (n = 42) Fig. 3 Kaplan–Meier plot of overall survival (n = 42) Table 6 Multivariate analysis of treatment-related factors for overall survival (OS)

The median follow-up period for 42 patients was 45 months (range, 8–143), and the 5-year PFS rate and 5-year OS rate were 67.2 % and 68.0 %, respectively. As to progression, the 5-year PFS rates for patients at stages I, II, and III were 53.3 %, 78.4 %, and 25.0 %, respectively, and the 5-year OS rates were 53.3 %, 79.2 %, and 25.0 %, respectively (Figs. 2, 3). In multivariate analysis of OS, lymph node metastasis before NAC [hazard radio (HR), 34.88; p = 0.0031] and response of NAC (HR, 30.58; p = 0.0014) were extracted as significant prognosis factors (Table 6).Fig. 2 Kaplan–Meier plot of progression-free survival (n = 42) Fig. 3 Kaplan–Meier plot of overall survival (n = 42) Table 6 Multivariate analysis of treatment-related factors for overall survival (OS) Factor Hazard ratio 95 % CI p value PS 0/1 0.11 0.002–2.693 0.1858 FIGO stage Stage I, II/III 5.75 0.224–90.468 0.2435 Tumor diameter >5/<5 0.25 0.009–3.177 0.2788 Pre-NAC lymph node metastasis Positive/negative 34.88 2.969–1152.9 0.0031 Tumor response CR, PR/SD, PD 30.59 3.675–447.9 0.0014 Pathological lymph node metastasis Positive/negative 1.21 0.182–7.932 0.836 Postoperative treatment Yes/no 6.81 0.718–174.323 0.0983 NAC neoadjuvant chemotherapy

Factor Hazard ratio 95 % CI p value PS 0/1 0.11 0.002–2.693 0.1858 FIGO stage Stage I, II/III 5.75 0.224–90.468 0.2435 Tumor diameter >5/<5 0.25 0.009–3.177 0.2788 Pre-NAC lymph node metastasis Positive/negative 34.88 2.969–1152.9 0.0031 Tumor response CR, PR/SD, PD 30.59 3.675–447.9 0.0014 Pathological lymph node metastasis Positive/negative 1.21 0.182–7.932 0.836 Postoperative treatment Yes/no 6.81 0.718–174.323 0.0983 NAC neoadjuvant chemotherapy Discussion Clinical studies of NAC for various forms of cancer are underway, but as yet there is little evidence of the usefulness of NAC in the field of gynecology including cervical, endometrial, and ovarian cancers. The clinical significance of NAC for cervical cancer is that the following can be expected: (1) tumor size reduction, which improves operative curability and safety and thus expands the surgical indications; and (2) inhibition of distant metastasis based on systemic effects being exerted on latent and lymph node micrometastatic lesions. As such, NAC for cervical cancer in Japan is currently performed to improve operative curability and safety as well as to increase the indications for surgery. However, there are no reports indicating that NAC prolongs survival.

s based on systemic effects being exerted on latent and lymph node micrometastatic lesions. As such, NAC for cervical cancer in Japan is currently performed to improve operative curability and safety as well as to increase the indications for surgery. However, there are no reports indicating that NAC prolongs survival. A key drug for progressive recurrent cervical cancer is CDDP; the results of a randomized study by the Gynecologic Oncology Group (GOG) demonstrated the usefulness of paclitaxel and topotecan in addition to CDDP, and dual therapy with CDDP has been recommended for recurrence and progressive cancer [12, 13]. Topotecan is widely used in Western countries, whereas mainly CPT-11, with a mechanism of action based on the same DNA type I topoisomerase inhibitory action, is used in Japan.

f paclitaxel and topotecan in addition to CDDP, and dual therapy with CDDP has been recommended for recurrence and progressive cancer [12, 13]. Topotecan is widely used in Western countries, whereas mainly CPT-11, with a mechanism of action based on the same DNA type I topoisomerase inhibitory action, is used in Japan. In a study of combination therapy with CDDP [CDDP 60 mg/m2 (day 1) + CPT-11 60 mg/m2 (days 1, 8, 15)], the response rate in patients with recurrent/progressive cervical cancer was 59.9 %, and the response rate when this regimen was administered as NAC was 78 % [6, 9]. To reduce the time to surgical therapy, the primary treatment, we conducted a phase II clinical study of NAC in combination with radical hysterectomy with a dosing schedule involving a 21-day cycle with a higher than usual CDDP dose intensity. The response rate in 42 patients was 83.3 %, better than the therapeutic results of Sugiyama et al. Notably, a response rate of 86.8 % was obtained in 38 patients with stage IB2 to IIB disease. However, the response rates were 50.0 % in 4 stage IIIB patients, 50 % in operated patients, and 25 % in patients who had completed radical hysterectomy. Our results indicate that NAC plus radical hysterectomy is not useful in stage III patients.

rate of 86.8 % was obtained in 38 patients with stage IB2 to IIB disease. However, the response rates were 50.0 % in 4 stage IIIB patients, 50 % in operated patients, and 25 % in patients who had completed radical hysterectomy. Our results indicate that NAC plus radical hysterectomy is not useful in stage III patients. With regard to adverse events, the incidence of grade 3 or more severe neutropenia was 54.8 %, although this adverse event could be managed with a G-CSF agent. In all cases with diarrhea characteristic of CPT-11, the severity was grade 2 or lower. A single dose of CPT-11 had to be reduced in this regimen because of administration in divided doses on days 1 and 8, thereby preventing grade 3 or more severe diarrhea. In 40 patients undergoing NAC plus radical hysterectomy, median operative time was 260 min and median surgical blood loss was 500 ml, similar to those findings in studies of radical hysterectomy for patients with stage IB2 to IIB with a bulky mass reported by He et al. and Wang et al. [14, 15]. No serious postoperative complications, such as intestinal obstruction and thrombosis, occurred. Accordingly, this study demonstrated the safety of NAC plus radical hysterectomy, with no major adverse effects for patients.

patients with stage IB2 to IIB with a bulky mass reported by He et al. and Wang et al. [14, 15]. No serious postoperative complications, such as intestinal obstruction and thrombosis, occurred. Accordingly, this study demonstrated the safety of NAC plus radical hysterectomy, with no major adverse effects for patients. We compared the treatment outcomes in our 38 IB2–IIB cases with the results reported by Uegaki et al. [16]. The response rate for our cases was 86.6 %, comparable to the rate reported by Uegaki et al. (86.2 %). The 5-year PFS (73.3 % vs. 62.2 %) and 5-year OS (78.9 % vs. 74.9 %) were better in our cases than in the cases reported by Uegaki et al. Comparisons were also made with respect to adjuvant therapy and recurrence sites. According to the report by Uegaki et al., recurrence was noted in 21 (32.3 %) of the 65 cases, and 15 of these 21 cases had received adjuvant therapy, but recurrence in the pelvic cavity was observed in only 4 cases (the other cases of recurrence had remote metastasis, including metastasis to the paraaortic lymph nodes). Of the 38 patients we managed, 10 patients (26.3 %) developed recurrence, including 8 patients who received adjuvant therapy (4 patients with intrapelvic recurrence and the other 4 patients with metastasis to lymph nodes, including the paraaortic lymph nodes). Postoperative adjuvant therapy by means of CCRT or radiation was performed in 27 (93.1 %) of the 29 cases reported by Uegaki et al. and 14 (50.0 %) of the 28 cases managed by us. Postoperative CCRT or radiation allowed control of intrapelvic recurrence to some extent, whereas postoperative chemotherapy tended to reduce the risk of remote recurrence. These results suggest that the site of recurrence depends on the type of postoperative adjuvant therapy. Furthermore, as recurrence was also observed in cases that did not receive postoperative adjuvant therapy, it seems advisable to perform postoperative adjuvant therapy in all cases of bulky tumors.

emote recurrence. These results suggest that the site of recurrence depends on the type of postoperative adjuvant therapy. Furthermore, as recurrence was also observed in cases that did not receive postoperative adjuvant therapy, it seems advisable to perform postoperative adjuvant therapy in all cases of bulky tumors. Six randomized clinical trials (1036 patients) including Study JCOG102 in Japan were analyzed in Cochrane Reviews in 2010, and NAC in combination with surgical therapy reportedly improved the disease-free survival rate as compared with initial surgery (HR, 0.76; p = 0.01). NAC plus radical hysterectomy was thus suggested to improve patient prognosis [17]. According to a multivariate analysis of OS, clinical stages, lymph node metastasis before NAC, and antitumor effects are significant prognostic factors. Therefore, it was suggested that NAC plus radical hysterectomy, for patients other than those with lymph node metastasis before NAC, may further improve the therapeutic effects of NAC. That is, the prognosis of patients with stage IB2 to IIB cervical cancer with a bulky mass might be improved by hysterectomy alone in those who have lymph node metastasis before NAC, and by NAC plus radical hysterectomy in those without lymph node metastasis. The next step we should take would be implementation of a phase II clinical study on the use of NAC plus radical hysterectomy plus adjuvant therapy, with details of postoperative adjuvant therapy set forth in advance, in patients having a bulky tumor (clinical stage IB2–IIB). The results from such a study would provide important data for planning a phase III clinical study on a national scale of NAC plus radical hysterectomy plus adjuvant therapy versus radical hysterectomy plus adjuvant therapy versus CCRT.

py set forth in advance, in patients having a bulky tumor (clinical stage IB2–IIB). The results from such a study would provide important data for planning a phase III clinical study on a national scale of NAC plus radical hysterectomy plus adjuvant therapy versus radical hysterectomy plus adjuvant therapy versus CCRT. Conclusions The results of this study suggest that NAC, that is, the CDDP/CPT-11 regimen, in combination with radical hysterectomy exerts high antitumor efficacy with manageable adverse reactions. Thus, NAC may facilitate safe radical hysterectomy. This treatment strategy is considered to be therapeutically useful, and an improved prognosis can also be expected. Therefore, we add our evidence of the usefulness of NAC plus radical hysterectomy in Japan to the world literature on cervical cancer treatment. Compliance with ethical standards Conflict of interest None of the authors of this manuscript has any conflicts of interest to declare.

Introduction Recent studies have shown that preoperative inflammation-based prognostic scores can predict the overall survival of patients with various cancers [1–3]. The systemic inflammatory response is associated with immune and coagulation processes, although the precise mechanisms that underlie this response, as well as the interaction between coagulation, inflammation, and carcinogenesis remain obscure. The red blood cell distribution width (RDW) is the coefficient of variation in red blood cell size, and an elevated RDW corresponds to anisocytosis [4]. Although its main clinical application has been limited to the diagnosis of iron deficiency anemia, fluctuations in RDW have recently been reported in many pathophysiological conditions, and elevated RDW is strongly associated with chronic inflammation, poor nutritional status, and age-associated diseases via changes in erythropoiesis. Cancer is known to evoke chronic inflammation and malnutrition, and cancer-associated inflammation is a key determinant of disease progression and survival in various cancers [5, 6].

RDW is strongly associated with chronic inflammation, poor nutritional status, and age-associated diseases via changes in erythropoiesis. Cancer is known to evoke chronic inflammation and malnutrition, and cancer-associated inflammation is a key determinant of disease progression and survival in various cancers [5, 6]. Mean platelet volume (MPV), a marker of platelet size, is a platelet volume index and reflects early platelet activation [7]. An elevated MPV is closely associated with thromboembolism in patients with ischemic stroke, myocardial infarction, and cerebrovascular thromboembolism [8–10]. In addition, platelets have an inflammatory role that is mediated by the secretion of pro-inflammatory factors, chemokines, and growth factors. They also have a role in cancer progression. Consequently, an inflammatory response significantly increases the risk of metastases at each cancer stage, and thus these hematological parameters predict a poor prognosis in, for example, gastric, lung, and renal cancer [11–13].

ory factors, chemokines, and growth factors. They also have a role in cancer progression. Consequently, an inflammatory response significantly increases the risk of metastases at each cancer stage, and thus these hematological parameters predict a poor prognosis in, for example, gastric, lung, and renal cancer [11–13]. Another indicator of platelet morphology is the platelet distribution width (PDW). PDW is a measure of variation in platelet size, and a high PDW can be a sign of active platelet release. The induction of platelet production leads to an increase in the average platelet size and consequently affects the PDW. Furthermore, an elevated platelet count and increased platelet aggregation have been shown to facilitate tumor progression by protecting tumor cells from the immune system [14]. PDW is therefore a potential prognostic indicator in cancer, although it should be noted that both PDW and MPV can also change in a number of benign conditions [15, 16]. It is now widely recognized that outcomes in cancer patients are not determined by their tumor characteristics alone, but also by non-tumor factors such as their general health [17]. There is a growing interest in establishing novel predictive biomarkers for various cancers. However, to the best of our knowledge, there has been no direct analysis of the predictive value of RDW, PDW, and MPV indices in esophageal cancer. In this study, we retrospectively analyzed the clinical data of esophageal cancer patients to evaluate the impact of RDW, PDW, and MPV on cancer-specific survival (CSS).

s. However, to the best of our knowledge, there has been no direct analysis of the predictive value of RDW, PDW, and MPV indices in esophageal cancer. In this study, we retrospectively analyzed the clinical data of esophageal cancer patients to evaluate the impact of RDW, PDW, and MPV on cancer-specific survival (CSS). Patients and methods Patients We retrospectively reviewed a database of 144 consecutive patients who underwent potentially curative esophagectomy with R0 resection for histologically verified esophageal squamous cell carcinoma at our institute between January 2006 and December 2014. R0 resection was defined as a complete resection without any microscopic resection margin involvement. Video-assisted or thoracoscopic esophagectomy with three-field lymph node dissection was performed for all patients, followed by elevation of the gastric conduit to the neck via the posterior mediastinal approach or the retrosternal approach with end-to-end anastomosis of the cervical esophagus and the gastric conduit. The clinical characteristics, laboratory data, treatment, and pathological data for the patients were obtained from their medical records. No patient had clinical signs of infection or other systemic inflammatory conditions preoperatively. We evaluated the CSS, in which the cause of death was determined from the case notes or computerized records. Two patients who died of a complication related to surgery within 60 days after esophagectomy were excluded from the analysis. We defined ‘elderly’ patients as those aged ≥70 years and ‘non-elderly’ as those aged <70 years [3].

CSS, in which the cause of death was determined from the case notes or computerized records. Two patients who died of a complication related to surgery within 60 days after esophagectomy were excluded from the analysis. We defined ‘elderly’ patients as those aged ≥70 years and ‘non-elderly’ as those aged <70 years [3]. The observation period started from the day of the operation and lasted for 5 years or until death, loss to follow-up, or withdrawal of consent. Permission to perform this retrospective study was obtained from the ethical board of our institution. Blood sample analysis Preoperative complete blood count (CBC) data were retrospectively extracted from the medical records. Only subjects for whom preoperative CBC and blood differential data were available were included in the study. All white blood cell and differential counts were taken within 1 week prior to surgery. RDW, PDW, and MPV CBC and hematological marker levels were measured using ethylenediaminetetraacetic acid-treated blood. Blood parameters, RDW, PDW, and MPV were analyzed using an automated hematology analyzer XE-5000 (Sysmex XE-5000 hematology analyzer; TOA Medical Electronics, Kobe, Japan). RDW, PDW, and MPV values were obtained directly from the CBC tests. TNM stage The pathological classification of the primary tumor, the degree of lymph node involvement, and the presence of organ metastasis were determined according to the TNM classification system in the 7th edition of the Cancer Staging Manual of the American Joint Committee on Cancer [18].

RDW, PDW, and MPV CBC and hematological marker levels were measured using ethylenediaminetetraacetic acid-treated blood. Blood parameters, RDW, PDW, and MPV were analyzed using an automated hematology analyzer XE-5000 (Sysmex XE-5000 hematology analyzer; TOA Medical Electronics, Kobe, Japan). RDW, PDW, and MPV values were obtained directly from the CBC tests. TNM stage The pathological classification of the primary tumor, the degree of lymph node involvement, and the presence of organ metastasis were determined according to the TNM classification system in the 7th edition of the Cancer Staging Manual of the American Joint Committee on Cancer [18]. Statistical analysis The means and standard deviations were calculated, and the differences were analyzed using Student’s t test. Differences between categories of each clinicopathological feature were analyzed using the chi-squared test. The routine reference cut-off values for RDW, PDW, and MPV used by our hospital laboratory were <50, <15.3, and <11.5, respectively. Patients with a RDW, PDW, or MPV greater than these cut-off values were considered to have a high RDW, PDW, and MPV, while the remaining patients were considered to have a low RDW, PDW, and MPV, respectively. The CSS was calculated using Kaplan–Meier statistics, and inter-group differences were assessed using the log-rank test. CSS was defined as the interval from the date of operation to the date of cancer specific death, or last follow-up.

e remaining patients were considered to have a low RDW, PDW, and MPV, respectively. The CSS was calculated using Kaplan–Meier statistics, and inter-group differences were assessed using the log-rank test. CSS was defined as the interval from the date of operation to the date of cancer specific death, or last follow-up. Univariate analyses were performed to determine variables associated with CSS. Variables with p values <0.05 in the univariate analysis were included in a multivariate logistic regression analysis. The potential prognostic factors for esophageal cancer were age (<70 vs ≥70 years), sex (female vs male), pathological stage (pStage; I/II vs III), tumor size (<3 cm vs ≥3 cm), operation time (<600 min vs ≥600 min), intraoperative blood loss (<500 mL vs ≥500 mL), serum squamous cell carcinoma antigen (SCC) value (<1.5 vs ≥1.5), RDW (<50 vs ≥50), PDW (<15.3 vs ≥15.3), and MPV (<11.5 vs ≥11.5). All statistical analyses were performed using the statistical software JMP (version 11 for Windows; SAS Institute, Cary, NC, USA), and p values <0.05 were considered statistically significant.

Univariate analyses were performed to determine variables associated with CSS. Variables with p values <0.05 in the univariate analysis were included in a multivariate logistic regression analysis. The potential prognostic factors for esophageal cancer were age (<70 vs ≥70 years), sex (female vs male), pathological stage (pStage; I/II vs III), tumor size (<3 cm vs ≥3 cm), operation time (<600 min vs ≥600 min), intraoperative blood loss (<500 mL vs ≥500 mL), serum squamous cell carcinoma antigen (SCC) value (<1.5 vs ≥1.5), RDW (<50 vs ≥50), PDW (<15.3 vs ≥15.3), and MPV (<11.5 vs ≥11.5). All statistical analyses were performed using the statistical software JMP (version 11 for Windows; SAS Institute, Cary, NC, USA), and p values <0.05 were considered statistically significant. Results Relationships between RDW, PDW, MPV, and clinicopathological features The relationships between RDW, PDW, MPV, and the clinicopathological features of 144 esophageal cancer patients are shown in Table 1. The mean and standard deviation of RDW, PDW, and MPV was 48.6 ± 6.9, 11.3 ± 1.9, and 10.1 ± 0.9, respectively. Standard values of these parameters were different by measuring instrument. The routine reference cut-off values for RDW, PDW, and MPV used by our hospital laboratory were <50, <15.3, and <11.5, respectively. Patients with a RDW, PDW, or MPV greater than these cut-off values were considered to have a high RDW, PDW, and MPV, while the remaining patients were considered to have a low RDW, PDW, and MPV, respectively.Table 1 Relationships between RDW, PDW, MPV, and clinicopathological features in 144 patients with esophageal cancer

ents with a RDW, PDW, or MPV greater than these cut-off values were considered to have a high RDW, PDW, and MPV, while the remaining patients were considered to have a low RDW, PDW, and MPV, respectively.Table 1 Relationships between RDW, PDW, MPV, and clinicopathological features in 144 patients with esophageal cancer Characteristics Total patients RDW RDW MPV <50 (n = 94) ≥50 (n = 50) p value <15.3 (n = 140) ≥15.3 (n = 4) p value <11.5 (n = 132) ≥11.5 (n = 12) p value Age (years) 66.3 ± 8.3 65.2 ± 8.3 0.433 65.8 ± 8.3 69.8 ± 4.6 0.352 65.8 ± 8.4 67.5 ± 6.9 0.498 Gender 0.206 0.333 0.084 Male 129 82 47 126 3 120 9 Female 15 12 3 14 1 12 3 Location of tumor 0.039 0.905 0.75 Ce 5 1 4 5 0 5 0 Ut 11 6 5 11 0 9 2 Mt 66 40 26 64 2 61 5 Lt 51 37 14 49 2 47 4 Ae 11 10 1 11 0 10 1 Tumor size (mm) 4.3 ± 2.6 4.6 ± 2.4 0.494 4.3 ± 2.5 6.9 ± 1.3 0.043 4.4 ± 2.5 4.6 ± 2.4 0.718 Vessel invasion 0.034 0.035 0.451 Negative 55 20 75 0 70 5 Positive 39 30 65 4 62 7 Lymphatic invasion 0.054 0.062 0.762 Negative 51 15 66 0 61 5 Positive 43 35 74 4 71 7 Differentiation 0.0013 0.28 0.986 Well 40 7 47 0 43 4 Moderate 48 35 80 3 76 7 Poor 6 8 13 13 1 Depth of tumor . <0.0001 0.196 0.06 T1a–1b 63 53 10 63 0 56 7 0.359 2 12 7 5 12 0 12 0 3 56 31 25 53 3 53 3 4a–4b 13 3 10 12 1 11 2 Lymph node metastasis 0.273 0.0002 0.502 N0 77 53 24 77 0 72 5 N1 42 25 17 42 0 39 3 N2 13 8 5 11 2 11 2 N3 12 8 4 10 2 10 2 Pathological stage 0.006 0.032 0.916 1a–1b 56 45 11 56 0 52 4 2a–2b 34 21 13 34 0 31 3 3a–3c 54 28 26 50 4 49 5 Operation time (min) 655.7 ± 158.2 600.8 ± 178.8 0.06 633.8 ± 166.0 738.0 ± 200.4 0.22 634.1 ± 167.5 664.5 ± 167.0 0.548 Intraoperative blood loss (mL) 550.1 ± 550.1 784.7 ± 647.7 0.024 635.8 ± 598.1 485.0 ± 479.8 0.619 633.5 ± 600.0 610.8 ± 550.6 0.9 SCC value 1.18 ± 1.16 1.28 ± 2.39 0.722 1.19 ± 1.69 2.28 ± 0.87 0.202 1.22 ± 1.73 1.13 ± 1.01 0.847

8.2 600.8 ± 178.8 0.06 633.8 ± 166.0 738.0 ± 200.4 0.22 634.1 ± 167.5 664.5 ± 167.0 0.548 Intraoperative blood loss (mL) 550.1 ± 550.1 784.7 ± 647.7 0.024 635.8 ± 598.1 485.0 ± 479.8 0.619 633.5 ± 600.0 610.8 ± 550.6 0.9 SCC value 1.18 ± 1.16 1.28 ± 2.39 0.722 1.19 ± 1.69 2.28 ± 0.87 0.202 1.22 ± 1.73 1.13 ± 1.01 0.847 Ce cervical esophagus, Ut upper thoracic esophagus, Mt middle thoracic esophagus, Lt lower thoracic esophagus, Ae abdominal esophagus There was a significant association between RDW and factors such as tumor location (p = 0.039), tumor depth (p < 0.0001), pStage (p = 0.006), and intraoperative blood loss (p = 0.024). The PDW also showed significant associations with tumor size (p = 0.043), lymph node metastasis (p = 0.0002), and pStage (p = 0.032). No significant associations were found between these clinicopathological features and MPV. Prognostic factors for CSS In this study, we did not analyze the relationship between PDW, MPV and prognostic value, because the size of the high PDW (n = 4) and the high MPV (n = 12) subgroup populations were too small to be compared with another group (n = 132).

There was a significant association between RDW and factors such as tumor location (p = 0.039), tumor depth (p < 0.0001), pStage (p = 0.006), and intraoperative blood loss (p = 0.024). The PDW also showed significant associations with tumor size (p = 0.043), lymph node metastasis (p = 0.0002), and pStage (p = 0.032). No significant associations were found between these clinicopathological features and MPV. Prognostic factors for CSS In this study, we did not analyze the relationship between PDW, MPV and prognostic value, because the size of the high PDW (n = 4) and the high MPV (n = 12) subgroup populations were too small to be compared with another group (n = 132). Univariate analyses demonstrated that pStage (hazard ratio [HR] 4.467; 95 % confidence interval [CI] 2.469–8.337; p < 0.0001), tumor size (HR 3.172; 95 % CI 1.511–7.076; p = 0.002), operation time (HR 0.497; 95 % CI 0.275–0.888; p = 0.018), and a high RDW (HR 2.332; 95 % CI 1.304–4.190; p = 0.005) were significant risk factors for a poor prognosis (Table 2). In multivariate analysis, pStage (HR 3.362; 95 % CI 1.772–6.643; p = 0.0002) and a high RDW (HR 1.684; 95 % CI 0.929–3.071; p = 0.0300) were found to be independently associated with poor survival.Table 2 Prognostic factors for cancer-specific survival in 144 patients with esophageal cancer after a curative esophagectomy

multivariate analysis, pStage (HR 3.362; 95 % CI 1.772–6.643; p = 0.0002) and a high RDW (HR 1.684; 95 % CI 0.929–3.071; p = 0.0300) were found to be independently associated with poor survival.Table 2 Prognostic factors for cancer-specific survival in 144 patients with esophageal cancer after a curative esophagectomy Variables Patients (n = 144) Category or characteristics Univariate Multivariate HR 95 % CI p value HR 95 % CI p value Gender 15/129 (Female/male) 1.209 0.460–2.642 0.672 Age (years) 47/97 (<70/≥70) 1.638 0.889–2.943 0.112 pStage 90/54 (I, II/III) 4.467 2.469–8.337 <0.0001 3.362 1.772–6.643 0.0002 Tumor size 45/99 (<3/≥3) 3.172 1.511–7.076 0.002 1.657 0.722–4.290 0.2427 Operation time 52/92 (<600/≥600) 0.497 0.275–0.888 0.018 0.634 0.348–1.145 0.1303 Intraoperative blood loss 71/73 (<500/≥500) 1.066 0.596–1.924 0.830 SCC value 112/32 (<1.5/≥1.5) 1.468 0.707–2.820 0.288 RDW 94/50 (<50/≥50) 2.332 1.304–4.190 0.005 1.684 0.929–3.071 0.0300 RDW, PDW, MPV, and CSS We excluded analysis of PDW and MPV because the number of patients in the high PDW and MPV subgroups was too small to show the correct data. Patients with a high RDW had a significantly poorer prognosis in terms of CSS than those with a low RDW (p = 0.004) (Fig. 1).Fig. 1 Postoperative cancer-specific survival based on RDW in 144 patients with esophageal cancer

RDW, PDW, MPV, and CSS We excluded analysis of PDW and MPV because the number of patients in the high PDW and MPV subgroups was too small to show the correct data. Patients with a high RDW had a significantly poorer prognosis in terms of CSS than those with a low RDW (p = 0.004) (Fig. 1).Fig. 1 Postoperative cancer-specific survival based on RDW in 144 patients with esophageal cancer Relationships between RDW, PDW, MPV, and clinicopathological features in non-elderly patients Significant associations were found between the RDW and factors such as tumor location (p = 0.022), tumor depth (p < 0.0001), pStage (p = 0.017), and intraoperative blood loss (p = 0.044) (Table 3), whilst the PDW only showed a significant association with lymph node metastasis (p = 0.008). None of the clinicopathological features were significantly associated with MPV.Table 3 Relationships between RDW, PDW, MPV, and clinicopathological features in 97 non-elderly patients with esophageal cancer

= 0.044) (Table 3), whilst the PDW only showed a significant association with lymph node metastasis (p = 0.008). None of the clinicopathological features were significantly associated with MPV.Table 3 Relationships between RDW, PDW, MPV, and clinicopathological features in 97 non-elderly patients with esophageal cancer Characteristics Total patients RDW PDW MPV <50 (n = 61) ≥50 (n = 36) p value 15.3< (n = 95) ≥15.3 (n = 2) p value 11.5< (n = 90) ≥11.5 (n = 7) p value Age (years) 61.6 ± 5.5 61.2 ± 5.5 0.353 61.3 ± 5.5 66.5 ± 3.5 0.191 61.3 ± 5.5 63.0 ± 5.1 0.22 Gender 0.805 0.648 0.636 Male 88 55 33 86 2 82 6 Female 9 6 3 9 0 8 1 Location of tumor 0.022 0.736 0.766 Ce 4 0 4 4 0 4 0 Ut 5 3 2 5 0 4 1 Mt 49 28 21 47 2 46 3 Lt 30 22 8 30 0 28 2 Ae 9 8 1 9 0 8 1 Tumor size (mm) 4.2 ± 2.8 4.8 ± 2.5 0.276 4.4 ± 2.7 8.0 ± 1.9 0.06 4.4 ± 2.7 5.2 ± 2.5 0.469 Vessel invasion 0.009 0.149 0.674 Negative 37 12 49 0 46 3 Positive 24 24 46 2 44 4 Lymphatic invasion 0.0007 0.175 0.802 Negative 37 9 46 0 43 3 Positive 24 27 49 2 47 4 Differentiation <0.0001 0.116 0.893 Well 28 2 30 0 28 2 Moderate 31 27 57 1 54 4 Poor 2 7 8 1 8 1 Depth of tumor <0.0001 0.284 0.278 T1a–1b 43 39 4 43 0 39 4 2 6 1 5 6 0 6 0 3 37 18 19 36 1 36 1 4a-4b 11 3 8 10 1 9 2 Lymph node metastasis 0.891 0.008 0.668 N0 54 35 19 54 0 50 4 N1 28 17 11 28 0 27 1 N2 6 3 3 5 1 5 1 N3 9 6 3 8 1 8 1 Pathological stage 0.017 0.191 0.884 1a–1b 39 31 8 39 0 36 3 2a–2b 21 12 9 21 0 20 1 3a–3c 37 18 19 35 2 34 3 Operation time (min) 645.1 ± 153.4 630.4 ± 149.3 0.676 638.2 ± 151.8 707.0 ± 149.9 0.527 638.2 ± 151.6 658.3 ± 158.0 0.737 Intraoperative blood loss (mL) 545.8 ± 515.5 736.9 ± 549.1 0.044 619.5 ± 535.7 485.0 ± 558.6 0.726 612.3 ± 531.8 674.3 ± 596.0 0.769 SCC value 1.14 ± 1.17 0.96 ± 0.67 0.803 1.06 ± 1.02 1.55 ± 0.35 0.504 1.09 ± 1.04 0.81 ± 0.60 0.485

630.4 ± 149.3 0.676 638.2 ± 151.8 707.0 ± 149.9 0.527 638.2 ± 151.6 658.3 ± 158.0 0.737 Intraoperative blood loss (mL) 545.8 ± 515.5 736.9 ± 549.1 0.044 619.5 ± 535.7 485.0 ± 558.6 0.726 612.3 ± 531.8 674.3 ± 596.0 0.769 SCC value 1.14 ± 1.17 0.96 ± 0.67 0.803 1.06 ± 1.02 1.55 ± 0.35 0.504 1.09 ± 1.04 0.81 ± 0.60 0.485 Ce cervical esophagus, Ut upper thoracic esophagus, Mt middle thoracic esophagus, Lt lower thoracic esophagus, Ae abdominal esophagus Prognostic factors for CSS in non-elderly patients Because the total number of non-elderly patients was small, we excluded analysis of PDW and MPV. Among non-elderly patients, univariate analysis demonstrated that pStage (HR 4.395; 95 % CI 2.059–9.933, p = 0.0001), tumor size (HR 5.275; 95 % CI 1.849–22.162; p = 0.0009), and a high RDW (HR 3.654; 95 % CI 1.716–8.241; p = 0.0007) were significantly associated with a worse prognosis (Table 4). Multivariate analysis demonstrated that pStage (HR 2.775; 95 % CI 1.247–6.617; p = 0.0120), and a high RDW (HR 2.759; 95 % CI 1.282–6.284; p = 0.0092) were independent risk factors for a worse prognosis in non-elderly patients.Table 4 Prognostic factors for cancer-specific survival in 97 non-elderly patients with esophageal cancer after a curative esophagectomy

age (HR 2.775; 95 % CI 1.247–6.617; p = 0.0120), and a high RDW (HR 2.759; 95 % CI 1.282–6.284; p = 0.0092) were independent risk factors for a worse prognosis in non-elderly patients.Table 4 Prognostic factors for cancer-specific survival in 97 non-elderly patients with esophageal cancer after a curative esophagectomy Variables Patients (n = 97) Category or characteristics Univariate Multivariate HR 95 % CI p value HR 95 % CI p value Gender 9/88 (Female/male) 0.526 0.202–1.794 0.272 pStage 60/37 (I, II/III) 4.395 2.059–9.933 0.0001 2.775 1.247–6.617 0.012 Tumor size 33/64 (<3/≥3) 5.275 1.849–22.162 0.0009 2.716 0.862–12.018 0.0919 Operation time 34/63 (<600/≥600) 0.487 0.228–1.027 0.059 Intraoperative blood loss 46/51 (<500/≥500) 1.415 0.670–3.116 0.365 SCC value 76/21 (<1.5/≥1.5) 1.051 0.351–2.575 0.92 RDW 61/36 (<50/≥50) 3.654 1.716–8.241 0.0007 2.759 1.282–6.284 0.0092 RDW and CSS in non-elderly patients Non-elderly patients with a high RDW had a significantly poorer prognosis in terms of CSS than those with a low RDW (p = 0.0003) (Fig. 2).Fig. 2 Postoperative cancer-specific survival based on RDW in 97 non-elderly patients with esophageal cancer

Variables Patients (n = 97) Category or characteristics Univariate Multivariate HR 95 % CI p value HR 95 % CI p value Gender 9/88 (Female/male) 0.526 0.202–1.794 0.272 pStage 60/37 (I, II/III) 4.395 2.059–9.933 0.0001 2.775 1.247–6.617 0.012 Tumor size 33/64 (<3/≥3) 5.275 1.849–22.162 0.0009 2.716 0.862–12.018 0.0919 Operation time 34/63 (<600/≥600) 0.487 0.228–1.027 0.059 Intraoperative blood loss 46/51 (<500/≥500) 1.415 0.670–3.116 0.365 SCC value 76/21 (<1.5/≥1.5) 1.051 0.351–2.575 0.92 RDW 61/36 (<50/≥50) 3.654 1.716–8.241 0.0007 2.759 1.282–6.284 0.0092 RDW and CSS in non-elderly patients Non-elderly patients with a high RDW had a significantly poorer prognosis in terms of CSS than those with a low RDW (p = 0.0003) (Fig. 2).Fig. 2 Postoperative cancer-specific survival based on RDW in 97 non-elderly patients with esophageal cancer Relationships between RDW, PDW, MPV and clinicopathological features in elderly patients There was a significant relationship between RDW and operation time (p = 0.015) (Table 5), and between PDW and lymph node metastasis (p = 0.022). However, there were no significant associations between any of the clinicopathological features and MPV.Table 5 Relationships between RDW, PDW, MPV, and clinicopathological features in 47 elderly patients with esophageal cancer

operation time (p = 0.015) (Table 5), and between PDW and lymph node metastasis (p = 0.022). However, there were no significant associations between any of the clinicopathological features and MPV.Table 5 Relationships between RDW, PDW, MPV, and clinicopathological features in 47 elderly patients with esophageal cancer Characteristics Total patients RDW PDW MPV <50 (n = 33) ≥50 (n = 14) p value <15.3 (n = 45) ≥15.3 (n = 2) p value <11.5 (n = 42) ≥11.5 (n = 5) p value Age (years) 75.1 ± 4.4 75.6 ± 4.2 0.634 75.3 ± 4.4 73.0 ± 2.8 0.461 75.4 ± 4.5 73.8 ± 2.3 0.781 Gender 0.088 0.107 0.054 Male 41 27 14 40 1 38 3 Female 6 6 0 5 1 4 2 Location of tumor 0.652 0.639 0.959 Ce 1 1 0 1 0 1 0 Ut 6 3 3 6 0 5 1 Mt 17 12 5 17 0 15 2 Lt 21 15 6 19 2 19 2 Ae 2 2 0 2 0 2 0 Tumor size (mm) 4.3 ± 2.2 3.9 ± 1.9 0.496 4.2 ± 2.1 5.8 ± 0.7 0.300 4.3 ± 2.1 3.9 ± 2.3 0.652 Vessel invasion 0.870 0.108 0.466 Negative 18 8 26 0 24 2 Positive 15 6 19 2 18 3 Lymphatic invasion 0.978 0.214 0.903 Negative 14 6 20 0 18 2 Positive 19 8 25 2 24 3 Differentiation 0.866 0.399 0.717 Well 12 5 17 0 15 2 Moderate 17 8 23 2 22 3 Poor 4 1 5 0 5 0 Depth of tumor 0.063 0.38 0.717 T1a–1b 20 14 6 20 0 17 3 2 6 6 0 6 0 6 0 3 19 13 6 17 2 17 2 4a–4b 13 0 2 2 0 2 0 Lymph node metastasis 0.598 0.022 0.411 N0 77 18 5 23 0 22 1 N1 42 8 6 14 0 12 2 N2 13 5 2 6 1 6 1 N3 12 2 1 2 1 2 1 Pathological stage 0.323 0.158 0.692 1a–1b 56 14 3 17 0 16 1 2a–2b 34 9 4 13 0 11 2 3a–3c 54 10 7 15 2 15 2 Operation time (min) 675.4 ± 167.3 524.6 ± 227.6 0.015 624.4 ± 194.1 769.0 ± 306.9 0.316 625.4 ± 199.1 673.2 ± 197.7 0.693 Intraoperative blood loss (mL) 558.1 ± 617.3 907.5 ± 864.4 0.062 670.0 ± 717.7 485.0 ± 615.2 0.722 678.9 ± 730.2 522.0 ± 533.0 0.322 SCC value 1.25 ± 1.15 2.12 ± 4.39 0.145 1.44 ± 2.60 3.00 ± 0.14 0.405 1.50 ± 2.67 1.56 ± 1.36 0.519

524.6 ± 227.6 0.015 624.4 ± 194.1 769.0 ± 306.9 0.316 625.4 ± 199.1 673.2 ± 197.7 0.693 Intraoperative blood loss (mL) 558.1 ± 617.3 907.5 ± 864.4 0.062 670.0 ± 717.7 485.0 ± 615.2 0.722 678.9 ± 730.2 522.0 ± 533.0 0.322 SCC value 1.25 ± 1.15 2.12 ± 4.39 0.145 1.44 ± 2.60 3.00 ± 0.14 0.405 1.50 ± 2.67 1.56 ± 1.36 0.519 Ce cervical esophagus, Ut upper thoracic esophagus, Mt middle thoracic esophagus, Lt lower thoracic esophagus, Ae abdominal esophagus Prognostic factors for CSS in elderly patients Because the total number of elderly patients was small, we excluded analysis of PDW and MPV. Univariate analysis demonstrated that pStage (p = 0.0008) was the only significant risk factor for a poor prognosis in elderly patients (Table 6). RDW showed no significant association with postoperative CSS in elderly patients (p = 0.664) (Fig. 3).Table 6 Prognostic factors for cancer-specific survival in 47 elderly patients with esophageal cancer after a curative esophagectomy Variables Patients (n = 47) Category or characteristics Univariate Multivariate HR 95 % CI p value HR 95 % CI p value Gender 6/41 (Female/male) 1.946 0.546–12.390 0.338 pStage 30/17 (I, II/III) 5.303 2.001–15.063 0.0008 5.303 2.001–15.063 0.0008 Tumor size 12/35 (<3/≥3) 1.496 0.535–5.288 0.463 Operation time 18/29 (<600/≥600) 0.54 0.206–1.372 0.193 Intraoperative blood loss 25/22 (<500/≥500) 0.666 0.251–1.709 0.396 SCC value 36/11 (<1.5/≥1.5) 2.139 0.731–5.657 0.156 RDW 33/14 (<50/≥50) 1.243 0.431–3.216 0.669 Fig. 3 Postoperative cancer-specific survival based on RDW in 47 elderly patients with esophageal cancer

Variables Patients (n = 47) Category or characteristics Univariate Multivariate HR 95 % CI p value HR 95 % CI p value Gender 6/41 (Female/male) 1.946 0.546–12.390 0.338 pStage 30/17 (I, II/III) 5.303 2.001–15.063 0.0008 5.303 2.001–15.063 0.0008 Tumor size 12/35 (<3/≥3) 1.496 0.535–5.288 0.463 Operation time 18/29 (<600/≥600) 0.54 0.206–1.372 0.193 Intraoperative blood loss 25/22 (<500/≥500) 0.666 0.251–1.709 0.396 SCC value 36/11 (<1.5/≥1.5) 2.139 0.731–5.657 0.156 RDW 33/14 (<50/≥50) 1.243 0.431–3.216 0.669 Fig. 3 Postoperative cancer-specific survival based on RDW in 47 elderly patients with esophageal cancer Discussion Esophageal cancer is primarily a disease of the elderly, with a peak in incidence in the eighth decade of life, and the elderly population is rapidly increasing worldwide [19]. Despite recent advances in early detection, surgical techniques, and chemoradiation therapies, the prognosis of esophageal cancer remains poor. Surgery is the mainstay of treatment for this malignancy, but a considerable proportion of patients with advanced esophageal cancer develop recurrence, even after curative resection. Therefore, reliable prognostic factors that permit more accurate patient stratification are needed to improve clinical decision making for this malignancy. In addition, esophageal cancer is the eighth most common incident cancer and sixth most common cause of cancer death worldwide [20]. It occurs predominantly in elderly people, the average age at the time of diagnosis continues to rise, with a peak in incidence between 70 and 75 years of age [21]. CSS is an important outcome measure in elderly patients, as they are more likely to die from other age-related diseases such as cardiovascular, renal, and pulmonary diseases. As few patients actually died from causes other than cancer in this analysis, the data had a limited impact on overall survival. In addition we divided the patients into two groups in order to calculate the tendency of prognostic value by age, because the correlation between prognostic value and age was unknown until now.

few patients actually died from causes other than cancer in this analysis, the data had a limited impact on overall survival. In addition we divided the patients into two groups in order to calculate the tendency of prognostic value by age, because the correlation between prognostic value and age was unknown until now. RDW, which is a measure of heterogeneity in erythrocyte size, is routinely examined as part of the CBC test. In the development of iron deficiency, an elevated RDW usually precedes other blood abnormalities, such as a low red blood cell (RBC) count, a low mean corpuscular volume, and low hemoglobin levels [22]. Furthermore, RDW is a more sensitive screening marker for iron deficiency than serum ferritin level, transferrin saturation, or serum iron level [23]. Since the variability of RBC size increases before overt anemia, elevated RDW is a sensitive and specific indicator of early iron deficiency. In this study, we investigated the relationship between hematological parameters and CSS in esophageal cancer patients. Among these hematological parameters, we included RDW as a marker of several conditions including anemia, inflammation, and early nutritional deficiencies [24]. Univariate and multivariate analyses demonstrated that a high RDW was independently associated with poor prognosis in esophageal cancer patients. These findings reflect the widely accepted hypothesis that cancer is both a cause and a result of chronic inflammation [25, 26].

ion, and early nutritional deficiencies [24]. Univariate and multivariate analyses demonstrated that a high RDW was independently associated with poor prognosis in esophageal cancer patients. These findings reflect the widely accepted hypothesis that cancer is both a cause and a result of chronic inflammation [25, 26]. Our findings also reveal that a high RDW is potentially an independent risk factor for a worse prognosis in non-elderly patients, but not in elderly patients. This was unexpected, but could reflect the prevalence of anemia and malnutrition in the latter. This would in turn lead to an elevated RDW, which would reduce its prognostic significance for cancer in elderly patients.

n independent risk factor for a worse prognosis in non-elderly patients, but not in elderly patients. This was unexpected, but could reflect the prevalence of anemia and malnutrition in the latter. This would in turn lead to an elevated RDW, which would reduce its prognostic significance for cancer in elderly patients. Platelets play a key role in the coagulation cascade. They also release growth factors that can contribute to tumor progression and metastasis, in part by mediating immunosuppression [16]. Changes in platelet count and platelet function have been identified as part of a paraneoplastic syndrome in many cancers [27], and a high platelet count was found to be closely associated with TNM stage, metastasis, and a high risk of recurrence in many types of cancer [28, 29]. Platelets release angiogenic growth factors, adhere to tumor microvessels, and undergo extravasation via increased vascular permeability, a process that leads to platelet activation. Increased platelet production is associated with bigger platelets, which could affect the PDW, and thus the PDW in turn may predict clinical outcome in cancer patients. However, to the best of our knowledge, the relationship between PDW and esophageal cancer has not yet been clarified. We therefore focused on PDW and CSS in esophageal cancer patients. However, we could not analyze the relationship between PDW and prognostic factors, because only a small proportion of patients had an elevated PDW, and it had a weak predictive value for clinical outcome. Further study of the predictive value of PDW is needed with a larger number of patients.

other groups, resulting in a recovery time of more than 100 days regardless of rash grade, compared with the before onset groups who had median time to recovery of 35–51 days depending on rash grade.Fig. 1 Time to recovery according to time to treatment initiation: grade 1 rash (a), grade 2 rash (b), grade ≥3 rash (c) Median time to recovery stratified by steroid strength and by grade of rash is shown in Fig. 2. Time to recovery in patients who were changed to strong- or higher-rank steroids (medium to strong) was longer than that observed in other groups. Median time to recovery of rash in the medium- to strong-rank steroids subgroup was 47, 98, and 103 days for those with grade 1, 2, and 3 rash, respectively; this is compared with 39, 53, and 73 days median recovery for grade 1, 2, and 3 rash, respectively, in patients in the strong-rank steroid subgroup. In patients who received medium-rank steroids for grade 1, 2, or 3 rash, 21.9 %, 36.5 %, and 47.5 % of patients, respectively, needed to have their treatment changed to strong-rank steroids.Fig. 2 Class effects of steroids for rash management: grade 1 rash (a), grade 2 rash (b), and grade ≥3 rash (c). Medium: patients treated with medium- or weak-rank steroids; medium to strong: patients initially treated with medium- or weak-rank steroids then changed to strong- or higher-rank steroids; strong: patients treated with strong- or higher-rank steroids

sophageal cancer patients. However, we could not analyze the relationship between PDW and prognostic factors, because only a small proportion of patients had an elevated PDW, and it had a weak predictive value for clinical outcome. Further study of the predictive value of PDW is needed with a larger number of patients. MPV is a platelet volume index and an early marker of platelet activation [1]. Elevated MPV is closely associated with the severity and prognosis of malignant tumors, as well as ischemic cardiovascular disorders [30]. Neoplasms are usually accompanied by thrombocytosis, which may lead to an elevated MPV and hence an increased risk of metastasis and a poor prognosis, as the tumor can produce or stimulate the production of cytokines including interleukins, interferon-, and tumor necrosis factor [31]. However, the prognostic effect of MPV has not been clarified in esophageal cancer patients after curative resection. Therefore, we focused on MPV and CSS in esophageal cancer patients but could not analyze the relationship between MPV and prognostic factor. As we determined the cut-off values for RDW, PDW, and MPV to our routine laboratory data, the number of patients in the high MPV group (n = 12) was too small to compare with another group (n = 132), similar to PDW. Unfortunately, in this study we could not evaluate the prognostic value of MPV. Although platelet count is closely related to a variety of pathophysiological conditions, such as myocardial infarction, stroke, chronic inflammation, and poor nutrition, these conditions are frequently found in the elderly regardless of whether they have cancer [32, 33]. Furthermore, platelets are known to facilitate tumor progression by protecting them from immune responses. The present study may have failed to demonstrate a prognostic significance for MPV and PDW, because the number of patients was too small to evaluate the prognostic value for esophageal cancer. However, several clinical studies have demonstrated that MPV was elevated in patients with various carcinomas, although other studies have contradicted this [11, 34]. Further investigations are required to elucidate the precise mechanisms through which circulating platelets affect the prognosis of esophageal cancer patients.

r, several clinical studies have demonstrated that MPV was elevated in patients with various carcinomas, although other studies have contradicted this [11, 34]. Further investigations are required to elucidate the precise mechanisms through which circulating platelets affect the prognosis of esophageal cancer patients. In this study, we confirmed that the RDW was associated with the CSS of esophageal cancer patients after curative esophagectomy. It is particularly noteworthy that a high RDW was a significant and independent predictor of poor survival. Furthermore, in non-elderly patients, a high RDW was also an independent risk factor for a worse prognosis. We showed that a high RDW was associated with poor survival; the mean RDW was 48.6 ± 6.9 in this analysis. A high RDW may by correlated with tumor depth and pathological stage in advanced-staged esophageal cancer patients. However, this study was retrospective analysis with a small sample size. As the RDW is convenient, cost-effective and readily available as part of the routine CBC, it could act as a marker of survival in this malignancy. Thus, larger prospective studies are needed to confirm these preliminary results.

cancer patients. However, this study was retrospective analysis with a small sample size. As the RDW is convenient, cost-effective and readily available as part of the routine CBC, it could act as a marker of survival in this malignancy. Thus, larger prospective studies are needed to confirm these preliminary results. It is important to explore the risk factors for postoperative complications after esophagectomy, because they are more likely to cause complications when comparing esophagectomy with other gastrointestinal surgery. However, we could not demonstrate the predictive significance of RDW, PDW, and MPV of postoperative complication risks in this study (data not shown). We think this is because many of the patients with esophageal cancer had preoperative complications. In addition, we observed many patients who showed a high RDW after recurrence. However, we were unable to demonstrate a relationship efficacy between chemoradiotherapy and RDW, PDW or MPV after recurrence. Further studies are warranted to confirm the changes in before and after surgery effects after recurrence.

ations. In addition, we observed many patients who showed a high RDW after recurrence. However, we were unable to demonstrate a relationship efficacy between chemoradiotherapy and RDW, PDW or MPV after recurrence. Further studies are warranted to confirm the changes in before and after surgery effects after recurrence. In conclusion, we confirmed that a high RDW was associated with the CSS of esophageal cancer patients after curative esophagectomy. However, a number of potential study limitations need to be taken into consideration. The cut-off value should be set by ROC analysis rather than by routine laboratory value in the data analysis. However, we performed analysis by routine laboratory values because the correct cut-off values of RDW, MPV, and PDW were uncertain, and we confirmed survival data using the hematological parameters of our routine laboratory values in this analysis. Therefore, these data should be carefully interpreted. Furthermore, this was a retrospective, single-institution design study with a small sample size and short follow-up period. Thus, larger prospective studies are required to elucidate the precise mechanisms that relate RDW, PDW and MPV to survival in esophageal cancer patients. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

Introduction Chemotherapy-induced nausea and vomiting (CINV) were the first and second most distressing adverse events in the past [1]. Nausea and vomiting severely impair the patient’s quality of life, which may lead to the discontinuance of therapy. However, the rank order of nausea and vomiting has dropped [2] since the development of 5-hydroxytryptamine-3 (5-HT3) receptor antagonists and the prevalence of the American Society of Clinical Oncology (ASCO) clinical practice guideline for antiemesis [3]. Subsequently, with the development of novel classes of antiemetic drugs such as neurokinin 1(NK1) receptor antagonists, including aprepitant, and palonosetron, a second-generation 5-HT3 receptor antagonist, the antiemetic guideline has been revised by several international societies for clinical oncology, including ASCO [4], Multinational Association of Supportive Care in Cancer (MASCC) [5], National Comprehensive Cancer Network (NCCN) [6], and Japanese Society of Clinical Oncology (JSCO) [7].

n 5-HT3 receptor antagonist, the antiemetic guideline has been revised by several international societies for clinical oncology, including ASCO [4], Multinational Association of Supportive Care in Cancer (MASCC) [5], National Comprehensive Cancer Network (NCCN) [6], and Japanese Society of Clinical Oncology (JSCO) [7]. However, guideline-consistent antiemetic medication is not always widely used in clinical practice, a so-called evidence–practice gap [8]. Gomez et al. [9] reported, in patients who received high emetic risk chemotherapy (HEC) or moderate emetic risk chemotherapy (MEC), that the use of 5-HT3 receptor antagonist with dexamethasone is 60–90 %, whereas the use of a NK1 antagonist is much less common (<10 %). Hori et al. [10] also reported by a nationwide survey of 9978 patients receiving 81,739 chemotherapy cycles from 39 Japanese hospitals that the rates of adherence to the JSCO guideline during acute and delayed periods are 28.1 % and 9.7 %, respectively, for HEC, and 7.2 % and 6.9 %, respectively, for MEC.

mmon (<10 %). Hori et al. [10] also reported by a nationwide survey of 9978 patients receiving 81,739 chemotherapy cycles from 39 Japanese hospitals that the rates of adherence to the JSCO guideline during acute and delayed periods are 28.1 % and 9.7 %, respectively, for HEC, and 7.2 % and 6.9 %, respectively, for MEC. It has been demonstrated that adherence to the antiemetic guideline improves the control of CINV [11–13]. Chan et al. [11] reported in 361 breast cancer patients receiving anthracycline-based chemotherapy that the adherence to the antiemetic guideline is 57.9 % and that the complete control of CINV is better in the guideline-consistent group than in the guideline-inconsistent group, indicating that adherent patients were more likely to achieve complete control of CINV [adjusted odds ratio (OR) 1.74, 95 % confidence interval (CI) 1.01–3.01, P = 0.048]. Aapro et al. [12] also reported in 991 patients receiving the first cycle of HEC or MEC that the complete response (no vomiting and no rescue) is significantly better in the guideline-consistent group than in the guideline-inconsistent group (59.9 % vs. 50.7 %, P = 0.008), resulting in the adjusted odds ratio of 1.43 (95 % CI 1.04–1.97, P = 0.027). We also previously reported in 125 colorectal cancer patients receiving the first cycle of MEC such as levofolinate, fluorouracil, oxaliplatin (mFOLFOX6) and levofolinate, fluorouracil, irinotecan (FOLFIRI) regimen that the complete control of nausea but not vomiting is significantly better in the guideline-consistent group than in the guideline-inconsistent (lack of dexamethasone on days 2 and 3) group (74 % vs. 56 % for nausea, P < 0.05; 94 % vs. 91 % for vomiting) [14].

levofolinate, fluorouracil, irinotecan (FOLFIRI) regimen that the complete control of nausea but not vomiting is significantly better in the guideline-consistent group than in the guideline-inconsistent (lack of dexamethasone on days 2 and 3) group (74 % vs. 56 % for nausea, P < 0.05; 94 % vs. 91 % for vomiting) [14]. On the other hand, cancer chemotherapy is currently shifting from inpatient admission to the outpatient setting. Chemotherapy-induced vomiting is almost preventable in the outpatient setting by the addition of NK1 receptor antagonists and 5-HT3 receptor antagonists to the standard medication, although nausea remains a distressing adverse drug reaction [15, 16]. In the present study, the antiemetic medication consistent with the JSCO guideline has been implemented in our outpatient chemotherapy clinic, and the rates of complete protection of CINV were subsequently investigated. Risk analysis for overall nausea was also carried out.

a distressing adverse drug reaction [15, 16]. In the present study, the antiemetic medication consistent with the JSCO guideline has been implemented in our outpatient chemotherapy clinic, and the rates of complete protection of CINV were subsequently investigated. Risk analysis for overall nausea was also carried out. Patients and methods Patients There were 9590 visits for cancer chemotherapy in our outpatient chemotherapy clinic during the 2 years from January 2013 to December 2014; among these, chemotherapy was carried out in 8206 visits, thereby indicating a 14.4 % discontinuance rate. The actual number of patients counted by the patient ID number was 779 and the number of chemotherapy cycles was 5577. Health professionals such as pharmacists and nurses were in charge of provision of drug information and safety precaution in daily life and of monitoring adverse drug reactions to all patients in our outpatient chemotherapy clinic. Patient consultation was carried out in 8206 visits (100 %), but the CINV data were obtained from 5511 chemotherapy cycles. Thus, data from 98.8 % of the chemotherapy cycles were subjected to analysis in the present study. The present study was carried out in accordance with the guidelines for the care for human study adopted by the Ethics Committee of the Gifu Graduate School of Medicine, and notified by the Japanese Government (approved no. 26-153 of the Institutional Review Board).

Patients and methods Patients There were 9590 visits for cancer chemotherapy in our outpatient chemotherapy clinic during the 2 years from January 2013 to December 2014; among these, chemotherapy was carried out in 8206 visits, thereby indicating a 14.4 % discontinuance rate. The actual number of patients counted by the patient ID number was 779 and the number of chemotherapy cycles was 5577. Health professionals such as pharmacists and nurses were in charge of provision of drug information and safety precaution in daily life and of monitoring adverse drug reactions to all patients in our outpatient chemotherapy clinic. Patient consultation was carried out in 8206 visits (100 %), but the CINV data were obtained from 5511 chemotherapy cycles. Thus, data from 98.8 % of the chemotherapy cycles were subjected to analysis in the present study. The present study was carried out in accordance with the guidelines for the care for human study adopted by the Ethics Committee of the Gifu Graduate School of Medicine, and notified by the Japanese Government (approved no. 26-153 of the Institutional Review Board). Adherence to the Japanese guideline for the use of antiemetic drugs According to the JSCO clinical practice guideline for antiemesis, the use of antiemetic drugs was promoted for prevention of CINV: the combination of 5-HT3 receptor antagonist, aprepitant, and dexamethasone was used before chemotherapy and a combination of aprepitant and dexamethasone was provided on days 2 and 3, and dexamethasone on day 4 for high emetic risk chemotherapy (HEC), a combination of 5-HT3 receptor antagonist and dexamethasone was taken before chemotherapy, and dexamethasone was prescribed on days 2 and 3 for moderate emetic risk chemotherapy (MEC), and dexamethasone was administered only before chemotherapy for low emetic risk chemotherapy. No routine antiemetic drug was provided for prevention of minimum risk chemotherapy. Health professionals made extensive efforts in facilitating the use of appropriate antiemetic premedication by inclusion of antiemetic regimens into the prescription order for chemotherapy regimens or by proposing the prescription of antiemetic drugs to physicians. Adherence to the Japanese antiemetic guideline in both acute and delayed periods was evaluated. In the case of the dose of dexamethasone, the guideline recommends 8 mg/day in the delayed period for HEC and MEC. In the present study, however, 4 mg/day on days 2–3 for MEC was regarded as positive for adherence because previous findings indicated that the control of delayed nausea is improved by 20 % by the addition of 4 mg/day of dexamethasone on days 2–3, when compared with no treatment with dexamethasone in patients receiving MEC [14].

sent study, however, 4 mg/day on days 2–3 for MEC was regarded as positive for adherence because previous findings indicated that the control of delayed nausea is improved by 20 % by the addition of 4 mg/day of dexamethasone on days 2–3, when compared with no treatment with dexamethasone in patients receiving MEC [14]. Evaluation of chemotherapy-induced nausea All patients were provided with a checklist for daily check of adverse events on their first visit to the outpatient chemotherapy clinic. Using the checklist, patients checked daily their nausea by numeric rating scale (0–10) and the number of vomiting episodes up to 7 days after chemotherapy. Pharmacists and nurses recorded the control of nausea and vomiting on the electronic medical record after verifying the data or hearing results from patients on the next visit. Complete protection from nausea (NRS scale <1) and vomiting (no episode) during acute (within 24 h after chemotherapy) and delayed (during 2–7 days after chemotherapy) periods was assessed in patients receiving the first cycle of chemotherapy or in those with overall cycles of chemotherapy.

patients on the next visit. Complete protection from nausea (NRS scale <1) and vomiting (no episode) during acute (within 24 h after chemotherapy) and delayed (during 2–7 days after chemotherapy) periods was assessed in patients receiving the first cycle of chemotherapy or in those with overall cycles of chemotherapy. Risk analysis for chemotherapy-induced nausea during overall period Demographics of patients were compared between patients who revealed acute and delayed nausea and those who showed complete protection from nausea. Subsequently, uni- as well as multivariate logistic regression analyses were carried out to determine the risk for incomplete protection from for nausea or vomiting during the overall period. Odds ratio (OR) and 95 % confidence interval (CI) were determined. The cutoff value of age was determined by the Youden index or the distance method in the receiver operating characteristic curve (ROC) analysis. In the Youden index, cutoff age was estimated from the maximum value of (sensitivity + specificity − 1), whereas in the distance method, cutoff value was predicted from the minimum value for square root [(1−sensitivity)2 + (1−specificity)2)], according to the method described earlier [17, 18].

tic curve (ROC) analysis. In the Youden index, cutoff age was estimated from the maximum value of (sensitivity + specificity − 1), whereas in the distance method, cutoff value was predicted from the minimum value for square root [(1−sensitivity)2 + (1−specificity)2)], according to the method described earlier [17, 18]. Statistical analyses Data were analyzed using IBM SPSS Statistics ver. 22 (IBM Japan Services, Tokyo, Japan) and Graph Pad Prism version 6.0 (Graph Pad Software, San Diego, CA, USA). Parametric variables were analyzed using the t test, and nonparametric data were analyzed by the Mann–Whitney U test or the chi-square test. Multiple comparisons for the control of nausea and vomiting were carried out by Kruskal–Wallis test followed by Scheffe’s test. A P value less than 0.05 was considered statistically significant.

ables were analyzed using the t test, and nonparametric data were analyzed by the Mann–Whitney U test or the chi-square test. Multiple comparisons for the control of nausea and vomiting were carried out by Kruskal–Wallis test followed by Scheffe’s test. A P value less than 0.05 was considered statistically significant. Results Demographics of patients Demographics of the patients are shown in Table 1. Seven hundred and seventy-nine patients received 5511 chemotherapy cycles during 2 years from January 2013 to December 2014 in our outpatient chemotherapy clinic. The average chemotherapy cycle was 8.3 cycles. The most common type of cancer was colorectal cancer (26 % of patients and 35 % of all chemotherapy cycles), followed by lung cancer (14 % and 12 %), breast cancer (14 % and 17 %), gastric cancer (13 % and 12 %), liver/gallbladder/pancreas cancer (10 % and 9 %), hematological cancer (9 % and 5 %), gynecological cancer (6 % and 5 %), and head and neck cancer (3 % and 2 %). The emetic risk of the chemotherapy regimens included HEC (291 cycles, 5 %), MEC (2184 cycles, 40 %), low risk (2162 cycles, 39 %), and minimum risk (874 cycles, 16 %).Table 1 Demographics of patients

d 9 %), hematological cancer (9 % and 5 %), gynecological cancer (6 % and 5 %), and head and neck cancer (3 % and 2 %). The emetic risk of the chemotherapy regimens included HEC (291 cycles, 5 %), MEC (2184 cycles, 40 %), low risk (2162 cycles, 39 %), and minimum risk (874 cycles, 16 %).Table 1 Demographics of patients Number of patients (male/female) 779 (391/388) Age (average, min/max) 63.4 (18/88) Body surface area (average, SD) 1.56 ± 0.40 Serum creatinine (mg/dl, average, SD) 0.72 ± 0.27 Number of chemotherapy courses 5511 Cancer type Number of patients % Number of courses % Colorectal 201 25.8 1923 34.9 Lung 107 13.7 645 11.7 Breast 105 13.5 935 17.0 Gastric 101 13.0 680 12.3 Liver/gallbladder/pancreas 81 10.4 510 9.3 Hematological 73 9.4 270 4.9 Gynecological 49 6.3 259 4.7 Head and neck 26 3.3 117 2.1 Esophageal 15 1.9 49 0.9 Urological 10 1.3 89 1.6 Brain 9 1.2 25 0.5 Sarcoma 1 0.1 1 0.02 Dermatological 1 0.1 8 0.1 Average chemotherapy courses (min/max) 8.3 (1/91) Emetic risk of chemotherapy n % High 291 5.3 Moderate 2184 39.6 Low 2162 39.2 Minimum 874 15.9

nagement: grade 1 rash (a), grade 2 rash (b), and grade ≥3 rash (c). Medium: patients treated with medium- or weak-rank steroids; medium to strong: patients initially treated with medium- or weak-rank steroids then changed to strong- or higher-rank steroids; strong: patients treated with strong- or higher-rank steroids The trend of patients in the medium to strong subgroup having longer recovery time than those responding to medium-rank steroids or those initiated on strong-rank steroids was also seen in patients who did not have erlotinib dose reduction or interruption (Supplementary Fig. 1). In patients with grade 2 rash (the most common grade of rash), there was again a trend of patients in the medium to strong subgroup having longer recovery times than the other two groups, regardless of time of steroid initiation (Supplementary Fig. 2). In patients with grade 2 rash, there was a trend of earlier initiation of steroid, resulting in shorter recovery time, regardless of steroid rank.

Gynecological 49 6.3 259 4.7 Head and neck 26 3.3 117 2.1 Esophageal 15 1.9 49 0.9 Urological 10 1.3 89 1.6 Brain 9 1.2 25 0.5 Sarcoma 1 0.1 1 0.02 Dermatological 1 0.1 8 0.1 Average chemotherapy courses (min/max) 8.3 (1/91) Emetic risk of chemotherapy n % High 291 5.3 Moderate 2184 39.6 Low 2162 39.2 Minimum 874 15.9 Control of nausea and vomiting The rates of complete protection from nausea and vomiting during acute and delayed periods in the first cycle and overall cycles are shown in Fig. 1. In the first cycle of chemotherapy, the rate of adherence to the Japanese clinical practice guideline for prevention of CINV ranged from 76 % (HEC) to 99 % (low risk). Under such a condition, patients receiving HEC showed poor control of nausea during acute and delayed periods, although vomiting was favorably controlled (83–85 % of complete protection). The rates of complete protection from acute and delayed nausea were 61 % and 44 %, respectively, for HEC, and 87 % and 68 %, respectively, for MEC, in which the rates increased in a manner dependent on the emetic risk of the chemotherapy. In the overall cycles, the control of nausea in HEC was greatly improved, in which the complete protection from acute and delayed nausea was 77 % and 62 %, respectively.Fig. 1 Complete protection from nausea and vomiting during acute and delayed periods in patients receiving high emetic risk chemotherapy (HEC), moderate emetic risk chemotherapy (MEC), low risk, or minimum risk of chemotherapy as the first cycle or the overall cycles in the outpatient chemotherapy clinic. Adherence to the Japanese antiemetic guideline is shown at bottom. **P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test

etic risk chemotherapy (HEC), moderate emetic risk chemotherapy (MEC), low risk, or minimum risk of chemotherapy as the first cycle or the overall cycles in the outpatient chemotherapy clinic. Adherence to the Japanese antiemetic guideline is shown at bottom. **P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test Comparison of demographics between patients who showed no nausea and those with nausea To determine the risk of chemotherapy-induced nausea, the demographics of patients were compared between those with and without complete protection from nausea in 608 patients who received the first cycle of chemotherapy. As shown in Table 2, significant differences in gender, age, proportion of HEC/MEC and anthracycline/cyclophosphamide (A/C) regimen, and type of cancer were observed between the two groups. Females were more often affected (67 % vs. 48 %, P < 0.001), patient age was younger (56.7 vs. 63.4 years), and the proportion of A/C regimen (18 % vs. 2 %, P < 0.001) and HEC/MEC (64 % vs. 36 %) in patients without complete protection from nausea. Moreover, the percentage of breast cancer patients was significantly higher (31 % vs. 18 %, P < 0.001) in patients with nausea than those without it.Table 2 Comparison of demographics of patients with nausea during overall period in 608 patients who received the first cycle of chemotherapy With nausea during overall period (n = 158) Without nausea during overall period (n = 450) P Ratio of female (female/male) 67.1 (106/52) 47.8 (215/235) <0.001a Age (years) 56.7 (18–84) 63.4 (35–88) <0.001a Height (cm) 160.1 ± 7.5 160.3 ± 8.5 0.775c

Comparison of demographics between patients who showed no nausea and those with nausea To determine the risk of chemotherapy-induced nausea, the demographics of patients were compared between those with and without complete protection from nausea in 608 patients who received the first cycle of chemotherapy. As shown in Table 2, significant differences in gender, age, proportion of HEC/MEC and anthracycline/cyclophosphamide (A/C) regimen, and type of cancer were observed between the two groups. Females were more often affected (67 % vs. 48 %, P < 0.001), patient age was younger (56.7 vs. 63.4 years), and the proportion of A/C regimen (18 % vs. 2 %, P < 0.001) and HEC/MEC (64 % vs. 36 %) in patients without complete protection from nausea. Moreover, the percentage of breast cancer patients was significantly higher (31 % vs. 18 %, P < 0.001) in patients with nausea than those without it.Table 2 Comparison of demographics of patients with nausea during overall period in 608 patients who received the first cycle of chemotherapy With nausea during overall period (n = 158) Without nausea during overall period (n = 450) P Ratio of female (female/male) 67.1 (106/52) 47.8 (215/235) <0.001a Age (years) 56.7 (18–84) 63.4 (35–88) <0.001a Height (cm) 160.1 ± 7.5 160.3 ± 8.5 0.775c Body weight (kg) 55.0 ± 10.7 55.8 ± 10.8 0.464c Serum creatinine (mg/dl) 0.69 ± 0.23 0.72 ± 0.23 0.170c n % n % P Regimens/anticancer drug A/C 28 17.7 9 2.0 <0.001a CHOP 3 1.9 5 1.1 0.733a Oxaliplatin 29 18.4 61 13.6 0.183a Irinotecan 17 10.8 35 7.8 0.323a Carboplatin 11 7.0 29 6.4 0.969a Cyclophosphamide 9 5.7 5 1.1 <0.001a

Height (cm) 160.1 ± 7.5 160.3 ± 8.5 0.775c Body weight (kg) 55.0 ± 10.7 55.8 ± 10.8 0.464c Serum creatinine (mg/dl) 0.69 ± 0.23 0.72 ± 0.23 0.170c n % n % P Regimens/anticancer drug A/C 28 17.7 9 2.0 <0.001a CHOP 3 1.9 5 1.1 0.733a Oxaliplatin 29 18.4 61 13.6 0.183a Irinotecan 17 10.8 35 7.8 0.323a Carboplatin 11 7.0 29 6.4 0.969a Cyclophosphamide 9 5.7 5 1.1 <0.001a Cisplatin (25–30 mg/m2) 2 1.3 14 3.1 0.338a Emetic risk HEC + MEC 101 63.9 162 36.0 <0.001a Low + minimum 57 36.1 288 64.0 Cancer type Breast 49 31.0 81 18.0 <0.001a Colorectal 42 26.6 100 22.2 0.315a Lung 6 3.8 82 18.2 <0.001a Gastric 23 14.6 63 14.0 0.968a Liver/gall bladder/pancreas 10 6.3 48 10.7 0.150a Hematological 6 3.8 17 3.8 1.000a Gynecological 16 10.1 26 5.8 0.095a Adherence to antiemetic guideline 146 92.4 420 93.3 0.831a aChi-square test bMann–Whitney U test c t test On the other hand, adherence to the antiemetic guideline was not different between the two groups (92.4 % vs. 93.6 %, P = 0.755).

Liver/gall bladder/pancreas 10 6.3 48 10.7 0.150a Hematological 6 3.8 17 3.8 1.000a Gynecological 16 10.1 26 5.8 0.095a Adherence to antiemetic guideline 146 92.4 420 93.3 0.831a aChi-square test bMann–Whitney U test c t test On the other hand, adherence to the antiemetic guideline was not different between the two groups (92.4 % vs. 93.6 %, P = 0.755). Risks for chemotherapy-induced overall nausea or vomiting Because age was significantly different between patients with and without overall nausea, an ROC curve was plotted for sensitivity versus 1−specificity. The area under the curve (AUC) was 0.658 (95 % CI, 0.607–0.709), indicating low accuracy prediction [17]. Using the ROC curve method, the cutoff age was predicted to be 58.5 years old (Youden index, 72.4 % sensitivity vs. 55.9 % specificity) or 61.5 years old (distance method, 63.6 % sensitivity vs. 62.7 % specificity). Thus, the cutoff age was set to 60 years for nausea. For vomiting, AUC of ROC curve was 0.721 (95 % CI, 0.629–0.813), indicating moderate accuracy prediction [17]. ROC analysis indicated that the cutoff age was 49.5 years old (Youden index as well as distance method, 84.5 % sensitivity vs. 56.4 % specificity). Thus, the cutoff age was set to 50 years old for vomiting.

a. For vomiting, AUC of ROC curve was 0.721 (95 % CI, 0.629–0.813), indicating moderate accuracy prediction [17]. ROC analysis indicated that the cutoff age was 49.5 years old (Youden index as well as distance method, 84.5 % sensitivity vs. 56.4 % specificity). Thus, the cutoff age was set to 50 years old for vomiting. As presented in Table 3, multivariate analysis showed that four factors such as female gender (OR 1.615; 95 % CI, 1.022–2.552; P = 0.004), age under 60 years (OR 2.303; 1.525–3.477; P < 0.001), inclusion of HEC/MEC (OR 2.321; 1.489–3.617; P < 0.001), and A/C regimen (OR 4.955; 1.863–13.18; P = 0.001) were significant risks for overall nausea.Table 3 Risk analysis for nausea and vomiting in 608 patients who underwent the first cycle of cancer chemotherapy Univariate analysis Multivariate analysis Odds ratio (OR) 95 % confidence interval P OR 95 % confidence interval P

As presented in Table 3, multivariate analysis showed that four factors such as female gender (OR 1.615; 95 % CI, 1.022–2.552; P = 0.004), age under 60 years (OR 2.303; 1.525–3.477; P < 0.001), inclusion of HEC/MEC (OR 2.321; 1.489–3.617; P < 0.001), and A/C regimen (OR 4.955; 1.863–13.18; P = 0.001) were significant risks for overall nausea.Table 3 Risk analysis for nausea and vomiting in 608 patients who underwent the first cycle of cancer chemotherapy Univariate analysis Multivariate analysis Odds ratio (OR) 95 % confidence interval P OR 95 % confidence interval P Nausea Female 2.228 (1.524–3.258) <0.001 1.615 (1.022–2.552) 0.040 Age < 60 years 3.007 (2.070–4.369) <0.001 2.303 (1.525–3.477) <0.001 HEC/MEC 3.150 (2.160–4.595) <0.001 2.321 (1.489–3.617) <0.001 A/C regimen 10.554 (4.857–22.93) <0.001 4.955 (1.863–13.18) 0.001 Breast cancer 2.048 (1.354–3.098) 0.001 0.700 (0.375–1.306) 0.262 Lung cancer 0.177 (0.076–0.415) <0.001 0.301 (0.125–0.725) 0.007 Adherence to antiemetic guideline 0.869 (0.434–1.742) 0.692 0.960 (0.444–2.076) 0.918 Vomiting Female 4.429 (1.923–10.20) <0.001 3.151 (1.213–8.183) 0.018 Age < 50 years 4.026 (1.997–8.117) <0.001 5.803 (2.667–12.63) <0.001 HEC/MEC 4.152 (1.985–8.683) <0.001 2.993 (1.245–7.195) 0.014 A/C regimen 8.205 (3.684–18.27) <0.001 2.987 (0.785–11.36) 0.108 Breast cancer 2.777 (1.420–5.427) 0.003 0.527 (0.167–1.667) 0.276 Lung cancer 0.304 (0.072–1.283) 0.105 0.759 (0.164–3.506) 0.724 Adherence to antiemetic guideline 0.883 (0.260–2.997) 0.842 0.539 (0.138–2.110) 0.375

8.683) <0.001 2.993 (1.245–7.195) 0.014 A/C regimen 8.205 (3.684–18.27) <0.001 2.987 (0.785–11.36) 0.108 Breast cancer 2.777 (1.420–5.427) 0.003 0.527 (0.167–1.667) 0.276 Lung cancer 0.304 (0.072–1.283) 0.105 0.759 (0.164–3.506) 0.724 Adherence to antiemetic guideline 0.883 (0.260–2.997) 0.842 0.539 (0.138–2.110) 0.375 On the other hand, female gender (OR 3.151; 95 % CI, 1.213–8.183; P = 0.018), age under 50 years (OR 5.803; 2.667–12.63, P < 0.001), and inclusion of HEC/MEC (OR 2.993; 1.245–7.195; P = 0.014) were found to be significant risks for overall vomiting by multivariate analysis. Adherence to the antiemetic guideline did not reduce the risk for overall nausea or vomiting (OR 0.960; 0.444–2.076, P = 0.918 for nausea; OR 0.539; 0.138–2.110, P = 0.375 for vomiting).

usion of HEC/MEC (OR 2.993; 1.245–7.195; P = 0.014) were found to be significant risks for overall vomiting by multivariate analysis. Adherence to the antiemetic guideline did not reduce the risk for overall nausea or vomiting (OR 0.960; 0.444–2.076, P = 0.918 for nausea; OR 0.539; 0.138–2.110, P = 0.375 for vomiting). Comparison of the control of nausea and vomiting among various HEC and MEC regimens As risk analysis indicated that HEC/MEC and A/C regimen were significant risks for chemotherapy-induced nausea, the rates of complete protection from nausea and vomiting during acute and delayed periods were compared among HEC and MEC regimens in patients who received the first cycle of chemotherapy. As shown in Fig. 2, the rate was significantly lower in A/C regimen (46 % and 24 % for acute and delayed periods, respectively) as compared with other regimens, regardless of 100 % adherence to the guideline-recommended antiemetic medication. In contrast, the control of nausea was favorable for a cisplatin (CDDP)-containing regimen (94 % and 88 % for acute and delayed periods, although the dose of CDDP was low (25 mg/m2 in CDDP/gemcitabine for gallbladder cancer or 30 mg/m2 in CDDP/irinotecan for gastric cancer).Fig. 2 Comparison of the rates of complete protection from nausea and vomiting among various chemotherapy regimens in patients receiving the first cycle of chemotherapy in the outpatient chemotherapy clinic. The number of patients (n) is shown in each pair of parentheses. Adherence to the Japanese antiemetic guideline during the overall period is represented at bottom of figure. Shaded columns represent HEC; open columns exhibit MEC. *P < 0.05, *P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test

hemotherapy clinic. The number of patients (n) is shown in each pair of parentheses. Adherence to the Japanese antiemetic guideline during the overall period is represented at bottom of figure. Shaded columns represent HEC; open columns exhibit MEC. *P < 0.05, *P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test Change in the control of nausea and vomiting in A/C regimen after repeated treatment cycles Although the complete protection from acute and delayed nausea in the first cycle of A/C chemotherapy was poor (46 % for acute nausea and 24 % for delayed nausea), the rates were improved in the second and third cycles of chemotherapy (Fig. 3). There were significant differences in the rates of delayed nausea and acute as well as delayed vomiting (Kruskal–Wallis test). On the other hand, dose reduction was not carried out in the first cycle in all patients but was performed in one patient in the second cycle (15 % reduction) as well as in the third cycle (15 % reduction).Fig. 3 Change in the control of nausea and vomiting in patients receiving three consecutive cycles of anthracycline/cyclophosphamide (A/C) regimen. *P < 0.05, *P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test Discussion In the present study, we surveyed the rate of control of CINV in 779 patients who received 5511 cycles of chemotherapy regimens in our outpatient cancer chemotherapy clinic. Among them, patients with gastrointestinal cancer were predominant (51 % of patients and 57 % of chemotherapy cycles).

Change in the control of nausea and vomiting in A/C regimen after repeated treatment cycles Although the complete protection from acute and delayed nausea in the first cycle of A/C chemotherapy was poor (46 % for acute nausea and 24 % for delayed nausea), the rates were improved in the second and third cycles of chemotherapy (Fig. 3). There were significant differences in the rates of delayed nausea and acute as well as delayed vomiting (Kruskal–Wallis test). On the other hand, dose reduction was not carried out in the first cycle in all patients but was performed in one patient in the second cycle (15 % reduction) as well as in the third cycle (15 % reduction).Fig. 3 Change in the control of nausea and vomiting in patients receiving three consecutive cycles of anthracycline/cyclophosphamide (A/C) regimen. *P < 0.05, *P < 0.01 by Kruskal–Wallis test followed by Scheffe’s test Discussion In the present study, we surveyed the rate of control of CINV in 779 patients who received 5511 cycles of chemotherapy regimens in our outpatient cancer chemotherapy clinic. Among them, patients with gastrointestinal cancer were predominant (51 % of patients and 57 % of chemotherapy cycles). According to the clinical practice guidelines for antiemesis formulated by ASCO [4], MASCC/ESMO [5], NCCN [6], and JSCO [7], palonosetron, a long-acting second-generation 5-HT3 receptor antagonist, is recommended for use in HEC and MEC; however, in the present study, the first generation of 5-HT3 receptor antagonists such as granisetron was predominantly prescribed. In the present study, health professionals, including pharmacists and nurses, interviewed all patients and monitored adverse drug reactions. In addition, we checked the prescription for antiemetic medication and aggressively promoted the appropriate use of antiemetic drugs. As a consequence, adherence to the clinical practice guideline for the use of antiemetic drugs was generally high (76 % for HEC, 88 % for MEC, 99 % for low-risk) for patients receiving the first cycle of chemotherapy, except for those undergoing CDDP-containing regimens (25 %). In the CDDP-containing regimens used in the present study, the dose of CDDP was low (25–30 mg/m2); thus, aprepitant was excluded from the standard medication for HEC. Even such an antiemetic medication effectively prevented nausea and vomiting, in which the overall control rate was 88 % for nausea and 94 % for vomiting. Very recently, Tamura et al. [19] reported the effectiveness of the antiemetic guideline by a multi-institutional prospective observational study, showing that adherence to the guideline is approximately 74 % for HEC and 95 % for MEC. They also reported that adherence (three antiemetics containing aprepitant, 5-HT3 receptor antagonist, and dexamethasone) for HEC decreases the risk for delayed vomiting as compared with two antiemetics (5-HT3 receptor antagonist and dexamethasone) without marked influence on the control of nausea. In the present study, the rate of guideline consistency was generally consistent with the data reported by Tamura et al. [19], although the non-adherence did not affect the control of overall nausea or vomiting.

antiemetics (5-HT3 receptor antagonist and dexamethasone) without marked influence on the control of nausea. In the present study, the rate of guideline consistency was generally consistent with the data reported by Tamura et al. [19], although the non-adherence did not affect the control of overall nausea or vomiting. Under the condition of roughly consistent with guideline-recommended antiemetic medication, vomiting was fairly well controlled, but complete protection from nausea was not sufficient for HEC and MEC. The poor control of nausea for HEC in the first cycle occurred primarily in the A/C regimen for breast cancer. Tamura et al. [19] also reported the high incidence of delayed nausea (49.4 % for HEC and 41.7 % for MEC), with a limited incidence of vomiting.

plete protection from nausea was not sufficient for HEC and MEC. The poor control of nausea for HEC in the first cycle occurred primarily in the A/C regimen for breast cancer. Tamura et al. [19] also reported the high incidence of delayed nausea (49.4 % for HEC and 41.7 % for MEC), with a limited incidence of vomiting. It was notable that the control of nausea and vomiting was generally higher in the overall cycles than in the first cycle. Particularly, marked improvement of the control rate of delayed nausea was observed at the second and third cycles of A/C chemotherapy. It is unlikely that improvement of the control of nausea results from decrease in the dose of anticancer drugs because, in the present study, the dose reduction was not observed in the first cycle but was carried out in one patient in the second cycle (15 % reduction) as well as in the third cycle (15 % reduction). Patients who showed failure in the control of CINV in the previous cycle were administered prochlorperazine or olanzapine in addition to an antianxiety drug such as lorazepam in the following cycles, which may be the predominant reason for the improvement of CINV control in the subsequent cycles. Moreover, in 608 patients who received the first cycle of HEC, MEC, low, or minimal risk of chemotherapy, the dose intensity was quite similar between patients with and without complete protection from nausea, in which the values were 97.0 ± 8.1 % (mean ± SD) in 450 patients who showed complete protection from nausea and 97.0 ± 7.5 % in 158 patients who did not. In addition, the complete protection from nausea was 71.4 % (60 of 84 patients) in patients with dose reduction and 74.4 % (390 of 524 patients) in those without dose reduction (P = 0.654 by χ2 test). Therefore, it is unlikely that the dose of anticancer drugs affected the control of chemotherapy-induced nausea in the present study.

he complete protection from nausea was 71.4 % (60 of 84 patients) in patients with dose reduction and 74.4 % (390 of 524 patients) in those without dose reduction (P = 0.654 by χ2 test). Therefore, it is unlikely that the dose of anticancer drugs affected the control of chemotherapy-induced nausea in the present study. Several investigators have shown the risk factors for CINV. Female gender, age, no history of drinking, and history of hyperemesis gravidarum are common as risks that lead to a loss of control of CINV [19–26]. Sekine et al. [21] showed in patients receiving HEC or MEC that female gender has high risk (OR 2.49) for failure in complete response (no vomiting and no rescue). It has also been reported that female patients are more likely to experience nausea and vomiting than male patients receiving HEC or MEC [22, 23]. On the other hand, Hesketh et al. [24] reported in patients receiving CDDP (≥70 mg/m2) that females are at high risk for the inability to complete response (OR 1.303) only when aprepitant is not included in the antiemetic medication. Younger age is also a risk for the loss of emetic control, but the cutoff age differs among studies, ranging from 40 to 65 years old [20–23]. Tamura et al. [19] also reported that older age is a decreased risk for CINV. However, the cutoff value of age that influences the control of CINV is still unclear. In the present study, the cutoff value of age was estimated from the ROC curve method, in which the AUC was 0.658 for nausea (low accuracy prediction) and 0.721 for vomiting (moderate accuracy prediction). The cutoff age was 58.5 years old as determined by the Youden index or 61.5 years old by the distance method and was set at 60 years old. Interestingly, the cutoff age for vomiting predicted by Youden index and distance method (49.5 years old) was younger than that for nausea. As a consequence, age under 60 years old was a significant risk for nausea (OR 2.303; 95 % CI, 1.525–3.477, P < 0.001), whereas age under 50 years old was a significant risk for vomiting (OR 5.803; 95 % CI, 2.667–12.63, P < 0.001). In addition, female gender (OR 1.615; 1.022–2.552, P = 0.04 for nausea; OR 3.151; 1.213–8.183, P = 0.018 for vomiting) and HEC/MEC (OR 2.321; 1.489–3.617, P < 0.001 for nausea; OR 2.993; 1.245–7.195, P = 0.014 for vomiting) were also significant risks for nausea or vomiting, although A/C chemotherapy was a significant risk for nausea but not for vomiting (OR 4.955; 1.863–13.18, P = 0.001).

OR 3.151; 1.213–8.183, P = 0.018 for vomiting) and HEC/MEC (OR 2.321; 1.489–3.617, P < 0.001 for nausea; OR 2.993; 1.245–7.195, P = 0.014 for vomiting) were also significant risks for nausea or vomiting, although A/C chemotherapy was a significant risk for nausea but not for vomiting (OR 4.955; 1.863–13.18, P = 0.001). Our present findings indicating the difference in the cutoff age between nausea and vomiting suggest that the differences in the cutoff age among studies may result from different indices of the control of CINV, including complete response, complete control, and complete protection from nausea or vomiting. These findings, taken together, suggested that extensive antiemetic medication using other types of antiemetic drugs such as olanzapine in addition to the standard medication is required for prevention of chemotherapy-induced nausea in patients who possess risks for poor control of CINV, including being female, younger age, and A/C chemotherapy. In the present study, no marked difference in the control of CINV among MEC except for cyclophosphamide-base regimens other than the A/C regimen, although there was a marked difference in the control of CINV among HEC, as mentioned earlier. The low rate of the control of CINV for cyclophosphamide-base regimens may be caused by the patient risks (female and young age) rather than the chemotherapy, because the cyclophosphamide-base regimens were used for the most part in breast cancer patients, whose average age was 48 years.

mong HEC, as mentioned earlier. The low rate of the control of CINV for cyclophosphamide-base regimens may be caused by the patient risks (female and young age) rather than the chemotherapy, because the cyclophosphamide-base regimens were used for the most part in breast cancer patients, whose average age was 48 years. In conclusion, the control of CINV was investigated in 779 patients receiving 5511 cycles of chemotherapy regimens in our outpatient cancer chemotherapy clinic. In spite of the high rate of adherence to the antiemetic guideline, the control of nausea, but not vomiting, was poor in patients receiving HEC and MEC. A multivariate logistic regression analysis indicated that female gender, age under 60 years, HEC/MEC, and A/C chemotherapy were significant risks for overall nausea. Care should be taken to prevent chemotherapy-induced nausea in high-risk patients. Compliance with ethical standards Conflict of Interest The authors declare that they have no conflict of interest.

Introduction Erlotinib is an orally administered epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor (TKI). The phase III BR.21 study showed that erlotinib treatment of non-small-cell lung cancer (NSCLC) in the second- or third-line setting achieved significant overall survival (OS), progression-free survival (PFS), and response rate benefit compared with best supportive care [1]. In addition, the OPTIMAL and EURTAC studies have reported significant PFS benefits with erlotinib as first-line treatment for patients with EGFR mutation-positive NSCLC compared with chemotherapy in Asian and European populations, respectively [2, 3]. Skin toxicities (especially acneiform rash) are the most common adverse reactions associated with erlotinib treatment [4, 5]. Across NSCLC phase III studies, the incidence of rash is 62–76 % [6]. This finding is not unexpected as EGFR is expressed in undifferentiated and proliferating keratinocytes of the skin, meaning that EGFR TKIs often result in skin toxicity [7]. The most common erlotinib-related skin toxicities are acneiform rash, xeroderma, paronychia, and pruritus [8–10].

cidence of rash is 62–76 % [6]. This finding is not unexpected as EGFR is expressed in undifferentiated and proliferating keratinocytes of the skin, meaning that EGFR TKIs often result in skin toxicity [7]. The most common erlotinib-related skin toxicities are acneiform rash, xeroderma, paronychia, and pruritus [8–10]. The incidence of skin rash in Japanese patients treated with erlotinib has been high, with up to 98.1 % of patients experiencing rash [8]. In phase II studies, 72.2 % of Japanese patients experienced xeroderma, which is characterized by dry, rough skin causing fissures and a scaling effect [9]. Pruritus, or skin itching, is common with the development of rash or xeroderma. Paronychia is a painful erythema around several fingernails or toenails, which can result in swelling, granulation, and bleeding.

ts experienced xeroderma, which is characterized by dry, rough skin causing fissures and a scaling effect [9]. Pruritus, or skin itching, is common with the development of rash or xeroderma. Paronychia is a painful erythema around several fingernails or toenails, which can result in swelling, granulation, and bleeding. Although most cases of skin toxicities are mild and transient, they can have a considerable impact on patients’ quality of life and can therefore reduce compliance with erlotinib therapy. A number of studies have reported evidence suggesting a correlation between the incidence and severity of rash with improved clinical outcomes, such as longer OS among erlotinib-treated patients [11–13]. In the BR.21 study, all grades of rash were associated with longer OS compared with patients who did not develop rash [grade 1 rash vs. no rash: hazard ratio (HR) 0.41; P < 0.001; grade ≥2 rash vs. no rash: HR 0.29; P < 0.001] [13]. An association between rash and prolonged OS has also been reported in Japanese patients (OS 8.8 months for patients with no rash compared with 16.6 months for patients with grade 2/3 rash) [14]. Considering the correlation between rash and survival outcomes, adequate rash management (prophylactic cleansing regimens, reducing the dose or interruption of erlotinib treatment, or use of concomitant treatment for rash) is of the utmost importance to ensure the continuation of erlotinib treatment and therefore the maximum benefit for patients.

between rash and survival outcomes, adequate rash management (prophylactic cleansing regimens, reducing the dose or interruption of erlotinib treatment, or use of concomitant treatment for rash) is of the utmost importance to ensure the continuation of erlotinib treatment and therefore the maximum benefit for patients. Kiyohara et al. developed an algorithm for rash management (i.e., treatment course for rash symptoms) consisting of the use of strong or higher-class steroids to ‘manage’ rash, allowing patients to continue erlotinib use [15]. The use of steroids is an option for rash management depending on the grade of rash. In Japan, a five-class ranking system for steroids is used ranging from the strongest to very strong, strong, medium, and weak. It is generally advised that only strong or higher-potency steroids are used to treat grade ≥2 erlotinib-related rash [15]. Topical steroids (strong or higher rank) for grade ≥2 toxicities are recommended to treat both xeroderma and pruritus. Strong or higher-rank steroids are recommended for the treatment of paronychia. This analysis was part of the POst-Launch All-patient Registration Surveillance in TARceva (POLARSTAR) study [10]. POLARSTAR is a large-scale surveillance study undertaken as a post-approval commitment to monitor the efficacy and safety of erlotinib in Japan. This current analysis evaluates the use of topical steroids as a treatment for rash. The frequency of skin toxicity-related adverse events (AEs), the interventions used, and their outcomes were analyzed.

urveillance study undertaken as a post-approval commitment to monitor the efficacy and safety of erlotinib in Japan. This current analysis evaluates the use of topical steroids as a treatment for rash. The frequency of skin toxicity-related adverse events (AEs), the interventions used, and their outcomes were analyzed. Methods Study design In this phase IV observational study, all patients with unresectable, recurrent, or advanced NSCLC who were treated with erlotinib were enrolled. The study was approved by the relevant ethics committees. Treatment schedule Patients receiving erlotinib daily were monitored until termination of erlotinib therapy or completion of 12 months of treatment. Erlotinib treatment delay, dose reduction, and discontinuation were permitted in actual clinical practice to manage rash. Assessments Demographic and baseline data were collected for each patient, including age, gender, body mass index, tumor histology, Eastern Cooperative Oncology Group (ECOG) performance status (PS), smoking history, and medical history (including hepatic dysfunction, renal dysfunction, cardiovascular disease, and lung disorders). Safety data were collected at 1, 6, and 12 months after the start of erlotinib therapy. All AE reports were collected, and AEs were graded using the National Cancer Institute Common Terminology Criteria for AEs version 3.0 and coded using the Medical Dictionary for Regulatory Activities version 14.1 thesaurus terms.

). Safety data were collected at 1, 6, and 12 months after the start of erlotinib therapy. All AE reports were collected, and AEs were graded using the National Cancer Institute Common Terminology Criteria for AEs version 3.0 and coded using the Medical Dictionary for Regulatory Activities version 14.1 thesaurus terms. Outcome measures Frequency of erlotinib-related skin toxicities (acneiform rash, xeroderma, pruritus, and paronychia), interventions for the symptoms, and the outcomes of these interventions were assessed by time to treatment initiation, recovery rate, and time to recovery. To avoid confounding factors, only the first event of skin toxicity was analyzed. For rash management interventions, standard Japanese ranking of steroids was used, defining steroids as strongest (e.g., clobetasol propionate), very strong (e.g., dexamethasone propionate), strong (e.g., betamethasone valerate), medium (e.g., hydrocortisone butyrate), and weak (e.g., hydrocortisone acetate) [16]. Patients were categorized into subgroups according to the topical steroid treatment they received (weak- or medium-rank steroids categorized as ‘medium,’ strong, or higher-rank steroids categorized as ‘strong,’ and those initiated on medium-rank or lower steroids then changed to strong-rank or higher-rank steroids were categorized as ‘medium to strong’). Time to recovery was estimated from Kaplan–Meier curves.

ived (weak- or medium-rank steroids categorized as ‘medium,’ strong, or higher-rank steroids categorized as ‘strong,’ and those initiated on medium-rank or lower steroids then changed to strong-rank or higher-rank steroids were categorized as ‘medium to strong’). Time to recovery was estimated from Kaplan–Meier curves. Results Patients A total of 10,708 patients were enrolled between December 2007 and October 2009 from 1027 institutions; of these, 9,909 patients were evaluated for this analysis. Baseline characteristics are shown in Table 1. Briefly, the median patient age was 66 years; the majority of patients (80.2 %) had adenocarcinoma histology; 44 % of patients had an ECOG PS of 1; and 29.7 % of patients had ECOG PS of 0.Table 1 Baseline characteristics of the analysis population (N = 9909) N % Gender Male 5300 53.5 Female 4609 46.5 Age (years) Median (range) 66 (14–95) – Histology Adenocarcinoma 7950 80.2 Other 1935 19.5 Unknown 24 0.24 Stage Relapsed 3316 33.5 IIIB 1387 14.0 IV 4917 49.6 Other 202 2.0 Unknown 87 0.9 ECOG PS 0 2935 29.6 1 4380 44.2 2 1786 18.0 3 604 6.1 4 186 1.9 Unknown 18 0.2 ECOG PS Eastern Cooperative Oncology Group performance status

N % Gender Male 5300 53.5 Female 4609 46.5 Age (years) Median (range) 66 (14–95) – Histology Adenocarcinoma 7950 80.2 Other 1935 19.5 Unknown 24 0.24 Stage Relapsed 3316 33.5 IIIB 1387 14.0 IV 4917 49.6 Other 202 2.0 Unknown 87 0.9 ECOG PS 0 2935 29.6 1 4380 44.2 2 1786 18.0 3 604 6.1 4 186 1.9 Unknown 18 0.2 ECOG PS Eastern Cooperative Oncology Group performance status Incidence of skin toxicity The most common skin toxicities were acneiform rash, xeroderma, and paronychia, observed in 60.9 %, 7.5 %, and 6.6 % of the study population, respectively. The majority of these skin toxicities were mild in severity, as grade 3/4 acneiform rash, xeroderma, and paronychia were reported in only 6.3 %, 0.3 %, and 0.7 % of patients, respectively (Table 2). Three grade 5 skin toxicities reported: one case of toxic skin eruption and two cases of Stevens–Johnson syndrome.Table 2 Erlotinib-related skin toxicities by grade (N = 9909) N (%) Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Total Acneiform rash 2415 (24.4) 2944 (29.7) 598 (6.0) 23 (0.2) 1 (0.0) 6032 (60.9) Xeroderma 422 (4.3) 286 (2.9) 24 (0.2) 1 (0.0) 0 738 (7.5) Paronychia 274 (2.8) 303 (3.1) 70 (0.7) 0 0 654 (6.6) The median time from erlotinib administration to onset of acneiform rash was within 2 weeks (9 days), xeroderma was within 3 weeks (16 days), and paronychia was approximately 5 weeks (34 days) from initial erlotinib administration.

N (%) Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Total Acneiform rash 2415 (24.4) 2944 (29.7) 598 (6.0) 23 (0.2) 1 (0.0) 6032 (60.9) Xeroderma 422 (4.3) 286 (2.9) 24 (0.2) 1 (0.0) 0 738 (7.5) Paronychia 274 (2.8) 303 (3.1) 70 (0.7) 0 0 654 (6.6) The median time from erlotinib administration to onset of acneiform rash was within 2 weeks (9 days), xeroderma was within 3 weeks (16 days), and paronychia was approximately 5 weeks (34 days) from initial erlotinib administration. Interventions for skin toxicity The most common intervention for the treatment of skin toxicities was topical steroids, with more than 75 % of patients who suffered from acneiform rash (the most common skin toxicity) receiving steroids within 4 days of diagnosis. Of the patients experiencing xeroderma, more than 75 % received steroids within 5 days of onset or diagnosis, and many patients with paronychia were already on steroids, for an average of 10 days, before diagnosis or onset of paronychia.

a trend of patients in the medium to strong subgroup having longer recovery times than the other two groups, regardless of time of steroid initiation (Supplementary Fig. 2). In patients with grade 2 rash, there was a trend of earlier initiation of steroid, resulting in shorter recovery time, regardless of steroid rank. Discussion This analysis focused on the incidence and management of rash in POLARSTAR, as skin toxicities are one of the most common AEs associated with erlotinib, with rash being the most common skin toxicity experienced. As rash can have an impact on patients’ quality of life and may lead to discontinuation of treatment, effective rash management is required to ensure patients remain on erlotinib for as much of the treatment course as possible to gain maximal benefit. As erlotinib treatment is often given for an extended period of time, particularly in patients with EGFR mutations, the development of effective rash management strategies to ameliorate ongoing rash symptoms is vital, particularly as prophylactic rash treatments have not yet been fully validated. Effective management strategies are especially important when considering that some data indicate a correlation between increased rash grade and erlotinib efficacy, meaning those with the most severe rash, who may wish to discontinue treatment, may actually be gaining the most benefit from erlotinib [11–14].

validated. Effective management strategies are especially important when considering that some data indicate a correlation between increased rash grade and erlotinib efficacy, meaning those with the most severe rash, who may wish to discontinue treatment, may actually be gaining the most benefit from erlotinib [11–14]. The majority of skin toxicities reported in the POLARSTAR Japanese surveillance study were grade 1/2. Acneiform rash was the most common skin toxicity observed, seen in 60.9 % of patients. Earlier initiation of topical steroid treatment for erlotinib-related rash resulted in reduced recovery time. Patients who were initiated on strong-rank steroids had a shorter recovery time than patients who failed to respond to medium-rank steroids and then progressed to strong-rank steroids, regardless of whether patients received additional erlotinib dose reduction or interruption for rash. These data suggest that earlier initiation of treatment (within 0–14 days of diagnosis) with strong-ranked or higher-rank steroids could be a suitable administration regimen for rash management in erlotinib-treated NSCLC patients. Patients who were initiated on medium-rank steroids had the shortest recovery time; however, some patients needed to change steroid rank (21.9 % of patients with grade 1 rash, 36.5 % with grade 2 rash, and 47.5 % with grade 3 rash needed to change from medium- or weaker-rank steroids to stronger-rank steroids). Furthermore, patients who needed to change steroid rank had the longest recovery time. This finding suggests there may be some risk of undertreatment with medium-rank steroids because it is difficult to determine precisely whether medium-rank steroids have enough intensity for each case before treatment initiation. Additionally, undertreatment with medium-rank steroids could lead to longer recovery time than initiation with strong-rank steroids. Therefore, if there is no adequate reason for avoiding administration of strong-rank steroids (e.g., concomitant skin infection), it might be more effective to initiate all patients on strong- or higher-rank steroids for maximal benefit and quicker recovery time.

recovery time than initiation with strong-rank steroids. Therefore, if there is no adequate reason for avoiding administration of strong-rank steroids (e.g., concomitant skin infection), it might be more effective to initiate all patients on strong- or higher-rank steroids for maximal benefit and quicker recovery time. There are various ways to manage erlotinib-related rash. A recent review by Kiyohara et al. highlighted very strong/strong class steroids as a recommended treatment for EGFR-related acneiform rash [15]. In addition to steroids ranging from hydrocortisone to methylprednisolone for varying grades of rash, patient education is also seen as important for prophylactic treatment (teaching patients about moisturization, reducing sun exposure, and avoiding products that dry the skin) [6]. Novel treatments, such as menadione lotion, retinoids, and alpha-hydroxy acids, are also being investigated as possible treatment options for erlotinib-related rash [6]. Earlier steroid treatments for skin toxicities and even pre-emptive regimens have been effective in reducing EGFR TKI-related rash. Lacouture et al. showed that pre-emptive steroid treatment reduced the incidence of grade ≥2 skin toxicities by 50 % compared with reactive treatment in colorectal cancer patients treated with panitumumab [29 % vs. 62 % of patients: odds ratio 0.3; 95 % confidence interval (CI), 0.1–0.6] [17]. However, these novel pre-emptive treatments are neither fully established nor validated; therefore, adequate reactive steroid treatment is still a key management strategy in the current scenario.

patients treated with panitumumab [29 % vs. 62 % of patients: odds ratio 0.3; 95 % confidence interval (CI), 0.1–0.6] [17]. However, these novel pre-emptive treatments are neither fully established nor validated; therefore, adequate reactive steroid treatment is still a key management strategy in the current scenario. To our knowledge, this is the first analysis focusing on the correlation between steroid rank, timing of initiation of steroid treatment, and recovery time of rash induced by erlotinib. However, there are a number of factors to consider when interpreting data from this analysis. As this was a single-arm surveillance study, there was no control group with which to directly compare results. The study design meant that in contrast to a clinical trial, there was no strict observation period, and the study lacked any patient selection criteria, as all patients treated with erlotinib in Japan in the post-approval period were enrolled. Conclusion As most current treatment algorithms are based on anecdotal evidence, and this study provides evidence to support the use of topical steroids for EGFR TKI-associated rash, further studies should be undertaken to corroborate our findings that strong topical steroids initiated early in skin toxicity diagnosis are a suitable regimen to treat these AEs to allow continuation of EGFR TKI therapy.

this study provides evidence to support the use of topical steroids for EGFR TKI-associated rash, further studies should be undertaken to corroborate our findings that strong topical steroids initiated early in skin toxicity diagnosis are a suitable regimen to treat these AEs to allow continuation of EGFR TKI therapy. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary Fig. S1 Class effects of steroids for rash management in patients who did not have an erlotinib dose reduction or interruption: grade 1 rash (a), grade 2 rash (b), and grade ≥ 3 rash (c). Medium: patients treated with medium- or weak-rank steroids; medium to strong: patients initially treated with medium- or weak-rank steroids then changed to strong- or higher-rank steroids; strong: patients treated with strong- or higher-rank steroids. (DOCX 13 kb) Supplementary Fig. S2 Time to recovery by rank of steroid and time to treatment initiation in patients with grade 2 rash. (TIFF 1237 kb) Supplementary material 3 (TIFF 1409 kb) Support for third-party writing assistance for this manuscript was provided by Joanna Musgrove of Gardiner–Caldwell Communications and was funded by Chugai Pharmaceutical Co. Ltd. The authors thank all the patients who participated in the study and the clinical personnel involved in data collection.

Supplementary material 3 (TIFF 1409 kb) Support for third-party writing assistance for this manuscript was provided by Joanna Musgrove of Gardiner–Caldwell Communications and was funded by Chugai Pharmaceutical Co. Ltd. The authors thank all the patients who participated in the study and the clinical personnel involved in data collection. Compliance with ethical standards Role of funding source This trial was designed, funded, and monitored by Chugai Pharmaceutical Co. Ltd. Data were gathered, analyzed, and interpreted by Chugai with input from all authors. The corresponding author had full access to the relevant data and took full responsibility for the final decision to submit the report for publication. Third-party writing assistance for the manuscript was funded by Chugai Pharmaceutical Co. Ltd. Conflict of interest Drs. Yamazaki, Kudoh, and Fukuoka received personal fees from Chugai as members of an Independent Advisory Board for erlotinib. Mr. Seki is an employee of Chugai Pharmaceuticals Co. Ltd. Dr. Kiyohara has no conflict of interest to declare.

Introduction Colorectal cancer (CRC) is the second most common type of cancer and the third most common cause of cancer mortality in Japan [1]. Surgical removal of metastatic CRC (mCRC) is usually difficult, making chemotherapy the first choice for treatment [2]. FOLFOX4, a bi-weekly regimen of intravenous bolus and infusional 5-fluorouracil/leucovorin (5-FU/LV) plus oxaliplatin, is widely used in patients with previously untreated mCRC [3]. Capecitabine, an oral fluoropyrimidine, has shown efficacy similar to bolus 5-FU/LV as a first-line treatment for mCRC [4, 5]. Oral fluoropyrimidines can replace the intravenous fluoropyrimidine component of combination regimens. Capecitabine and a 3-week dose of oxaliplatin (the XELOX regimen) have also been shown to have efficacy similar to 5-FU/LV plus oxaliplatin (FOLFOX4 or FOLFOX6) for first-line treatment of mCRC patients [6, 7]. A pivotal phase III study (NO16966) reported that adding bevacizumab to oxaliplatin-based chemotherapy significantly improved progression-free survival (PFS) by 1.4 months when used as first-line treatment for mCRC [8]. XELOX plus bevacizumab is an effective treatment strategy with a manageable tolerability profile in Japanese patients with mCRC [9]. However, there are no prospective data in clinical practice concerning XELOX plus bevacizumab in Japan. The present study was designed to evaluate the efficacy and safety of XELOX plus bevacizumab in a Japanese mCRC population that included elderly patients.

able tolerability profile in Japanese patients with mCRC [9]. However, there are no prospective data in clinical practice concerning XELOX plus bevacizumab in Japan. The present study was designed to evaluate the efficacy and safety of XELOX plus bevacizumab in a Japanese mCRC population that included elderly patients. Patients and methods Study design This was a prospective, multicenter, single-arm, open-label study that was conducted to evaluate the efficacy and safety of the commonly used doses of XELOX plus bevacizumab in a Japanese mCRC population that included elderly patients. The primary endpoint was the objective response rate (ORR), and the secondary endpoints were PFS, overall survival (OS), and safety.

open-label study that was conducted to evaluate the efficacy and safety of the commonly used doses of XELOX plus bevacizumab in a Japanese mCRC population that included elderly patients. The primary endpoint was the objective response rate (ORR), and the secondary endpoints were PFS, overall survival (OS), and safety. Eligibility Patients with histologically proven, unresectable, advanced CRC or mCRC that was previously untreated were eligible for this study if they met the following criteria—measurable lesions based on Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 [10]; age ≥20 years; Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤2; life expectancy ≥3 months; no prior systemic chemotherapy for mCRC; no progression within 6 months of completion of adjuvant chemotherapy; adequate bone marrow function (neutrophil count ≥1,500/mm3, platelet count ≥100,000/mm3, and hemoglobin ≥9.0 g/dL); adequate hepatic function [total bilirubin ≤2.0 mg/dL, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5 or 5 (in cases with liver metastases) times the institutional upper limit of normal]; adequate renal function (creatinine ≤1.5 mg/dL and protein urea ≤ grade 1); and written informed consent before enrollment in the study. The exclusion criteria included brain metastases; clinically significant ascites and pleural effusion; major surgery, open biopsy, or significant traumatic injury within 4 weeks before enrollment; fine-needle aspiration biopsy or central venous line placement within 1 week before enrollment; bleeding diathesis or coagulopathy; non-healing bone fracture; diarrhea grade ≥2; uncontrolled hypertension or peptic ulcer; clinically significant cardiovascular disease; daily treatment with high-dose aspirin (≥325 mg/day) or non-steroidal anti-inflammatory medications; immune suppressive or steroidal medications; and peripheral neuropathy grade ≥1.

y; non-healing bone fracture; diarrhea grade ≥2; uncontrolled hypertension or peptic ulcer; clinically significant cardiovascular disease; daily treatment with high-dose aspirin (≥325 mg/day) or non-steroidal anti-inflammatory medications; immune suppressive or steroidal medications; and peripheral neuropathy grade ≥1. Treatment schedule XELOX treatment included a 2-h intravenous infusion of 130 mg/m2 oxaliplatin (Yakult Honsha Co., Ltd., Tokyo, Japan) on day 1 plus 1,000 mg/m2 oral capecitabine (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) twice daily for 2 weeks of a 3-week cycle. The first dose of capecitabine was administered in the evening on day 1, and the last dose was given in the morning on day 15. Patients received 7.5 mg/kg bevacizumab (Chugai Pharmaceutical Co., Ltd.) as a 30–90-min intravenous infusion before oxaliplatin treatment on day 1 of the 3-week cycle. Treatment continued until disease progression, intolerable adverse events, or withdrawal of consent.

the last dose was given in the morning on day 15. Patients received 7.5 mg/kg bevacizumab (Chugai Pharmaceutical Co., Ltd.) as a 30–90-min intravenous infusion before oxaliplatin treatment on day 1 of the 3-week cycle. Treatment continued until disease progression, intolerable adverse events, or withdrawal of consent. Treatment was interrupted if grade 2–4 toxicities were observed, and the delay continued until recovery in patients with neutrophil counts <1,500/mm3, febrile neutropenia, platelet counts <75,000/mm3, or significant persistent non-hematological toxicities. Oxaliplatin was skipped if grade ≥2 neurotoxicity was observed and bevacizumab was skipped if grade ≥2 protein urea was observed. Doses were modified based on hematological parameters and the degree of non-hematological toxicities. The capecitabine dose was reduced to 800 mg/m2 (or further to 600 mg/m2) if patients experienced grade 3 or 4 diarrhea, stomatitis, nausea or vomiting, anorexia, dermatitis, grade 4 neutropenia, or grade 3 or 4 thrombocytopenia. Oxaliplatin was also reduced to 100 mg/m2 (or further to 85 mg/m2) for all of the above conditions except for dermatitis, and was also reduced in cases of persistent (≥15 days) grade 2 neurotoxicity or temporary (8–14 days) grade 3 neurotoxicity. In patients with persistent (≥15 days) grade 3 neurotoxicity or temporary grade 4 neurotoxicity, oxaliplatin was omitted from the regimen. No dose modifications were performed for bevacizumab. This treatment plan was almost identical to that of the study NO16966 [8].

city or temporary (8–14 days) grade 3 neurotoxicity. In patients with persistent (≥15 days) grade 3 neurotoxicity or temporary grade 4 neurotoxicity, oxaliplatin was omitted from the regimen. No dose modifications were performed for bevacizumab. This treatment plan was almost identical to that of the study NO16966 [8]. If oxaliplatin and/or bevacizumab were discontinued, treatment with the remaining components could be continued. For example, capecitabine could be administered with or without bevacizumab after discontinuation of oxaliplatin, and XELOX or capecitabine could be given after discontinuation of bevacizumab. However, continuation of oxaliplatin or bevacizumab without capecitabine was not permitted. Efficacy and safety evaluation Computed tomography scans were performed to assess tumors, starting within 4 weeks prior to study registration and repeated every 6 weeks. The investigators evaluated response rates according to RECIST, version 1.1 [10]. An independent review committee (IRC) confirmed the tumor responses. PFS was defined as the time from the date of registration to the date that disease progression was first confirmed, as determined by the IRC, or to the date of death from any cause. Data were censored at the last tumor assessment if a patient withdrew before progression was observed. OS was defined as the time from the date of registration to death. Treatment continued until disease progression, unacceptable toxicity, or patient withdrawal. All eligible patients were included in the response and survival analyses (N = 46).

last tumor assessment if a patient withdrew before progression was observed. OS was defined as the time from the date of registration to death. Treatment continued until disease progression, unacceptable toxicity, or patient withdrawal. All eligible patients were included in the response and survival analyses (N = 46). Adverse events were assessed for all enrolled patients (N = 47) according to the Common Terminology Criteria for Adverse Events, version 4.0. Relative dose intensity Relative dose intensity can be decreased by reducing, delaying or skipping the chemotherapy dose and was calculated according to the following equation: [total actual administered dose/actual administration period (final day of the treatment course − day of the administration + 1 days)/total planned dose/planned administration period (21 days)] × 100.

creased by reducing, delaying or skipping the chemotherapy dose and was calculated according to the following equation: [total actual administered dose/actual administration period (final day of the treatment course − day of the administration + 1 days)/total planned dose/planned administration period (21 days)] × 100. Statistical consideration We examined whether the combination of XELOX plus bevacizumab could achieve a higher ORR than other chemotherapy regimens, as has been observed in other countries, in Japanese patients. In view of previous studies, we assumed a threshold ORR of 30 % and an expected value of at least 50 %. Under these assumptions, we determined that 39 patients were needed to provide a one-sided alpha of 0.05 and 80 % power. Factoring in a 5 % dropout rate and the possibility of ineligible patients, we set a target sample size of 41 patients. Registration was scheduled to continue for 12 months, and we planned to follow-up the patients for 36 months after the last registration. The 95 % CIs for the response rates were estimated by the Clopper−Pearson exact method. The PFS and OS curves were estimated by the Kaplan–Meier method, and their CIs were estimated using the Brookmeyer and Crowley method. All statistical analyses were performed using the SAS for Windows, release 9.3 (SAS Institute, Cary, NC, USA).

for the response rates were estimated by the Clopper−Pearson exact method. The PFS and OS curves were estimated by the Kaplan–Meier method, and their CIs were estimated using the Brookmeyer and Crowley method. All statistical analyses were performed using the SAS for Windows, release 9.3 (SAS Institute, Cary, NC, USA). Ethics The ethical, medical, and scientific aspects of the study were reviewed and approved by the ethics committee of each participating institution in the UMIN clinical trials registry (UMIN000003915). The study was conducted in accordance with the Declaration of Helsinki of 1975, revised in 2000. Results Patient characteristics A total of 47 patients were enrolled in the study between May 2010 and March 2011. One patient did not meet the eligibility criteria. Therefore, ORR, OS, and PFS were evaluated in 46 patients, while toxicity was evaluated in 47 patients treated with XELOX plus bevacizumab. The characteristics of the 47 patients are described in Table 1. The population included 30 male and 17 female patients with a median age of 69 years (range 38–81). Twenty of the 47 patients (42 %) were ≥70 years old, and 10 patients (21 %) were ≥75 years old. The ECOG PS was 0 in 40 patients (85 %), 1 in 5 patients (11 %), and 2 in 2 patients (4 %). Affected organs were the liver in 35 patients (75 %), the lung in 10 patients (21 %), the lymph nodes in 16 patients (34 %), and the peritoneum in 7 patients (15 %). The liver was the most common site of metastasis.Table 1 Patient characteristics (N = 47) Factor Characteristic N

The characteristics of the 47 patients are described in Table 1. The population included 30 male and 17 female patients with a median age of 69 years (range 38–81). Twenty of the 47 patients (42 %) were ≥70 years old, and 10 patients (21 %) were ≥75 years old. The ECOG PS was 0 in 40 patients (85 %), 1 in 5 patients (11 %), and 2 in 2 patients (4 %). Affected organs were the liver in 35 patients (75 %), the lung in 10 patients (21 %), the lymph nodes in 16 patients (34 %), and the peritoneum in 7 patients (15 %). The liver was the most common site of metastasis.Table 1 Patient characteristics (N = 47) Factor Characteristic N Sex Male 30 (64 %) Female 17 (36 %) Age Median 69 Range 38–81 Performance status (ECOG) 0 40 (85 %) 1 5 (11 %) 2 2 (4 %) Primary tumor histology Well-differentiated adenocarcinoma 7 (15 %) Moderately differentiated adenocarcinoma 23 (49 %) Poorly differentiated adenocarcinoma 6 (13 %) Other 2 (4 %) Unknown 9 (19 %) Affected organs Liver 35 (74 %) Lung 10 (21 %) Lymph nodes 16 (34 %) Peritoneum 7 (15 %) Local recurrence 3 (6 %) Other 5 (11 %) ECOG Eastern Cooperative Oncology Group

Sex Male 30 (64 %) Female 17 (36 %) Age Median 69 Range 38–81 Performance status (ECOG) 0 40 (85 %) 1 5 (11 %) 2 2 (4 %) Primary tumor histology Well-differentiated adenocarcinoma 7 (15 %) Moderately differentiated adenocarcinoma 23 (49 %) Poorly differentiated adenocarcinoma 6 (13 %) Other 2 (4 %) Unknown 9 (19 %) Affected organs Liver 35 (74 %) Lung 10 (21 %) Lymph nodes 16 (34 %) Peritoneum 7 (15 %) Local recurrence 3 (6 %) Other 5 (11 %) ECOG Eastern Cooperative Oncology Group Treatment duration The median duration of treatment was 5.0 months (range 0.7–20.0) with a median of 6.0 treatment cycles (range 1–28). XELOX plus bevacizumab combination therapy was administered for a median of 5 cycles (range 1–16). After discontinuing oxaliplatin, 3 patients (6.4 %) continued with capecitabine and bevacizumab combination therapy and a received a median of 5 cycles (range 3–20). A total of 22 patients (46.8 %) received XELOX therapy for a median of 1 cycle (range 1–4) during permanent or temporary discontinuation of bevacizumab. Based on the planned dose intensities of 1,000 mg/m2 capecitabine twice daily for 2 weeks of a 3-week cycle, 130 mg/m2 oxaliplatin per 3-week cycle, and 7.5 mg/kg bevacizumab per 3-week cycle, the median relative dose intensities of oxaliplatin and bevacizumab were 79.0 % (95 % CI 42.4–98.1) and 75.9 % (95 % CI 41.6–96.2), respectively.

ed dose intensities of 1,000 mg/m2 capecitabine twice daily for 2 weeks of a 3-week cycle, 130 mg/m2 oxaliplatin per 3-week cycle, and 7.5 mg/kg bevacizumab per 3-week cycle, the median relative dose intensities of oxaliplatin and bevacizumab were 79.0 % (95 % CI 42.4–98.1) and 75.9 % (95 % CI 41.6–96.2), respectively. Efficacy The results of the tumor response analysis are shown in Table 2. A complete response (CR) was observed in 1 patient (2.2 %) and a partial response (PR) was observed in 23 patients (50.0 %) giving an overall response rate (CR + PR) of 52.2 % (95 % CI 37.0–67.1). Stable disease (SD) was observed in 15 additional patients (32.6 %). Therefore, the overall disease control rate (CR + PR + SD) was 84.8 % (95 % CI 71.1–93.7). The response rate across all time points without confirmation was 67.4 % (95 % CI 52.0–80.5).Table 2 Tumor responses (N = 46) RECIST-confirmed N (%) CR 1 (2.2) PR 23 (50.0) SD 15 (32.6) PD 2 (4.3) NE 5 (10.9) ORR 24 (52.2) 90 % CI 39.2–65.0 95 % CI 37.0–67.1 DCR 39 (84.8) 95 % CI 71.1–93.7 OPR = (CR + PR), DCR = (CR + PR + SD) CI confidence interval, CR complete response, DCR disease control rate, NE not evaluable, ORR overall response rate, PD progressive disease, PR partial response, RECIST Response Evaluation Criteria in Solid Tumors, SD stable disease

RECIST-confirmed N (%) CR 1 (2.2) PR 23 (50.0) SD 15 (32.6) PD 2 (4.3) NE 5 (10.9) ORR 24 (52.2) 90 % CI 39.2–65.0 95 % CI 37.0–67.1 DCR 39 (84.8) 95 % CI 71.1–93.7 OPR = (CR + PR), DCR = (CR + PR + SD) CI confidence interval, CR complete response, DCR disease control rate, NE not evaluable, ORR overall response rate, PD progressive disease, PR partial response, RECIST Response Evaluation Criteria in Solid Tumors, SD stable disease The cut-off date for PFS and OS was April 2014. The median follow-up period was 34.4 months. Median PFS was 10.0 months (95 % CI 7.8–12.3; Fig. 1). A total of 27 of the 46 eligible patients died due to progression of advanced colorectal cancer. At the time of analysis, the median OS was 34.6 months (95 % CI 19.9–not estimable; Fig. 2).Fig. 1 Kaplan–Meier estimate for progression-free survival (PFS). After a median follow-up time of 34.4 months, the median PFS was 10.0 months (95 % CI 7.8–12.3) Fig. 2 Kaplan–Meier estimate for overall survival (OS). The median OS was 34.6 months (95 % CI 19.9–not estimable)

The cut-off date for PFS and OS was April 2014. The median follow-up period was 34.4 months. Median PFS was 10.0 months (95 % CI 7.8–12.3; Fig. 1). A total of 27 of the 46 eligible patients died due to progression of advanced colorectal cancer. At the time of analysis, the median OS was 34.6 months (95 % CI 19.9–not estimable; Fig. 2).Fig. 1 Kaplan–Meier estimate for progression-free survival (PFS). After a median follow-up time of 34.4 months, the median PFS was 10.0 months (95 % CI 7.8–12.3) Fig. 2 Kaplan–Meier estimate for overall survival (OS). The median OS was 34.6 months (95 % CI 19.9–not estimable) Safety Toxicity data are available for 47 patients treated with a median of 6.0 chemotherapy cycles (range 1–28). Toxicities are summarized in Table 3. Frequently encountered non-hematological toxicities included peripheral neuropathy, hand-foot syndrome, skin hyperpigmentation, fatigue, and gastrointestinal adverse effects such as diarrhea. Most of the non-hematological toxicities were grade 1 or 2. Frequently encountered adverse effects included grade 3 or 4 anorexia and fatigue, which were recorded in 6 (12.8 %) and 4 (8.5 %) of the 47 patients, respectively. No grade 3 or 4 peripheral neuropathy was observed. Other grade 3 or 4 non-hematological toxicities were febrile neutropenia, nausea, hand-foot syndrome, and oral mucositis, each of which occurred in 1 patient. With regard to hematological toxicities, including laboratory disorders, frequently encountered toxicities were grade 3 or 4 neutropenia, leukopenia, thrombocytopenia, anemia, and elevation of AST and ALT, which were recorded in 5 (10.6 %), 2 (4.3 %), 2 (4.3 %), 3 (6.4 %), 11 (23.4 %), and 10 (21.3 %) of the 47 patients, respectively. Frequently encountered bevacizumab-related toxicities were hypertension in 22 patients (46.8 %) and proteinuria in 20 patients (47.6 % of 42 patients). Other bevacizumab-related toxicities included a thromboembolic event, gastrointestinal hemorrhage, and gastrointestinal perforation, each of which occurred in 1 patient (2.1 %).Table 3 Adverse events related to treatment (N = 47)

pertension in 22 patients (46.8 %) and proteinuria in 20 patients (47.6 % of 42 patients). Other bevacizumab-related toxicities included a thromboembolic event, gastrointestinal hemorrhage, and gastrointestinal perforation, each of which occurred in 1 patient (2.1 %).Table 3 Adverse events related to treatment (N = 47) Adverse event Grade 1 Grade 2 Grade 3 Grade 4 Grade 3/4 Febrile neutropenia 0 (0.0 %) 0 (0.0 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Fatigue 17 (36.2 %) 5 (10.6 %) 4 (8.5 %) 0 (0.0 %) 4 (8.5 %) Diarrhea 14 (29.8 %) 7 (14.9 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Nausea 15 (31.9 %) 9 (19.1 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Vomiting 8 (17.0 %) 2 (4.3 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Anorexia 16 (34.0 %) 9 (19.1 %) 6 (12.8 %) 0 (0.0 %) 6 (12.8 %) Alopecia 1 (2.1 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Hand-foot syndrome 18 (38.3 %) 6 (12.8 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Oral mucositis 14 (29.8 %) 1 (2.1 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Dysgeusia 13 (27.7 %) 3 (6.4 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Skin hyperpigmentation 20 (42.6 %) 1 (2.1 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Hypersensitivity 1 (2.1 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Peripheral neuropathy 21 (44.7 %) 18 (38.3 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Blood bilirubin increased 10 (21.3 %) 7 (14.9 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) AST increased 19 (40.4 %) 4 (8.5 %) 10 (21.3 %) 1 (2.1 %) 11 (23.4 %) ALT increased 14 (29.8 %) 4 (8.5 %) 9 (19.1 %) 1 (2.1 %) 10 (21.3 %) ALP increased (N = 45) 19 (42.2 %) 2 (4.4 %) 1 (2.2 %) 0 (0.0 %) 1 (2.2 %) Creatinine increased 10 (21.3 %) 2 (4.3 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Leukopenia 15 (31.9 %) 14 (29.8 %) 2 (4.3 %) 0 (0.0 %) 2 (4.3 %) Neutropenia 8 (17.0 %) 23 (48.9 %) 5 (10.6 %) 0 (0.0 %) 5 (10.6 %) Thrombocytopenia 23 (48.9 %) 7 (14.9 %) 2 (4.3 %) 0 (0.0 %) 2 (4.3 %) Anemia 28 (59.6 %) 12 (25.5 %) 2 (4.3 %) 1 (2.1 %) 3 (6.4 %) Hypertension 9 (19.1 %) 11 (23.4 %) 2 (4.3 %) 0 (0.0 %) 2 (4.3 %) Thromboembolic event 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) 1 (2.1 %) 1 (2.1 %) Proteinuria (N = 42) 14 (33.3 %) 6 (14.3 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Gastrointestinal hemorrhage 0 (0.0 %) 0 (0.0 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Gastrointestinal perforation 0 (0.0 %) 1 (2.1 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %)

(0.0 %) 2 (4.3 %) Thromboembolic event 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) 1 (2.1 %) 1 (2.1 %) Proteinuria (N = 42) 14 (33.3 %) 6 (14.3 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) Gastrointestinal hemorrhage 0 (0.0 %) 0 (0.0 %) 1 (2.1 %) 0 (0.0 %) 1 (2.1 %) Gastrointestinal perforation 0 (0.0 %) 1 (2.1 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %) ALT alanine aminotransferase, AST aspartate aminotransferase, ALT alkaline phosphatase A total of 15 patients (31.9 %) discontinued the protocol because of adverse events—5 patients with neurosensory toxicity, 4 patients with anorexia, 4 patients with fatigue, 2 patients with neutropenia, 2 patients with hypertension, 1 patient with gastrointestinal hemorrhage, 1 patient with diarrhea, 1 patient with a thromboembolic event, and 1 patient with hand-foot syndrome. Post-treatment After treatment with XELOX plus bevacizumab, 18 patients (38.3 %) underwent surgery and 4 patients (8.5 %) received radiation therapy. Thirty-six of the 47 patients (76.6 %) were treated with second-line chemotherapy, and 21 of those 36 patients (58.3 %) received bevacizumab continuously. Two patients received combination chemotherapy with anti-epidermal growth factor receptor (EGFR) antibody as second-line treatment, and 13 patients received the anti-EGFR antibody alone or in combination with cytotoxic agents after second-line chemotherapy. Seven patients (14.9 %) were treated with best supportive care.

wo patients received combination chemotherapy with anti-epidermal growth factor receptor (EGFR) antibody as second-line treatment, and 13 patients received the anti-EGFR antibody alone or in combination with cytotoxic agents after second-line chemotherapy. Seven patients (14.9 %) were treated with best supportive care. Efficacy analyses according to age A summary of efficacy according to patient age (≥70 vs <70 years) is shown in Table 4. There were no significant differences in ORR (42.1 vs 59.3 %, p = 0.370), PFS (9.7 vs 12.3 months, p = 0.179), or OS (23.2 months vs not reached, p = 0.069) based on patient age. Table 4 Summary of treatment efficacy according to age Responses (RECIST-confirmed) N Events ORR (%) 95 % CI p value (fisher’s exact test) ≥70 years 19 8 42.1 20.3–66.5 0.3695 <70 years 27 16 59.3 38.8–77.6 PFS N Median PFS (months) 95 % CI p value (log-rank test) ≥70 years 19 9.7 6.4–11.4 0.1788 <70 years 27 12.3 7.2–14.5 OS N Median OS (months) 95 % CI p value (log-rank test) ≥70 years 19 23.2 11.6–38.9 0.0688 <70 years 27 NE 19.9–NE CI confidence interval, NE not estimable, ORR overall response rate, OS overall survival, PFS progression-free survival, RECIST Response Evaluation Criteria in Solid Tumors Safety analyses according to age A summary of safety according to age (≥70 vs <70 years) is shown in Table 5. The toxicity profile in elderly patients was similar to that in younger patients, with the exceptions of neutropenia and leukopenia; elderly patients had lower rates of neutropenia and leukopenia than younger patients.Table 5 Summary of safety according to age

ty according to age (≥70 vs <70 years) is shown in Table 5. The toxicity profile in elderly patients was similar to that in younger patients, with the exceptions of neutropenia and leukopenia; elderly patients had lower rates of neutropenia and leukopenia than younger patients.Table 5 Summary of safety according to age ≥70 years (N = 20) p value <70 years (N = 27) All grades Grade 3/4 All grades All grades Grade 3/4 Leukopenia 10 (50.0 %) 1 (5.0 %) 0.0650 21 (77.8 %) 1 (3.7 %) Neutropenia 11 (55.0 %) 3 (15.0 %) 0.0044 25 (92.6 %) 2 (7.4 %) Thrombocytopenia 13 (65.0 %) 1 (5.0 %) 0.7583 19 (70.4 %) 1 (3.7 %) Anemia 20 (100.0 %) 2 (10.0 %) 0.1256 23 (85.2 %) 1 (3.7 %) Fatigue 12 (60.0 %) 2 (10.0 %) 0.7674 14 (51.9 %) 2 (7.4 %) Diarrhea 10 (50.0 %) 0 (0.0 %) 0.5661 11 (40.7 %) 0 (0.0 %) Anorexia 14 (70.0 %) 4 (20.0 %) 0.7583 17 (63.0 %) 2 (7.4 %) Nausea 10 (50.0 %) 1 (5.0 %) 0.7729 15 (55.6 %) 0 (0.0 %)

19 (70.4 %) 1 (3.7 %) Anemia 20 (100.0 %) 2 (10.0 %) 0.1256 23 (85.2 %) 1 (3.7 %) Fatigue 12 (60.0 %) 2 (10.0 %) 0.7674 14 (51.9 %) 2 (7.4 %) Diarrhea 10 (50.0 %) 0 (0.0 %) 0.5661 11 (40.7 %) 0 (0.0 %) Anorexia 14 (70.0 %) 4 (20.0 %) 0.7583 17 (63.0 %) 2 (7.4 %) Nausea 10 (50.0 %) 1 (5.0 %) 0.7729 15 (55.6 %) 0 (0.0 %) Discussion Previous randomized and observational trials that have included the XELOX plus bevacizumab regimen as first-line therapy have been mainly conducted in North America and Europe [8, 11, 12]. The NO16966 study [8, 13] reported longer median PFS (9.3 vs 7.4 months; hazard ratio 0.77; 95 % CI 0.6–0.94; p = 0.0026) and median OS (21.6 vs 18.8 months) in the XELOX plus bevacizumab arm than in the XELOX plus placebo arm in a subgroup analysis. Furthermore, another phase III trial (CAIRO2) reported an ORR of 50.0 %, a median PFS of 10.7 months, and a median OS of 20.3 months in patients receiving XELOX plus bevacizumab [11]. In this prospective clinical study of Japanese mCRC patients that included elderly patients, XELOX plus bevacizumab achieved similar efficacy and safety as previous pivotal phase III studies of XELOX plus bevacizumab [8, 13], even in elderly patients. The ORR was 52.2 % (95 % CI 37.0–67.1). The median PFS and median OS were 10.0 months (95 % CI 7.8–13.8) and 34.6 months (95 % CI 19.9–not estimable), respectively. These outcomes are also similar to the favorable results from the phase I/II Japanese clinical trial of XELOX plus bevacizumab in mCRC patients (median PFS 11.0 months; median OS 27.4 months) [9] and to the results from a retrospective analysis of XELOX plus bevacizumab in clinical practice for Japanese mCRC patients (median PFS 290 days; median OS 816 days) [14].

le results from the phase I/II Japanese clinical trial of XELOX plus bevacizumab in mCRC patients (median PFS 11.0 months; median OS 27.4 months) [9] and to the results from a retrospective analysis of XELOX plus bevacizumab in clinical practice for Japanese mCRC patients (median PFS 290 days; median OS 816 days) [14]. Recent phase III trials as first-line chemotherapy plus biologics for mCRC patients yielded a favorable median OS of approximately 30 months [15, 16]. The OS in the present study was also favorable (34.6 months), as was the phase I/II Japanese clinical trial [9]. One reason for the favorable OS in our study may be that most patients received sequential therapy such as chemotherapy, radiotherapy, and surgery. A total of 21 patients (58.3 %) received bevacizumab-containing regimens as second-line chemotherapy in our study. Therefore, improved OS might partially depend on the sequential use of bevacizumab beyond disease progression [17], as it reportedly resulted in statistically significant increases in OS as second-line therapy in combination with irinotecan or oxaliplatin-based regimens. Phase III studies of another class of biologics, anti-EGFR antibodies (cetuximab and panitumumab), showed improved OS in patients with mCRC for whom other treatments had failed [18, 19]. In our study, 2 patients received anti-EGFR antibody-containing regimens as second-line therapy, and 13 patients received an anti-EGFR antibody alone or in combination with cytotoxic agents after second-line therapy. The anti-EGFR antibodies may have partially contributed to the improvement in OS.

had failed [18, 19]. In our study, 2 patients received anti-EGFR antibody-containing regimens as second-line therapy, and 13 patients received an anti-EGFR antibody alone or in combination with cytotoxic agents after second-line therapy. The anti-EGFR antibodies may have partially contributed to the improvement in OS. The toxicity profiles in our study were generally predictable and manageable. Elevated AST, increased ALT, and neutropenia were the most common grade 3 and 4 hematologic toxicities; these events were observed in 23.4, 21.3, and 10.6 % of the patients, respectively. Anorexia and fatigue were the most common grade 3 and 4 non-hematologic toxicities; these events occurred in 12.8 and 8.5 % of the patients, respectively. The incidence of other grade 3 and 4 toxicities including neurotoxicity was extremely low. The incidences of grade 3 or 4 neurotoxicity were reportedly 15 [14] and 17 % [9] in the Japanese trials with XELOX plus bevacizumab. In a number of trials of oxaliplatin-based therapies, neurotoxicity was the most frequently encountered adverse event that led to discontinuation of treatment. In our study, no grade 3 or 4 neurotoxicity was observed, because an administration criterion of oxaliplatin was grade 0 or 1 neurotoxicity. However, the neurotoxicity led to discontinuation of treatment in 5 of 47 patients (10.6 %). The overall rate of protocol discontinuation due to adverse events was 31.9 %, which might be acceptable.

ade 3 or 4 neurotoxicity was observed, because an administration criterion of oxaliplatin was grade 0 or 1 neurotoxicity. However, the neurotoxicity led to discontinuation of treatment in 5 of 47 patients (10.6 %). The overall rate of protocol discontinuation due to adverse events was 31.9 %, which might be acceptable. The treatment schedule and doses selected for our study were identical to those in the NO16966 study [8, 13]. The median relative dose intensities with consideration of both dose reduction and treatment delay/skip in this study were acceptable. These could not be accurately compared to those in the XELOX plus bevacizumab arm of the NO16966 study (ratio of dose received to dose planned), because there might be a difference in calculation method between the studies.

ith consideration of both dose reduction and treatment delay/skip in this study were acceptable. These could not be accurately compared to those in the XELOX plus bevacizumab arm of the NO16966 study (ratio of dose received to dose planned), because there might be a difference in calculation method between the studies. It is notable that 20 of the 47 patients enrolled in our trial were ≥70 years. Previous studies have established the efficacy and safety of combination chemotherapy in elderly patients [20–22]. The addition of bevacizumab to chemotherapy in geriatric populations has also been shown to be effective in observational cohort studies, subgroup analyses, and pooled analyses of cohorts from other randomized trials [23–25]. A phase II study reported that an alternative XELOX plus bevacizumab (AXELOX) regimen was an effective and safe combination for elderly patients (age >70 years) with mCRC [26]. AXELOX treatment includes intravenous infusion of 5 mg/kg bevacizumab, 85 mg/m2 oxaliplatin on day 1, and 750 mg/m2 oral capecitabine twice daily for 1 week of a 2-week cycle. The AXELOX regimen achieved favorable efficacy (ORR 46.8 %; PFS 7.9 months; OS 20.1 months) and low toxicity (incidence of all grade 3 or 4 adverse events <10 %). The low toxicity of AXELOX is thought to be attributable to the low dose intensity of capecitabine, which is 56 % of the international standard dose of XELOX plus bevacizumab used in our study. However, in our study, the efficacy and safety for elderly patients ≥70 years were favorable in a manner similar to results achieved with younger patients <70 years. Our results suggest that the international standard dose of XELOX plus bevacizumab might be effective and safe for both younger and elderly Japanese patients with mCRC. Moreover, the XELOX regimen requires only one visit per 3-week cycle for a 2- or 3-h infusion, and no portable pump is required, which may provide a marked advantage over the FOLFOX regimen in terms of patient convenience.

bevacizumab might be effective and safe for both younger and elderly Japanese patients with mCRC. Moreover, the XELOX regimen requires only one visit per 3-week cycle for a 2- or 3-h infusion, and no portable pump is required, which may provide a marked advantage over the FOLFOX regimen in terms of patient convenience. In conclusion, this study found that first-line XELOX plus bevacizumab showed good tolerability and efficacy in clinical practice for the treatment of mCRC in a Japanese population that included elderly patients. XELOX plus bevacizumab may be considered a routine first-line treatment option for mCRC patients. We thank the participated patients and their families very much. We are indebted to the physicians, all other co-medical staff and Independent Data Monitoring Committee (Shuji Nakano, Kuniaki Shirao and Kenji Sugio) who contributed to this study. We also thank Ms. Sakamoto, Ms. Shimamoto and the other staff at the Clinical Research Support Center Kyushu (CReS Kyushu) for their excellent collection and management of data, secretarial assistance, and any other supports.

toring Committee (Shuji Nakano, Kuniaki Shirao and Kenji Sugio) who contributed to this study. We also thank Ms. Sakamoto, Ms. Shimamoto and the other staff at the Clinical Research Support Center Kyushu (CReS Kyushu) for their excellent collection and management of data, secretarial assistance, and any other supports. Compliance with ethical standards Conflict of interest No disclosures—EO, MS, TTa, HS, NS, TK, TTo, MK and KS; college course financially maintained by private donations that Chugai Pharmaceutical Co., Ltd and Yakult Honsha Co. provide—YM; acceptance such as the researchers from Chugai Pharmaceutical Co., Ltd—YM; grant research funding from Chugai Pharmaceutical Co., Ltd.—HB, YO and YM; grant research funding from Yakult Honsha Co., Ltd.—HB and YM; lecturer’s fee from Chugai Pharmaceutical Co., Ltd.—YE, HB and YM; lecturer’s fee from Yakult Honsha Co., Ltd.—HB and YM.

Introduction In non-small-cell lung cancer (NSCLC), platinum doublet chemotherapy followed by second-line docetaxel monotherapy [1] or pemetrexed maintenance therapy following first-line platinum doublet chemotherapy [2] prolongs survival outcomes for patients with non-squamous NSCLC. Based on the efficacy of these treatments, it has been anticipated that they will improve long-term survival of patients with epidermal growth factor receptor (EGFR) mutation-positive NSCLC after the administration of EGFR tyrosine kinase inhibitors (TKIs). The treatment of NSCLC has changed considerably in recent years. Following the discovery of the pivotal oncogenic role of EGFR in unselected NSCLC [3, 4], the subsequent development of EGFR TKIs provided new therapeutic options for the treatment of this disease. Greater understanding of tumor biology has since led to the discovery that tumors with sensitizing EGFR mutations, particularly the somatic mutations in EGFR exons 19 and 21, respond favorably to EGFR TKIs compared with chemotherapy [5]. To reflect this, EGFR TKIs are recommended in clinical treatment guidelines for NSCLC.

ter understanding of tumor biology has since led to the discovery that tumors with sensitizing EGFR mutations, particularly the somatic mutations in EGFR exons 19 and 21, respond favorably to EGFR TKIs compared with chemotherapy [5]. To reflect this, EGFR TKIs are recommended in clinical treatment guidelines for NSCLC. Currently, gefitinib, erlotinib and afatinib are the only EGFR TKIs approved (US Food and Drug Administration, EU and Japan) for the treatment of EGFR mutation-positive NSCLC [6, 7]. These approvals were supported by data from several phase III clinical trials, which consistently reported that EGFR TKIs demonstrate significant progression-free survival (PFS) benefits compared with standard chemotherapy [8]. Median PFS with first-line gefitinib in EGFR mutation-positive NSCLC ranged between 9.6 and 10.4 months in the pan-Asian IPASS study of gefitinib versus carboplatin/paclitaxel [9], the Japanese NEJ002 study of gefitinib versus carboplatin-paclitaxel [10], and the WJTOG3405 study of gefitinib versus cisplatin/docetaxel [11]. However, despite similar PFS results with gefitinib in these studies, median OS was not consistent; the IPASS study reported a median OS of 21.6 months with gefitinib [9], whereas a longer median OS of 27.7 months was published in the NEJ002 study [10] and a median OS of 34.8 months was reported with gefitinib in the Japanese WJTOG3405 study [11].

results with gefitinib in these studies, median OS was not consistent; the IPASS study reported a median OS of 21.6 months with gefitinib [9], whereas a longer median OS of 27.7 months was published in the NEJ002 study [10] and a median OS of 34.8 months was reported with gefitinib in the Japanese WJTOG3405 study [11]. Median OS with erlotinib in EGFR mutation-positive NSCLC was 22.7 months in the phase III OPTIMAL study of erlotinib versus gemcitabine plus carboplatin [12], and 22.9 months in the phase III EURTAC study of erlotinib versus chemotherapy [13]. However, as these two studies were conducted outside of Japan, the median OS with erlotinib in Japanese patients with EGFR mutation-positive NSCLC is currently unknown. PFS for the single-agent erlotinib arm of the Japanese phase II JO25567 study was 9.7 months [14], which was similar to the 11.8 months median PFS (primary endpoint) reported for the phase II Japanese JO22903 study [15]. Here, we report final OS data with erlotinib monotherapy in the JO22903 study and present exploratory analyses of OS with respect to EGFR mutation subtype. We also evaluated whether OS was impacted by the use of post-progression therapy.

dian PFS (primary endpoint) reported for the phase II Japanese JO22903 study [15]. Here, we report final OS data with erlotinib monotherapy in the JO22903 study and present exploratory analyses of OS with respect to EGFR mutation subtype. We also evaluated whether OS was impacted by the use of post-progression therapy. Patients and methods Study design and patients JO22903 (JapicCTI-101085) was a phase II, single-arm, multicenter, open-label, non-randomized study of first-line erlotinib monotherapy for the treatment of EGFR mutation-positive NSCLC. Full study design information has been previously published [15]. Briefly, the study was conducted at 25 centers in Japan. Patients were aged ≥20 years with stage IIIB/IV or recurrent NSCLC, with no prior chemotherapy, Eastern Cooperative Oncology Group performance status of 0 or 1, and tumors harboring confirmed activating mutations of EGFR (exon 19 deletions or L858R point mutations in exon 21). Patients were excluded if they had symptomatic brain metastases or if they had co-existence or history of interstitial lung disease (ILD). After discontinuation of the protocol treatment, patients were treated at the investigators discretion. JO22903 was carried out in accordance with the Declaration of Helsinki and also the Japanese Good Clinical Practice Guidelines. All patients provided written informed consent for study participation. The study protocol was approved by the local ethics committees.

Patients and methods Study design and patients JO22903 (JapicCTI-101085) was a phase II, single-arm, multicenter, open-label, non-randomized study of first-line erlotinib monotherapy for the treatment of EGFR mutation-positive NSCLC. Full study design information has been previously published [15]. Briefly, the study was conducted at 25 centers in Japan. Patients were aged ≥20 years with stage IIIB/IV or recurrent NSCLC, with no prior chemotherapy, Eastern Cooperative Oncology Group performance status of 0 or 1, and tumors harboring confirmed activating mutations of EGFR (exon 19 deletions or L858R point mutations in exon 21). Patients were excluded if they had symptomatic brain metastases or if they had co-existence or history of interstitial lung disease (ILD). After discontinuation of the protocol treatment, patients were treated at the investigators discretion. JO22903 was carried out in accordance with the Declaration of Helsinki and also the Japanese Good Clinical Practice Guidelines. All patients provided written informed consent for study participation. The study protocol was approved by the local ethics committees. Procedures Full treatment procedures have been published previously [15]. Briefly, patients received oral erlotinib 150 mg/day until disease progression (PD) or unacceptable toxicity. Treatment was interrupted if ILD was suspected; for patients with confirmed ILD diagnosis, erlotinib was discontinued immediately. In cases of gastrointestinal perforation or any grade 4 adverse events (AEs), erlotinib was discontinued. Patients were screened for EGFR mutations in a local or central laboratory; EGFR mutation status was determined using Scorpion ARMS as described previously [15]. Lung and abdominal scans [computed tomography (CT)/magnetic resonance imaging (MRI)] were mandatory at baseline and during treatment until PD. Brain scans were mandatory at baseline (CT/MRI).

utations in a local or central laboratory; EGFR mutation status was determined using Scorpion ARMS as described previously [15]. Lung and abdominal scans [computed tomography (CT)/magnetic resonance imaging (MRI)] were mandatory at baseline and during treatment until PD. Brain scans were mandatory at baseline (CT/MRI). Assessments Tumor response was assessed by an independent review committee (IRC) using Response Evaluable Criteria in Solid Tumours (RECIST) version 1.0. The analysis of safety parameters was descriptive; safety was assessed according to the Medical Dictionary for Regulatory Activities (version 14.0) preferred terms and tabulated by grade. All patients who received at least one dose of study treatment were included in the safety population. A modified intent-to-treat (ITT) population was used for the efficacy analysis, which included all patients from the safety population without major protocol violations. Study endpoints The co-primary endpoints were PFS in the modified ITT population as assessed by IRC according to RECIST version 1.0, and safety. Secondary endpoints included OS and overall response rate. Statistical analyses Kaplan–Meier methodology was used to estimate median and 95 % confidence intervals (CI) for OS, and hazard ratios (HR) were estimated by the use of a Cox model. CI limits were calculated according to the Greenwood method.

Study endpoints The co-primary endpoints were PFS in the modified ITT population as assessed by IRC according to RECIST version 1.0, and safety. Secondary endpoints included OS and overall response rate. Statistical analyses Kaplan–Meier methodology was used to estimate median and 95 % confidence intervals (CI) for OS, and hazard ratios (HR) were estimated by the use of a Cox model. CI limits were calculated according to the Greenwood method. Results Patients Patients were enrolled between April 2010 and October 2010. Median follow-up was 32.2 months. At the time of this final analysis, 103 patients with confirmed EGFR mutations were included in the study. The safety population comprised all 103 patients whilst the modified ITT population comprised 102 patients; one patient was excluded due to a major protocol violation (receipt of incorrect study medication) after enrolment. Baseline patient characteristics have been previously published [15]. Briefly, the majority of patients were female (n = 70), with stage IV disease (n = 74), adenocarcinoma histology (n = 102), and were never-smokers (n = 59).

Results Patients Patients were enrolled between April 2010 and October 2010. Median follow-up was 32.2 months. At the time of this final analysis, 103 patients with confirmed EGFR mutations were included in the study. The safety population comprised all 103 patients whilst the modified ITT population comprised 102 patients; one patient was excluded due to a major protocol violation (receipt of incorrect study medication) after enrolment. Baseline patient characteristics have been previously published [15]. Briefly, the majority of patients were female (n = 70), with stage IV disease (n = 74), adenocarcinoma histology (n = 102), and were never-smokers (n = 59). Efficacy analyses In the modified ITT population at the updated data cut-off, median OS with first-line erlotinib was 36.3 months (95 % CI: 29.4–not reached [NR]) based on the occurrence of 50 events. The 1-year survival rate was 92 % (95 % CI 87–97), the 2-year survival rate was 69 % (95 % CI 60–78) and the 30-month survival rate was 57 % (95 % CI 47–67) (Fig. 1a). Univariate subgroup analyses showed shorter OS in patients with brain metastases at baseline (median OS 22.7 months, 95 % CI 19.6–29.4) and in those with a T790M EGFR mutation (median OS 20.0 months, 95 % CI 15.8–24.2) (Table 1). When analyzed by the specific type of EGFR mutation, median OS was 36.3 months (95 % CI 29.9–NR) versus 34.0 months (95 % CI 24.2–NR), respectively, for patients with exon 19 deletion versus exon 21 L858R point mutation (HR 0.77, 95 % CI 0.44–1.35, p = 0.3662) (Fig. 1b).Fig. 1 Overall survival with a erlotinib monotherapy in the modified ITT population and b by EGFR mutation type

was 36.3 months (95 % CI 29.9–NR) versus 34.0 months (95 % CI 24.2–NR), respectively, for patients with exon 19 deletion versus exon 21 L858R point mutation (HR 0.77, 95 % CI 0.44–1.35, p = 0.3662) (Fig. 1b).Fig. 1 Overall survival with a erlotinib monotherapy in the modified ITT population and b by EGFR mutation type Table 1 Subgroup analysis of median overall survival Characteristics n Events Median OS (months) 95 % CI Gender Female 69 33 36.3 29.4–NR Male 33 17 34.0 23.4–NR Age <75 years 88 43 36.3 28.3–NR ≥75 years 14 7 31.2 18.6–NR Stage IIIB/IV 77 43 31.2 26.5–NR Recurrence 25 7 NR 28.3–NR Smoking status Yes 44 24 31.2 23.4–NR No 58 26 36.3 29.8–NR EGFR mutation status Exon 19 deletion 50 24 36.3 29.8–NR L858R 50 24 34.0 22.7–NR L858R + T790M 2 2 20.0 15.8–24.2 Brain metastases Yes 21 16 22.7 19.6–29.4 No 81 34 NR 32.4–NR CI confidence interval, EGFR epidermal growth factor receptor, OS overall survival, NR not reached Four patients had PD with central nervous system (CNS) progression (Table 2; Fig. 2). Median OS was shorter in patients with CNS PD compared with those without (12.9 months [95 % CI 8.7–27.0] versus 36.3 months [95 % CI 22.9–NR]).Table 2 Characteristics of patients who had CNS progression in erlotinib treatment Number Age (years) Gender ECOG PS EGFR mutation Baseline CNS metastases Erlotinib dose at PD (mg) PFS (days) OS (days) 1 50 M 1 19 del No 100 106 420 2 55 M 1 19 del Yes 150 335 823 3 55 F 0 L858R No 150 168 363 4 73 F 1 L858R Yes 150 80 266

Four patients had PD with central nervous system (CNS) progression (Table 2; Fig. 2). Median OS was shorter in patients with CNS PD compared with those without (12.9 months [95 % CI 8.7–27.0] versus 36.3 months [95 % CI 22.9–NR]).Table 2 Characteristics of patients who had CNS progression in erlotinib treatment Number Age (years) Gender ECOG PS EGFR mutation Baseline CNS metastases Erlotinib dose at PD (mg) PFS (days) OS (days) 1 50 M 1 19 del No 100 106 420 2 55 M 1 19 del Yes 150 335 823 3 55 F 0 L858R No 150 168 363 4 73 F 1 L858R Yes 150 80 266 CNS central nervous system, del deletion, ECOG PS Eastern Cooperative Oncology Group performance status, EGFR epidermal growth factor receptor, F female, M male, OS overall survival, PD progressive disease, PFS progression-free survival Fig. 2 Overall survival by CNS progression

Number Age (years) Gender ECOG PS EGFR mutation Baseline CNS metastases Erlotinib dose at PD (mg) PFS (days) OS (days) 1 50 M 1 19 del No 100 106 420 2 55 M 1 19 del Yes 150 335 823 3 55 F 0 L858R No 150 168 363 4 73 F 1 L858R Yes 150 80 266 CNS central nervous system, del deletion, ECOG PS Eastern Cooperative Oncology Group performance status, EGFR epidermal growth factor receptor, F female, M male, OS overall survival, PD progressive disease, PFS progression-free survival Fig. 2 Overall survival by CNS progression Post-progression therapy Following PD, the majority of patients went on to receive either platinum doublet chemotherapy, with or without bevacizumab (n = 60), further EGFR TKIs (n = 35), or single-agent chemotherapy (n = 39) (Table 3). In terms of second-line therapy, median OS was similar in patients who were treated with platinum doublet chemotherapy or other types of therapy [median OS 33.1 months (95 % CI 27.0–NR) versus NR, respectively; Fig. 3a]. The use of further EGFR TKIs in any line of treatment following PD also had no apparent impact on OS compared with not using an EGFR TKI as post-PD therapy in any line [median OS 32.8 months (95 % CI 26.6–NR) versus 36.3 months (95 % CI 27.8–NR), respectively; Fig. 3b].Table 3 Therapies given upon disease progression (eight patients were receiving study treatment at data collection. Information was unavailable for ten patients)

ith not using an EGFR TKI as post-PD therapy in any line [median OS 32.8 months (95 % CI 26.6–NR) versus 36.3 months (95 % CI 27.8–NR), respectively; Fig. 3b].Table 3 Therapies given upon disease progression (eight patients were receiving study treatment at data collection. Information was unavailable for ten patients) Therapy (n) Second-line therapy All lines of treatment Platinum doublet 46 60 Without bevacizumab 31 41 With bevacizumab 15 21 EGFR TKI 30 35 Erlotinib 21 25 Gefitinib 8 14 Erlotinib + tivantinib 1 1 Erlotinib + pemetrexed 0 1 Gefitinib + pemetrexed 0 1 Single-agent chemotherapy 7 39 Docetaxel + bevacizumab 3 4 Pemetrexed 3 15 Docetaxel 1 24 Pemetrexed + bevacizumab 0 1 Platinum doublet + EGFR TKI 1 2 With erlotinib 1 2 Others 0 16 EGFR TKI epidermal growth factor receptor tyrosine kinase inhibitor Fig. 3 Overall survival by post-PD therapy with a second-line platinum doublet chemotherapy and b EGFR TKI in any line Safety The safety profile of erlotinib did not change at this data update (Table 4) and was as previously reported [15]. The most common all grade treatment-related AEs were rash (82.5 %) and diarrhea (79.6 %), and the most common grade ≥3 treatment-related AEs were rash (14.6 %) and an increase in alanine aminotransferase (8.7 %).Table 4 Treatment-related adverse events, all grades (≥30 %) and grade ≥3 (≥5 %) All grades (≥30 %) Grade ≥3 (≥5 %)

Safety The safety profile of erlotinib did not change at this data update (Table 4) and was as previously reported [15]. The most common all grade treatment-related AEs were rash (82.5 %) and diarrhea (79.6 %), and the most common grade ≥3 treatment-related AEs were rash (14.6 %) and an increase in alanine aminotransferase (8.7 %).Table 4 Treatment-related adverse events, all grades (≥30 %) and grade ≥3 (≥5 %) All grades (≥30 %) Grade ≥3 (≥5 %) n (%) n (%) Rash 85 (82.5) 15 (14.6) Diarrhea 82 (79.6) 0 (0.0) Dry skin 82 (79.6) 0 (0.0) Paronychia 69 (67.0) 0 (0.0) Stomatitis 65 (63.1) 0 (0.0) Pruritus 67 (65.0) 0 (0.0) Decreased appetite 35 (34.0) 0 (0.0) ALT increased 33 (32.0) 15 (14.6) ALT alanine aminotransaminase

Safety The safety profile of erlotinib did not change at this data update (Table 4) and was as previously reported [15]. The most common all grade treatment-related AEs were rash (82.5 %) and diarrhea (79.6 %), and the most common grade ≥3 treatment-related AEs were rash (14.6 %) and an increase in alanine aminotransferase (8.7 %).Table 4 Treatment-related adverse events, all grades (≥30 %) and grade ≥3 (≥5 %) All grades (≥30 %) Grade ≥3 (≥5 %) n (%) n (%) Rash 85 (82.5) 15 (14.6) Diarrhea 82 (79.6) 0 (0.0) Dry skin 82 (79.6) 0 (0.0) Paronychia 69 (67.0) 0 (0.0) Stomatitis 65 (63.1) 0 (0.0) Pruritus 67 (65.0) 0 (0.0) Decreased appetite 35 (34.0) 0 (0.0) ALT increased 33 (32.0) 15 (14.6) ALT alanine aminotransaminase Discussion EGFR TKIs are the standard of care for the first-line treatment of EGFR mutation-positive NSCLC [6, 7]. In Japan, the phase II, single-arm JO22903 study demonstrated efficacy of erlotinib monotherapy in EGFR mutation-positive NSCLC, with a reported median PFS of 11.8 months [15]. In this updated analysis of the JO22903 study, the 30-month OS rate was 57 % (95 % CI 47–67) and median OS was 36.3 months (95 % CI 29.4–NR). These findings represent a more favorable OS than observed in previous studies of first-line erlotinib in EGFR mutation-positive NSCLC outside Japan (median OS range 22.9–26.3 months [12, 13, 16]), and are in line with results from prospective studies of other EGFR TKIs in Japanese populations (median OS range 27.7–34.8 months [10, 11]). Recently, a median OS of 46.9 months was reported for Japanese patients who received afatinib in the LUX-Lung 3 study [17], which was longer than that observed in the entire study population [18]. Across these studies, the median PFS values observed in Japanese and global populations were very similar, at approximately 1 year [10–13, 16–18]. Thus, it seems that the current treatment landscape in Japan may be contributing to a longer OS compared with non-Japanese populations, and that OS in Japanese populations can reasonably be expected to reach beyond 3 years.

ed in Japanese and global populations were very similar, at approximately 1 year [10–13, 16–18]. Thus, it seems that the current treatment landscape in Japan may be contributing to a longer OS compared with non-Japanese populations, and that OS in Japanese populations can reasonably be expected to reach beyond 3 years. Although patients with brain metastases have a poor prognosis, which is reflected by the shorter median OS for this subgroup, the findings of this present analysis suggest that erlotinib could be considered effective for patients with brain metastases, as only four patients had CNS progression. This finding is consistent with the phase II ASPIRATION study in Asian patients, which reported that just 4.3 % of patients treated with post-PD erlotinib had new brain lesions [19]. This role for EGFR TKIs has also been observed in populations not restricted to Japanese or Asian patients [20–22]. A case series of 15 patients with NSCLC with EGFR mutations and CNS metastases who received cerebrospinal fluid concentration (CSF) examinations during EGFR TKI treatment provides further evidence to support this conclusion. In this case series, CNS response rate was 57 % with a favorable penetration rate of erlotinib in the CSF [23]. The penetration rate of erlotinib may be dependent on its affinity for p-glycoprotein, which pumps drugs out of the CNS. These findings suggest that erlotinib has a favorable pharmacokinetic profile as a treatment option for patients with brain metastases.

with a favorable penetration rate of erlotinib in the CSF [23]. The penetration rate of erlotinib may be dependent on its affinity for p-glycoprotein, which pumps drugs out of the CNS. These findings suggest that erlotinib has a favorable pharmacokinetic profile as a treatment option for patients with brain metastases. Patients with an exon 19 deletion appeared to have longer OS in our analysis than those with exon 21 L858R EGFR mutation-positive NSCLC. This is similar to the results of a meta-analysis of seven trials (n = 1649), which concluded that patients with an exon 19 deletion had better efficacy outcomes than patients with exon 21 L858R EGFR mutation-positive NSCLC, regardless of which EGFR TKI they received [24]. These data suggest that patients with exon 19 deletion and exon 21 L858R EGFR mutation are clinically distinct populations that should be evaluated further.

th an exon 19 deletion had better efficacy outcomes than patients with exon 21 L858R EGFR mutation-positive NSCLC, regardless of which EGFR TKI they received [24]. These data suggest that patients with exon 19 deletion and exon 21 L858R EGFR mutation are clinically distinct populations that should be evaluated further. In the present study, there was no apparent difference in OS according to subsequent treatments. Median OS was similar for patients who received EGFR TKIs as post-PD therapy (n = 36), which were mainly continuous erlotinib administration following RECIST PD (n = 21) (Table 3), and for those who did not. In contrast to our findings, in a retrospective study of patients with activating EGFR mutations (n = 123) who were treated with EGFR TKIs, OS showed a trend in favor of continuing versus discontinuing EGFR TKI treatment following RECIST PD (33.0 versus 21.2 months, respectively; p = 0.054) [25]. Furthermore, a retrospective clinical modeling study that evaluated the usefulness of EGFR TKI failure pattern for selecting subsequent management, suggested that the efficacy of EGFR TKI continuation differed between patients with gradual progression, local progression, and dramatic progression [26]. Thus, one hypothesis for the inconsistency between studies is the difference in the EGFR TKI failure pattern. Meanwhile, in the present study, various EGFR TKIs were used as post-PD therapy (i.e., erlotinib beyond progression, erlotinib re-challenge after another treatment, and other therapies), which should be noted as one of the limitations. As effective post-PD therapy options are important for patients with disease recurrence, any benefit of EGFR TKI re-administration or continuation after PD requires further study.

b beyond progression, erlotinib re-challenge after another treatment, and other therapies), which should be noted as one of the limitations. As effective post-PD therapy options are important for patients with disease recurrence, any benefit of EGFR TKI re-administration or continuation after PD requires further study. At this updated analysis, no new safety signals for erlotinib were observed; single-agent erlotinib was well tolerated and had an acceptable and manageable safety profile in EGFR mutation-positive NSCLC. The safety profile of erlotinib was also in line with previous studies of first-line erlotinib [13], with the most common AEs being rash and diarrhea. In conclusion, single-agent erlotinib resulted in a median OS of 36.3 months in the first-line treatment of EGFR mutation-positive NSCLC. Subgroup analyses of OS suggested that the presence of brain metastases was a negative prognostic factor, as these patients had shorter median OS compared with other subgroups. No further differences in OS between specific EGFR subgroups were observed. Although many patients went on to receive additional EGFR TKI therapy following progression, there was no significant difference in median OS for patients who received EGFR TKI as post-PD therapy compared with those who did not. The findings of this single-arm study should be validated in randomized controlled trials. An erratum to this article is available at http://dx.doi.org/10.1007/s10147-016-1052-3.

In conclusion, single-agent erlotinib resulted in a median OS of 36.3 months in the first-line treatment of EGFR mutation-positive NSCLC. Subgroup analyses of OS suggested that the presence of brain metastases was a negative prognostic factor, as these patients had shorter median OS compared with other subgroups. No further differences in OS between specific EGFR subgroups were observed. Although many patients went on to receive additional EGFR TKI therapy following progression, there was no significant difference in median OS for patients who received EGFR TKI as post-PD therapy compared with those who did not. The findings of this single-arm study should be validated in randomized controlled trials. An erratum to this article is available at http://dx.doi.org/10.1007/s10147-016-1052-3. The authors would like to thank all participating physicians, registered patients, Tomomi Shimura for data analysis and Gardiner-Caldwell Communications for medical writing assistance. Medical writing assistance was funded by Chugai Pharmaceutical Co. Ltd.

An erratum to this article is available at http://dx.doi.org/10.1007/s10147-016-1052-3. The authors would like to thank all participating physicians, registered patients, Tomomi Shimura for data analysis and Gardiner-Caldwell Communications for medical writing assistance. Medical writing assistance was funded by Chugai Pharmaceutical Co. Ltd. Compliance with ethical standards Conflict of interest Noboru Yamamoto received honoraria from AstraZeneca, Eli Lilly, Pfizer and Chugai Pharmaceutical. He also received research funding from Daiichi-Sankyo, Kyowa-Kirin, Chugai Pharmaceutical, Eli Lilly, Takeda, Quintiles, Bristol-Myers Squibb, Astellas, Taiho, Pfizer, Novartis and Eisai. Koichi Goto received research funding from Chugai Pharmaceutical. Makoto Nishio received honoraria from Chugai Pharmaceutical, Boehringer Ingelheim and AstraZeneca. He also received research funding from Chugai Pharmaceutical and AstraZeneca. Kenichi Chikamori received honoraria from Chugai Pharmaceutical. He also received research funding from Chugai Pharmaceutical and Bristol-Myers Squibb. Toyoaki Hida received honoraria from Chugai Pharmaceutical, Taiho, AstraZeneca and Boehringer Ingelheim. He also received research funding from Chugai Pharmaceutical, Taiho, AstraZeneca, Boehringer Ingelheim, Clovis Oncology and Astellas. Makoto Maemondo received honoraria from Chugai Pharmaceutical, AstraZeneca and Boehringer Ingelheim. He also received research funding from Chugai Pharmaceutical, AstraZeneca and Boehringer Ingelheim. Nobuyuki Katakami received honoraria and research funding from Chugai Pharmaceutical. Toshiyuki Kozuki received honoraria from Chugai Pharmaceutical, AstraZeneca, Eli Lilly, Pfizer, Kyowa Kirin, Sanofi, Taiho and Roche. He also received research funding from Chugai Pharmaceutical, Bristol-Myers Squibb and Pfizer. Hiroshige Yoshioka received honoraria from Boehringer Ingelheim, Eli Lilly and Chugai Pharmaceutical. He also received research funding from Chugai Pharmaceutical, Novartis, Takeda, Pfizer, Merck-Serono, Eli Lilly and Kyowa Kirin. Takashi Seto received honoraria and lecture fees from Chugai Pharmaceutical. Kosei Tajima is an employee of Chugai Pharmaceutical. Tomohide Tamura received honoraria from Chugai Pharmaceutical, Taiho, Ono, Eli Lilly, Eisai, Yakult Honsha, Boehringer Ingelheim and Bristol-Myers Squibb.

Introduction Colorectal cancer is the third most common malignant disease and ranks as the third leading cause of cancer-related death [1]. The standard treatment for colorectal cancer is surgical resection. Patients with postoperative complications have been reported to have poor long-term outcomes [2, 3]. The perioperative complications is associated with not only short-term disadvantages such as a compromised quality of life, but also with increased medical costs, delayed initiation of postoperative adjuvant chemotherapy [4], high recurrence rates [5], and poor long-term outcomes [5–7]. Rectal cancer has higher incidences of infectious complications and anastomotic leakage than colon cancer [8, 9]. In particular, lower rectal cancer is an important risk factor for anastomotic leakage. The development of anastomotic leakage has been reported to increase the rate of local recurrence [10]. The international standard treatment for rectal cancer is multidisciplinary treatment, including preoperative chemoradiotherapy [11]. Surgical procedures can be selected from a number of options, including sphincter-preserving surgery, transanal local excision, and defunctioning stomas. The ability to predict the risk of complications before treatment would most likely facilitate selection of the treatment policy optimally suited for individual patients.

ical procedures can be selected from a number of options, including sphincter-preserving surgery, transanal local excision, and defunctioning stomas. The ability to predict the risk of complications before treatment would most likely facilitate selection of the treatment policy optimally suited for individual patients. To date, various risk scores have been proposed for patients to undergo elective surgery, including the Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score (E-PASS CRS) [12], Surgical Apgar Score (SAS) [13], the Prognostic Nutritional Index (PNI) proposed by Onodera et al. [14], the Neutrophil-to-lymphocyte Ratio (NLR) [15], and the Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM) [16]. In addition, Colorectal-POSSUM (CR-POSSUM) [19] has been proposed for patients to undergo elective surgery for colorectal cancer. In the present study, we evaluated risk scores as a means of predicting perioperative complications in patients who underwent radical surgery for rectal cancer. Patients and methods From January 2003 through December 2013, a total of 392 patients underwent radical surgery for rectal adenocarcinoma in our hospital. We excluded 131 patients who underwent emergency surgery or laparoscopic surgery and studied the remaining 260 patients. Data on these patients were retrospectively collected to estimate the incidences of complications within 30 days after surgery and to compare the value of each risk score for predicting the probability of complications.

ents who underwent emergency surgery or laparoscopic surgery and studied the remaining 260 patients. Data on these patients were retrospectively collected to estimate the incidences of complications within 30 days after surgery and to compare the value of each risk score for predicting the probability of complications. Risk evaluation scores We studied the following risk evaluation scores: Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score (E-PASS CRS) [12], Surgical Apgar Score (SAS) [13], Prognostic Nutritional Index (PNI) [14], Colorectal POSSUM (CR-POSSUM) [16], and Neutrophil-to-lymphocyte Ratio (NLR) [15]. The preoperative general condition, concomitant diseases, and complications of each patient were examined from their medical records. Surgical information, such as intraoperative vital signs and bleeding volume, was obtained from each patient’s surgical and anesthesiologic records.

ophil-to-lymphocyte Ratio (NLR) [15]. The preoperative general condition, concomitant diseases, and complications of each patient were examined from their medical records. Surgical information, such as intraoperative vital signs and bleeding volume, was obtained from each patient’s surgical and anesthesiologic records. E-PASS CRS was calculated as described by Haga et al. [12]. The preoperative risk score, reflecting the patient’s physiological status before surgery, the surgical stress score, reflecting the degree of surgical invasion, and the comprehensive risk score, representing the overall risk associated with preoperative risk and surgical stress, were calculated for each patient. SAS was calculated from the intraoperative bleeding volume, the minimal heart rate, and the minimal mean blood pressure, as described by Gawande et al. [13]. PNI was calculated by the following formula, proposed by Onodera et al. [14]: PNI = 10 × serum albumin level (g/dL) + 0.05 × total lymphocyte count (mm3). CR-POSSUM was calculated as reported by Tekkis et al. [16, 17] on the basis of the Physiological Score (PS), derived from age and the results of preoperative assessments of cardiac dysfunction, systolic blood pressure, heart rate, serum hemoglobin concentration, and urea nitrogen concentration, and the Operative Severity Score (OS), derived from surgical invasion, Duke’s classification, and intraoperative findings. The CR-POSSUM score was the sum of PS and OS. NLR was calculated using blood samples obtained at initial presentation. In patients who received preoperative chemoradiotherapy, the score was calculated before chemoradiotherapy.

ore (OS), derived from surgical invasion, Duke’s classification, and intraoperative findings. The CR-POSSUM score was the sum of PS and OS. NLR was calculated using blood samples obtained at initial presentation. In patients who received preoperative chemoradiotherapy, the score was calculated before chemoradiotherapy. Classification and severity of complications We studied the following 4 types of complications occurring within 30 days after surgery: all complications, infectious complications (wound infection, inflammation of the pelvic dead space, and intraabdominal abscess), anastomotic leakage, and intestinal obstruction. All complications included exacerbation of underlying disease. The diagnosis of anastomotic leakage was based on the properties of drainage fluid or radiographic findings. The severity of complications was evaluated according to the Clavien-Dindo classification [18]. All complications, infectious complications, and intestinal obstruction of Clavien-Dindo grade 3a or higher that required surgical intervention and anastomotic leakage of Clavien-Dindo grade 3b or higher that required reoperation were defined as complications.

aluated according to the Clavien-Dindo classification [18]. All complications, infectious complications, and intestinal obstruction of Clavien-Dindo grade 3a or higher that required surgical intervention and anastomotic leakage of Clavien-Dindo grade 3b or higher that required reoperation were defined as complications. Evaluation of risk factors for anastomotic leakage To investigate the risk factors for anastomotic leakage, we excluded 76 patients with a diverting stoma at initial surgery from the 187 patients who underwent low anterior resection and studied the remaining 111 patients. To compare the accuracy of each score for predicting the risk of anastomotic leakage, we calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy rate for each score. We then compared the values among the different scores. A multivariate analysis was then performed to identify risk factors for anastomotic leakage in patients without a diverting stoma at initial surgery. The model included factors that were significantly related to anastomotic leakage in our study, as well as sex, body-mass index (BMI), smoking history, the American Society of Anesthesiologists (ASA) classification, tumor location, and preoperative chemoradiotherapy, ypStage, which have been reported to be risk factors for anastomotic leakage in patients with rectal cancer [19, 20].

stomotic leakage in our study, as well as sex, body-mass index (BMI), smoking history, the American Society of Anesthesiologists (ASA) classification, tumor location, and preoperative chemoradiotherapy, ypStage, which have been reported to be risk factors for anastomotic leakage in patients with rectal cancer [19, 20]. Statistical analysis The cutoff value (COV) for each score was calculated by risk evaluation analysis, performed using receiver operating characteristic curves (ROC) in which the presence of complications was considered a positive result. The patients were divided into 2 groups according to whether their score was less than the COV or equal to or greater than the COV, and the incidence of complications was compared. For risk evaluation analysis, the COV of the PNI was set at 40, as recommended by Onodera et al. [14]. The 2 groups were compared with the use of the Chi square test. Multiple logistic regression analysis was performed. P values of less than 0.05 were considered to indicate statistical significance. All statistical analyses were performed with the use of JMP® 10 software (SAS Institute Inc., Cary, NC, USA). This study was approved by the institutional review board of Tokai University (15R-217). Results The patients’ characteristics are shown in Table 1. The surgical procedure was lower anterior resection (LAR) in 187 patients and abdominoperineal resection (APR) in 73 patients. A total of 202 patients (77%) received preoperative chemoradiotherapy.Table 1 Patients’ characteristics

This study was approved by the institutional review board of Tokai University (15R-217). Results The patients’ characteristics are shown in Table 1. The surgical procedure was lower anterior resection (LAR) in 187 patients and abdominoperineal resection (APR) in 73 patients. A total of 202 patients (77%) received preoperative chemoradiotherapy.Table 1 Patients’ characteristics Variable n (%) Sex Male 190 (73) Female 70 (28) Age (year) Range 28–92 Median 63 Location of the tumor Upper and middle 112 (43) Lower 148 (57) Neoadjuvant CRT Yes 202 (77) No 58 (23) Concurrent chemotherapy UFT 37 (19) S-1 165 (81) Radiation 40 or 45 Gy 183 (91) 20 Gy and IOR 19 (9) Surgical procedure LAR 187 (70) APR 73 (30) Histological type Well 129 (50) Moderate 92 (35) Poor 1 (0.3) Mucinous 12 (5) pCR 26 (10) Lymphatic invasion Positive 121 (46) Negative 138 (53) Unknown 1 (0.3) Venous invasion Positive 126 (48) Negative 133 (51) Unknown 1 (0.3) Pathological stage (include ypStage) 0 29 (11) I 79 (30) II 78 (31) III 73 (28) CRT chemoradiotherapy; LAR: low anterior resection; APR abdominoperineal resection; pCR pathological complete response We used preoperative chemoradiotherapy for patients with clinical Stage II or III locally advanced rectal adenocarcinoma according to the NCCN guideline [11]. Tumor location was defined according to the Japanese criteria. The detail was reported previously [21].

CRT chemoradiotherapy; LAR: low anterior resection; APR abdominoperineal resection; pCR pathological complete response We used preoperative chemoradiotherapy for patients with clinical Stage II or III locally advanced rectal adenocarcinoma according to the NCCN guideline [11]. Tumor location was defined according to the Japanese criteria. The detail was reported previously [21]. One or more complication developed in 56 patients (21.5%). Nineteen patients (7.3%) had infectious complications, 16 (6.1%) had intestinal obstruction, and 12 (4.6%) had other complications. Anastomotic leakage was occurred in 12 patients (10.8%) out of 111 patients who received low anterior resection without diverting stomas (Table 2).Table 2 Postoperative complications according to the Cravien-Dindo grade Complication C–D grade n (%) Infectious complication 3a 18 (27.6) 3b 1 (1.5) 4 0 (0) Anastomotic leakage 3a 6 (27.2) 3b 11 (55.0) 4 1 (5.0) Bowel obstruction 3a 8 (26.6) 3b 8 (26.6) 4 0 (0) C–D grade Cravien-Dindo grade

One or more complication developed in 56 patients (21.5%). Nineteen patients (7.3%) had infectious complications, 16 (6.1%) had intestinal obstruction, and 12 (4.6%) had other complications. Anastomotic leakage was occurred in 12 patients (10.8%) out of 111 patients who received low anterior resection without diverting stomas (Table 2).Table 2 Postoperative complications according to the Cravien-Dindo grade Complication C–D grade n (%) Infectious complication 3a 18 (27.6) 3b 1 (1.5) 4 0 (0) Anastomotic leakage 3a 6 (27.2) 3b 11 (55.0) 4 1 (5.0) Bowel obstruction 3a 8 (26.6) 3b 8 (26.6) 4 0 (0) C–D grade Cravien-Dindo grade Evaluation of risk scores and the incidences of complications (Table 3) E-PASS CRS was significantly related to the incidences of all complications, infectious complications, and anastomotic leakage. PNI was significantly related to the incidences of all complications and intestinal obstruction. NLR was significantly related to the incidences of all complications, infectious complications, and anastomotic leakage. SAS was significantly related to the incidence of infectious complications. CR-POSSUM was significantly related to the incidences of all complications, infectious complications, and intestinal obstruction.Table 3 Relation between the predictive scoring systems and the incidence of complication

and anastomotic leakage. SAS was significantly related to the incidence of infectious complications. CR-POSSUM was significantly related to the incidences of all complications, infectious complications, and intestinal obstruction.Table 3 Relation between the predictive scoring systems and the incidence of complication Complication Cut-off value Incidence (%) OR 95% CI p value E-PASS CRS All complication <0.294 16/134 (11.9) Reference ≥0.294 40/126 (31.7) 3.45 1.84–6.73 <0.0001 Infectious complication <0.294 5/134 (3.7) Reference ≥0.294 14/126 (11.1) 3.23 1.19–10.23 0.0202 Anastomotic leakage <0.294 4/72 (5.5) Reference ≥0.294 8/39 (20.5) 4.38 1.28–17.46 0.0183 Bowel obstruction <0.294 5/134 (3.7) Reference ≥0.294 11/126 (8.7) 2.46 0.86–8.02 0.0906 PNI All complication ≥41 29/179 (16.1) Reference <40 27/81 (33.3) 2.60 1.41–4.80 0.0022 Infectious complication ≥41 12/179 (6.7) Reference <40 7/81 (8.6) 1.32 0.47–3.42 0.5750 Anastomotic leakage ≥41 8/85 (9.4) Reference <40 4/26 (15.4) 1.75 0.48–6.35 0.3907 Bowel obstruction ≥41 7/179 (3.9) Reference ≥40 9/81 (11.1) 3.08 1.10–8.94 0.0311 NLR All complication <2.21 17/123 (13.8) Reference ≥2.21 39/137 (28.5) 2.50 1.35–4.81 0.0033 Infectious complication <2.21 4/123 (3.2) Reference ≥2.21 15/137 (10.9) 3.65 1.28–13.11 0.0138 Anastomotic leakage <2.21 10/62 (16.1) Reference ≥2.21 2/49 (4.08) 4.51 1.11–30.38 0.0329 Bowel obstruction <2.21 5/123 (4.0) Reference ≥2.21 11/137 (8.0) 2.06 0.69–6.10 0.1842 Surgcal apgar score All complication ≥5 48/239 (20.0) Reference <5 8/21 (38.1) 2.46 0.92–6.18 0.0692 Infectious complication ≥5 14/239 (5.8) Reference <5 5/21 (23.8) 5.02 1.47–15.09 0.0119 Anastomotic leakage ≥5 12/108 (11.1) <5 0/3 – – 0.5410 Bowel obstruction ≥5 15/239 (6.3) Reference <5 1/21 (4.8) 0.74 0.09–5.94 0.7819 CR-POSSUM All complication <18 18/124 (14.5) Reference ≥18 38/136 (27.9) 2.26 1.22–4.29 0.0086 Infectious complication <18 3/124 (2.4) Reference ≥18 16/136 (11.8) 5.37 1.52–18.93 0.0038 Anastomotic leakage <18 7/75 (9.3) Reference ≥18 5/36 (13.8) 1.30 0.37–4.24 0.6660 Bowel obstruction <18 5/124 (4.0) Reference ≥18 11/136 (8.1) 2.09 1.73–6.81 0.1681

18/124 (14.5) Reference ≥18 38/136 (27.9) 2.26 1.22–4.29 0.0086 Infectious complication <18 3/124 (2.4) Reference ≥18 16/136 (11.8) 5.37 1.52–18.93 0.0038 Anastomotic leakage <18 7/75 (9.3) Reference ≥18 5/36 (13.8) 1.30 0.37–4.24 0.6660 Bowel obstruction <18 5/124 (4.0) Reference ≥18 11/136 (8.1) 2.09 1.73–6.81 0.1681 OR odds ratio; 95 % CI 95% confidence interval; E-PASS CRS Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score; SAS Surgical Apgar Score; PNI Onodera’s prognostic nutritional index; NLR neutrophilic lymphocytes ratio; CR-POSSUM colorectal physiological and operative severity score for the enumeration of mortality and morbidity Evaluation of risk factors for anastomotic leakage Univariate analysis showed that E-PASS CRS and NLR were risk factors related to anastomotic leakage (Table 4). The ASA classification is included in E-PASS CRS and was therefore excluded. A multivariate analysis was performed, including 8 variables, i.e., 6 variables that have been reported to be risk factors for suture failure in patients with rectal cancer (sex, BMI, smoking status, tumor location, pStage, and the presence or absence of preoperative chemoradiotherapy) in addition to E-PASS CRS and NLR. The results showed that E-PASS CRS (p = 0.0075, odds ratio = 6.85), and NLR (p = 0.0089, odds ratio = 8.24) were independent risk factors for anastomotic leakage (Table 5).Table 4 Univariate analysis of anastomotic leakage

and the presence or absence of preoperative chemoradiotherapy) in addition to E-PASS CRS and NLR. The results showed that E-PASS CRS (p = 0.0075, odds ratio = 6.85), and NLR (p = 0.0089, odds ratio = 8.24) were independent risk factors for anastomotic leakage (Table 5).Table 4 Univariate analysis of anastomotic leakage Variable Patients with leakage Patients without leakage p value Sex Male 11 122 Female 1 53 0.1045 Age Range 43–77 28–92 Median 64.5 63 0.5691 BMI ≥25 4 32 <25 8 143 0.2009 Smoking history No 4 86 Yes 8 89 0.3168 Location of the tumor Upper or middle 7 104 Lower 5 71 0.9404 pStage 0 4 21 I 3 54 II 2 49 III 3 51 0.2083 CRT No 2 40 Yes 10 135 0.6191 E-PASS CRS <0.294 4 104 ≥0.294 8 71 0.0767 NLR <2.21 2 86 ≥2.21 10 89 0.0292 BMI body mass index, CRT chemoradiotherapy, E-PASS CRS Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score, NLR neutrophilic lymphocytes ratio Table 5 Multivariate logistic regression analysis of anastomotic leakage OR 95% CI p value Sex Female Reference Male 3.66 0.60–71.08 0.1778 BMI 1.31 1.02–1.72 0.0775 Smoking history No Reference Yes 1.76 0.47–7.72 0.4018 Location of the tumor Upper or middle Reference Lower 1.59 0.42–6.61 0.4903 CRT No Reference Yes 2.13 0.42–16.82 0.3808 pStage 0 Reference 1 5.44 0.59–60.06 0.1317 2 7.60 0.90–84.88 0.0617 3 4.92 0.61–48.42 0.1322 E-PASS CRS <0.294 Reference ≥0.294 6.85 1.63–39.63 0.0075 NLR <2.21 Reference ≥2.21 8.24 1.61–76.07 0.0089

Location of the tumor Upper or middle Reference Lower 1.59 0.42–6.61 0.4903 CRT No Reference Yes 2.13 0.42–16.82 0.3808 pStage 0 Reference 1 5.44 0.59–60.06 0.1317 2 7.60 0.90–84.88 0.0617 3 4.92 0.61–48.42 0.1322 E-PASS CRS <0.294 Reference ≥0.294 6.85 1.63–39.63 0.0075 NLR <2.21 Reference ≥2.21 8.24 1.61–76.07 0.0089 OR odds ratio, 95 % CI 95% confidence interval, BMI body mass index, CRT chemoradiotherapy, E-PASS CRS Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score, NLR neutrophilic lymphocytes ratio The sensitivity, specificity, PPV, NPV, and accuracy rate of the 5 scores for the prediction of anastomotic leakage were calculated (Table 6). E-PASS CRS and NLR had higher PPV, NPV, and accuracy rates than the other scores.Table 6 Accuracy rate of anastomotic leakage according to predictive scoring systems Score COV OR 95% CI p value Accuracy rate (%) Sensitivity (%) Specificity (%) PPV (%) NPV (%) E-PASS CRS 0.294 4.38 1.28–17.46 0.0183 68.4 66.6 68.6 20.5 94.4 PNI 40 1.75 0.48–6.35 0.3907 27.0 66.6 22.2 9.4 84.6 NLR 2.21 4.51 1.11–30.38 0.0329 51.3 83.3 47.4 16.1 95.9 SAS 5 0 – 0.5410 13.5 100 96.9 11.0 100 CR-POSSUM 18 1.30 0.37–4.24 0.6660 34.2 58.3 31.3 9.3 86.1

ensitivity (%) Specificity (%) PPV (%) NPV (%) E-PASS CRS 0.294 4.38 1.28–17.46 0.0183 68.4 66.6 68.6 20.5 94.4 PNI 40 1.75 0.48–6.35 0.3907 27.0 66.6 22.2 9.4 84.6 NLR 2.21 4.51 1.11–30.38 0.0329 51.3 83.3 47.4 16.1 95.9 SAS 5 0 – 0.5410 13.5 100 96.9 11.0 100 CR-POSSUM 18 1.30 0.37–4.24 0.6660 34.2 58.3 31.3 9.3 86.1 COV cut off value; OR odds ratio; 95 % CI 95% confidence interval; PPV positive predictive value; NPV negative predictive value; E-PASS CRS Estimation of Physiologic Ability and Surgical Stress Comprehensive Risk Score; SAS Surgical Apgar Score; PNI Onodera’s prognostic nutritional index; NLR neutrophilic lymphocytes ratio; CR-POSSUM Colorectal physiological and operative severity score for the enumeration of mortality and morbidity Discussion The development of perioperative complications in patients with rectal cancer has been reported to delay the start of adjuvant chemotherapy [4], potentially leading to poor long-term outcomes [5–7]. The ability to predict the likelihood of postoperative complications before starting treatment would thus facilitate the design of personalized treatment strategies for individual patients, including the selection of surgical procedures such as diverting colostomy. Complications following rectal cancer surgery consisted several categories, such as cardiovascular, respiratory, urinary, wound infection, intraabdominal abscess and anastomotic leakage. However, we selected infectious complications, anastomotic leakage, intestinal obstruction and overall complications in the present study.

ications following rectal cancer surgery consisted several categories, such as cardiovascular, respiratory, urinary, wound infection, intraabdominal abscess and anastomotic leakage. However, we selected infectious complications, anastomotic leakage, intestinal obstruction and overall complications in the present study. E-PASS is a severity score quantifying general condition and surgical risk. It has been reported to be related to postoperative complications and overall survival in elderly patients with colon cancer and those with gastric cancer [22, 23]. Haga et al. reported that E-PASS is useful for predicting the risk of anastomotic leakage in patients who have undergone gastrointestinal surgery [24, 25]. The blood lymphocyte count is an index of immune status that is used to calculate several scores. The PNI proposed by Onodera et al. is calculated from the serum albumin concentration and lymphocyte count and is a useful index of immune and nutritional status in patients with gastrointestinal cancer [14]. Patients with colorectal cancer and a low PNI have a poor prognosis [26]. A PNI of less than the COV of 45.5 has been reported to be an independent risk factor for serious complications, such as myocardial infarction and pulmonary embolism [27].

mune and nutritional status in patients with gastrointestinal cancer [14]. Patients with colorectal cancer and a low PNI have a poor prognosis [26]. A PNI of less than the COV of 45.5 has been reported to be an independent risk factor for serious complications, such as myocardial infarction and pulmonary embolism [27]. NLR is a useful prognostic factor in patients with colorectal cancer [15, 28]. A low NLR before surgery is related to disease-free survival and overall survival [29, 30]. NLR on postoperative day 1 is a risk factor for infectious complications [31, 32]. SAS is related to surgical outcomes and is calculated on the basis of bleeding volume, intraoperative minimal blood pressure, and minimal heart rate, and is thus simpler to use than CR-POSSUM and E-PASS. Patients with a high SAS after colectomy have a low incidence of complications after discharge within 30 days after surgery [33]. The modified Surgical Apgar Score (mSAS), which uses a different COV for intraoperative bleeding volume from the SAS, has been reported to be useful for predicting complications after gastrectomy [34].

th a high SAS after colectomy have a low incidence of complications after discharge within 30 days after surgery [33]. The modified Surgical Apgar Score (mSAS), which uses a different COV for intraoperative bleeding volume from the SAS, has been reported to be useful for predicting complications after gastrectomy [34]. CR-POSSUM is a modified score based on POSSUM [11], a severity score that quantifies general condition and surgical risk in patients with colorectal disease. CR-POSSUM is useful for predicting the risk of death within 30 days after surgery for colorectal cancer [17]. In our study, E-PASS CRS, SAS, PNI, NLR, and CR-POSSUM were useful methods for evaluating the risk of complications in patients who underwent radical surgery for rectal cancer. A multivariate analysis was performed including the 8 variables of E-PASS CRS and NLR, found to be significantly related to anastomotic leakage in our study, as well as sex, BMI, smoking history, tumor location, and the presence or absence of preoperative chemoradiotherapy, pStage which that have been reported to be risk factors for anastomotic leakage in patients with rectal cancer. The results showed that E-PASS CRS, and NLR were independent risk factors for anastomotic leakage. McDermott et al. reported co-morbidity is a risk factor for colorectal anastomotic leakage [19]. E-PASS CRS is calculated from factors including co-morbidity. NLR has not been reported to associate with anastomotic leakage in colorectal cancer up to now. In addition, E-PASS CRS and NLR had higher PPV, NPV, and accuracy rates than the other scores.

CR-POSSUM is a modified score based on POSSUM [11], a severity score that quantifies general condition and surgical risk in patients with colorectal disease. CR-POSSUM is useful for predicting the risk of death within 30 days after surgery for colorectal cancer [17]. In our study, E-PASS CRS, SAS, PNI, NLR, and CR-POSSUM were useful methods for evaluating the risk of complications in patients who underwent radical surgery for rectal cancer. A multivariate analysis was performed including the 8 variables of E-PASS CRS and NLR, found to be significantly related to anastomotic leakage in our study, as well as sex, BMI, smoking history, tumor location, and the presence or absence of preoperative chemoradiotherapy, pStage which that have been reported to be risk factors for anastomotic leakage in patients with rectal cancer. The results showed that E-PASS CRS, and NLR were independent risk factors for anastomotic leakage. McDermott et al. reported co-morbidity is a risk factor for colorectal anastomotic leakage [19]. E-PASS CRS is calculated from factors including co-morbidity. NLR has not been reported to associate with anastomotic leakage in colorectal cancer up to now. In addition, E-PASS CRS and NLR had higher PPV, NPV, and accuracy rates than the other scores. All 5 evaluation scores assessed in our study can be calculated from general laboratory data and surgical course. However, E-PASS CRS, SAS, and CR-POSSUM include variables measured during surgery and therefore cannot be calculated until after surgery. Only PNI and NLR can be used to preoperatively evaluate risk. NLR was significantly related to anastomotic leakage and was an independent risk factor for anastomotic leakage. NLR was the only score for predicting the risk of anastomotic leakage that could be calculated preoperatively. There are many risk factors reported contributing to anastomotic leakage, such as anastomotic level from the anal verge, comorbidity, high ligation of the inferior mesenteric artery, male sex, and intraoperative complications [35].

for predicting the risk of anastomotic leakage that could be calculated preoperatively. There are many risk factors reported contributing to anastomotic leakage, such as anastomotic level from the anal verge, comorbidity, high ligation of the inferior mesenteric artery, male sex, and intraoperative complications [35]. The number of patients in the present study was small, therefore, further studies are in larger numbers of patients are needed. However, our results suggest that NLR can be used to predict the risk of anastomotic leakage preoperatively and may be helpful in determining the need for surgical procedures such as a diverting stoma. Conclusions Five types of risk evaluation scores were useful for predicting perioperative complications in patients with rectal cancer who received radical surgery. E-PASS CRS and NLR were risk scores related to anastomotic leakage. NLR was the only score for predicting the risk of anastomotic leakage that could be calculated preoperatively, suggesting that it is useful for assessing the need for a diverting stoma. Compliance with ethical standards Conflict of interest All authors have no conflict of interest to declare in association with this study.

Prostate-specific antigen (PSA) and the controversy in PSA screening In 2015, prostate cancer (PC) was the most commonly diagnosed male malignancy, not only in western countries [1], but also in Japan [2]. PSA is the most commonly used biomarker for the early detection of PC. After the introduction of PSA testing, the rate of PC diagnosis increased and PC-associated mortality decreased. Elevated PSA levels are associated with an increased risk of PC, a higher pathological grade, and an increased risk of metastatic disease [3]. However, the use of PSA as a biomarker has a number of limitations. First, PSA is not a PC-specific biomarker. In addition, PSA levels are influenced by several factors, including age, acute prostatitis, ejaculation, catheterization, and certain medications. Furthermore, there is no precise value indicative of a lack of PC risk, and PSA levels cannot distinguish between indolent and aggressive disease, particularly at PSA levels below 20 ng/mL. In one study, the conventional cutoff value of 4 ng/mL PSA predicted PC in 10- or 12-core needle biopsies in only 30–40 % of patients [4]. In addition, ~15 % of men with serum PSA levels below 4 ng/mL are reported to be at risk for PC [5]. A precise PSA cut-off value that can facilitate the early detection of PC with high sensitivity and specificity in healthy men has not yet been defined. In addition, the ideal age at which to initiate or discontinue PSA testing, and the appropriate frequency of testing remain unclear. Two recent randomized trials evaluating the effect of PSA-based screening on mortality reduction reported conflicting results [6, 7]. Together, these findings prompted the United States Preventive Services Task Force to recommend against the use of PSA-based screening in 2012 [8]. Nevertheless, as there are no other reliable PC biomarkers to replace PSA, PSA screening remains the first-line assay for PC detection. As this approach is the subject of much debate and controversy, there is an unmet need for the identification of novel biomarkers with high sensitivity and specificity for detecting PC and predicting aggressive disease.

reliable PC biomarkers to replace PSA, PSA screening remains the first-line assay for PC detection. As this approach is the subject of much debate and controversy, there is an unmet need for the identification of novel biomarkers with high sensitivity and specificity for detecting PC and predicting aggressive disease. Recently identified putative PC biomarkers are described in Table 1.Table 1 Recently identified putative prostate cancer biomarkers

reliable PC biomarkers to replace PSA, PSA screening remains the first-line assay for PC detection. As this approach is the subject of much debate and controversy, there is an unmet need for the identification of novel biomarkers with high sensitivity and specificity for detecting PC and predicting aggressive disease. Recently identified putative PC biomarkers are described in Table 1.Table 1 Recently identified putative prostate cancer biomarkers Biomarker Biomaterial Applications Marker description PHI Serum Diagnostic Total PSA, [−2]proPSA, fPSA PCA3 Urine Diagnostic PSA and PCA3 mRNA 4K score Plasma Diagnostic Total PSA, fPSA, intact PSA S2, 3PSA Serum Diagnostic Aberrant glycosylation in serum PSA TMPRSS2-ERG Urine, blood tissue Diagnostic Fusion gene of ERG and transmembrane protease, serine 2 Mi-Prostate score Urine Diagnostic PSA, PCA3 and TMPRSS2-ERG mRNAs miRNA Serum, plasma, urine Diagnostic/aggressiveness Altered miRNA expression profiles (miR-141, -375, -21, -107, 221, etc.) Oncotype DX Tissue Aggressiveness 12 Cancer-related genes: androgen pathway (AZGP1, KLK2, SRD5A2, RAM13C), proliferation (TPX2), cellular organization (FLNC, GSN, TPM2, GSTM2) and stromal response (BGN, COL1A1 and SFRP4). ProMark Tissue Aggressiveness 8 Proteins (DERL1, CUL2, SMAD4, PDSS2, HSPA9, FUS, phosphorylated S6, YBOX1) Prolaris Tissue Aggressiveness 31 Cell cycle progression genes and 15 housekeeping genes in combination with PSA and Gleason score Decipher GC Tissue Aggressiveness (metastasis) 22 RNAs form tissue after radical prostatectomy GCNT1 Urine, tissue Aggressiveness Overexpression of the enzyme that forms core 2-branched O-glycans

is Tissue Aggressiveness 31 Cell cycle progression genes and 15 housekeeping genes in combination with PSA and Gleason score Decipher GC Tissue Aggressiveness (metastasis) 22 RNAs form tissue after radical prostatectomy GCNT1 Urine, tissue Aggressiveness Overexpression of the enzyme that forms core 2-branched O-glycans N-glycans Serum Aggressiveness Aberrant glycosylation in serum N-glycans AR-V7 Blood Aggressiveness AR-V7 expression in CTCs

is Tissue Aggressiveness 31 Cell cycle progression genes and 15 housekeeping genes in combination with PSA and Gleason score Decipher GC Tissue Aggressiveness (metastasis) 22 RNAs form tissue after radical prostatectomy GCNT1 Urine, tissue Aggressiveness Overexpression of the enzyme that forms core 2-branched O-glycans N-glycans Serum Aggressiveness Aberrant glycosylation in serum N-glycans AR-V7 Blood Aggressiveness AR-V7 expression in CTCs PSA isoforms Serum PSA predominantly exists in a complex with α-1-antichymotrypsin. Levels of unbound PSA, referred to as free PSA (fPSA), are calculated using the following formula fPSA = total PSA − α-1-antichymotrypsin-bound PSA. %fPSA is associated with a greater specificity for detecting PC in men with a total PSA between 4 and 10 ng/mL [9, 10]. Three isoforms of fPSA are found in the serum: (1) proPSA, (2) intact PSA, and (3) benign PSA. There are also several truncated isoforms of proPSA, including [−2]proPSA, [−5]proPSA, and [−7]proPSA. [−2]proPSA is the predominant proPSA isoform in tumor extracts [11], suggesting that it has the potential to play a role in the early detection of PC and the prediction of aggressive disease [12] (Fig. 1).Fig. 1 PSA synthesis and PSA isoforms. PSA synthesis begins with the cleavage of the proenzyme PSA (proPSA) leader sequence from preproPSA. There are several truncated isoforms of proPSA, including [−7]proPSA, [−5]proPSA, and [−2]proPSA. [−2]proPSA is the predominant proPSA isoform in tumor extracts, indicating that it has the potential to play a role in the early detection of PC and the prediction of aggressive disease. The cleavage of the propeptide in proPSA by human kallikrein 2 (hk2) generates the mature PSA molecule. Benign PSA (BPSA), intact PSA, and [−2]proPSA exist as free PSA molecules in the serum. cPSA complexed PSA

at it has the potential to play a role in the early detection of PC and the prediction of aggressive disease. The cleavage of the propeptide in proPSA by human kallikrein 2 (hk2) generates the mature PSA molecule. Benign PSA (BPSA), intact PSA, and [−2]proPSA exist as free PSA molecules in the serum. cPSA complexed PSA Prostate health index (PHI) The prostate health index (PHI) is an assessment of the three PSA isoforms. PHI is calculated using the following formula: ([−2]proPSA/fPSA) × PSA1/2. The test was developed by Beckman Coulter in partnership with the NCI Early Detection Research Network. The aim of PHI is to distinguish between malignant and benign prostate conditions in men 50 years or older with normal digital rectal exam (DRE) results and PSA levels of 4–10 ng/mL, and to ultimately reduce the number of unnecessary biopsies performed. Several studies have suggested that PHI significantly improves prostate cancer detection in high-risk cases [11, 13] and that is associated with PC aggressiveness [12]. PHI and [−2]proPSA were approved by the US Food and Drug Administration (FDA) in 2012. PHI has been gaining acceptance worldwide and has been approved for clinical use in more than 50 countries. However, it is not yet recommended as a first-line screening method for all patients due to a lack of sufficient data. Further prospective analysis of this approach is needed to support its use as a first-line screening tool for PC in all patients.

dwide and has been approved for clinical use in more than 50 countries. However, it is not yet recommended as a first-line screening method for all patients due to a lack of sufficient data. Further prospective analysis of this approach is needed to support its use as a first-line screening tool for PC in all patients. 4K score The 4K score is an assessment of kallikrein-related peptide 2 (hK2) and the three PSA isoforms included in the PHI (total PSA, fPSA, and intact PSA). The 4K score also incorporates clinical information, including age and history of prior negative biopsy, to provide an estimate of the probability of biopsy-confirmed PC. Retrospective studies reported that the 4K score was more accurate in predicting clinically diagnosed PC [14] and aggressive disease [15] compared with PSA and age. A recent meta-analysis reported that the 4K score is associated with a improvement of 8–10 % in predicting biopsy-confirmed PC, indicating that the use of the 4K score could potentially eliminate the number of prostate biopsies currently conducted by an estimated 48–56 % [16]. Although the accuracy of the 4K score highlights the drawbacks of PSA screening, it is not yet FDA-approved or included in current guideline recommendations. Furthermore, as no comparative study has been reported, it is unclear if the predictive accuracy of the PHI and the 4K score differs. Therefore, additional prospective studies are needed to evaluate their use as screening tools for the early detection of PC.

DA-approved or included in current guideline recommendations. Furthermore, as no comparative study has been reported, it is unclear if the predictive accuracy of the PHI and the 4K score differs. Therefore, additional prospective studies are needed to evaluate their use as screening tools for the early detection of PC. Genomic biomarkers in urine Progensa PCA3 assay Prostate cancer gene 3 (PCA3) is prostate-specific noncoding mRNA that is strongly expressed in patients with PC. The PCA3 assay measures the concentration of PCA3 and PSA RNA molecules. The PCA3 score is calculated as the ratio of PCA3 RNA molecules to PSA RNA molecules (PCA3 score) in post-DRE urine specimens. A PCA3 score less than 25 indicates a low risk of PC [17]. The test was approved by the FDA in 2012. It is used to help determine if a repeat biopsy is necessary for men with a previous negative biopsy. A meta-analysis of 11 clinical studies reported that the sensitivity of the PCA3 assay ranged from 53 to 69 % and the specificity ranged from 71 to 83 % [18]. A more recent meta-analysis of 11 studies reported that a sensitivity and specificity of 72 and 53 %, respectively, was associated with a PCA3 score cut-off of 20 [19]. These findings indicate that the PCA3 test might be more accurate compared with other methods used for the early detection of PC. The Progensa PCA3 assay has been included in the EAU guidelines for repeat biopsy decision-making.

cificity of 72 and 53 %, respectively, was associated with a PCA3 score cut-off of 20 [19]. These findings indicate that the PCA3 test might be more accurate compared with other methods used for the early detection of PC. The Progensa PCA3 assay has been included in the EAU guidelines for repeat biopsy decision-making. The TMPRSS2-ERG fusion gene Gene rearrangements have been observed in multiple cancers, and they are especially prevalent in leukemia (e.g., the Philadelphia chromosome). The TMPRSS2-ERG fusion gene, comprising the androgen-responsive genes transmembrane protease, serine 2 (TMPRSS2), and estrogen-regulated gene (ERG), was observed in ~40–80 % of prostate cancers in 2005 [20]. Both genes are located on chromosome 21. The TMPRSS2-ERG score is calculated using the following formula: (TMPRSS2-ERG mRNA/PSA RNA copies) × 100,000. Levels of urine TMPRSS2-ERG appear to be associated with clinically significant PC [21]; however, the prognostic significance of TMPRSS2-ERG is unclear. A recent meta-analysis suggested that TMPRSS2-ERG overexpression is associated with tumor stage, but that it is not associated with disease recurrence or mortality in men treated with radical prostatectomy (RP) [22].

ed with clinically significant PC [21]; however, the prognostic significance of TMPRSS2-ERG is unclear. A recent meta-analysis suggested that TMPRSS2-ERG overexpression is associated with tumor stage, but that it is not associated with disease recurrence or mortality in men treated with radical prostatectomy (RP) [22]. Mi-Prostate score (MiPS) TMPRSS2-ERG is a PC-specific fusion gene. However, most prostate tumors have multiple foci and the expression of TMPRSS2-ERG is thought to be heterogeneous. The MiPS overcomes this limitation by assessing multiple PC-associated parameters. MiPS combines the prognostic significance of urine TMPRSS2-ERG and PCA3 with serum PSA to generate a PC risk assessment score. In a recent validation study with 1225 patients, MiPS was superior to serum PSA alone in predicting biopsy-confirmed PC and high-grade PC [23]. However, this test is not yet FDA-approved.

ters. MiPS combines the prognostic significance of urine TMPRSS2-ERG and PCA3 with serum PSA to generate a PC risk assessment score. In a recent validation study with 1225 patients, MiPS was superior to serum PSA alone in predicting biopsy-confirmed PC and high-grade PC [23]. However, this test is not yet FDA-approved. Genomic and protein biomarkers in tissue Oncotype DX test The Oncotype DX test is a multi-gene expression assay developed for formalin-fixed paraffin-embedded diagnostic prostate needle biopsies. The assay measures the expression of 12 cancer-related genes representing 4 distinct biological functions: androgen signaling (AZGP1, KLK2, SRD5A2, and RAM13C), proliferation (TPX2), cytoskeletal organization (FLNC, GSN, TPM2, GSTM2), and the stromal response (BGN, COL1A1, and SFRP4). Five reference genes have been included to normalize the data and control for variability. The gene expression levels are algorithmically combined to calculate a genomic prostate score (GPS). The Oncotype DX test has been validated as a predictor of prostate cancer aggressiveness, and it facilitates prostate cancer risk stratification to help guide treatment decision-making [24]. It is included as a potential option in the National Comprehensive Cancer Network (NCCN) guidelines of 2015, with the disclaimer that further studies of the assay are still needed [25].

f prostate cancer aggressiveness, and it facilitates prostate cancer risk stratification to help guide treatment decision-making [24]. It is included as a potential option in the National Comprehensive Cancer Network (NCCN) guidelines of 2015, with the disclaimer that further studies of the assay are still needed [25]. Prolaris test The Prolaris test is a multi-gene expression assay (46 genes) developed for formalin-fixed paraffin-embedded tissue derived from prostate needle biopsies. It is used in conjunction with the Gleason score and serum PSA to predict prostate cancer aggressiveness. The Prolaris test assesses disease progression by quantitatively analyzing the expression of 31 genes associated with cell cycle progression and 15 housekeeping genes. Low expression levels of these genes are associated with a low risk of disease progression, whereas high expression levels are indicative of a higher risk of disease progression [26]. The Prolaris test is included in the NCCN 2015 guidelines.

of 31 genes associated with cell cycle progression and 15 housekeeping genes. Low expression levels of these genes are associated with a low risk of disease progression, whereas high expression levels are indicative of a higher risk of disease progression [26]. The Prolaris test is included in the NCCN 2015 guidelines. Decipher genomic classifier (Decipher GC) Decipher is a genomic classifier (GC) test that uses a whole-transcriptome microarray assay to analyze the expression of 22 genes in formalin-fixed paraffin-embedded prostate cancer specimens obtained from RP. Decipher GC can predict the risk of metastasis following RP. In a clinical validation study, Decipher was a more accurate predictor of metastasis post-RP compared with individual clinical variables, with an area under the curve (AUC) of 0.79 for predicting 5-year metastasis risk [27]. A genomic risk stratification assay using the primary tumor can identify patients at a high risk for metastasis and potentially lethal prostate cancer, thereby providing information that can improve treatment decision-making post-RP. The Decipher score is included in the NCCN 2015 guidelines as a post-RP prognostic marker. The specific genes evaluated and the formula used to calculate the Decipher GC score have not been published.

d potentially lethal prostate cancer, thereby providing information that can improve treatment decision-making post-RP. The Decipher score is included in the NCCN 2015 guidelines as a post-RP prognostic marker. The specific genes evaluated and the formula used to calculate the Decipher GC score have not been published. ProMark ProMark is a prognostic assay that analyzes the expression of 8 protein biomarkers in formalin-fixed paraffin-embedded tissue obtained from prostate needle biopsies. It is used to predict prostate cancer aggressiveness, particularly in patients with a Gleason score 3 + 3 or 3 + 4. ProMark quantitatively analyzes the levels of 8 proteins (DERL1, CUL2, SMAD4, PDSS2, HSPA9, FUS, phosphorylated S6, and YBOX1) in biopsy tissue sections using an automated immunofluorescence method. The protein levels are used to calculate a risk score that has been clinically validated as an independent predictor of prostate cancer aggressiveness [28]. The ProMark score can help distinguish patients that should be actively monitored from those that require therapeutic intervention. Cancer-associated glycan biomarkers Cancer-associated glycan aberrations are frequently observed in tumors. The majority of tumor markers, including PSA, are glycoproteins that have glycosylation sites in their amino acid sequence. Importantly, each glycan exhibits specific cancer-associated carbohydrate aberrations compared with its normal counterpart, and these aberrations can be detected using specific monoclonal antibodies or lectin.

of tumor markers, including PSA, are glycoproteins that have glycosylation sites in their amino acid sequence. Importantly, each glycan exhibits specific cancer-associated carbohydrate aberrations compared with its normal counterpart, and these aberrations can be detected using specific monoclonal antibodies or lectin. Aberrant serum PSA glycosylation (S2,3PSA) PSA is a glycoprotein with a single N-glycosylation site at an asparagine (N) residue 45 amino acids from the N-terminus. In patients with PC, the terminal N-glycan structure of PSA is rich in sialic acid α2,3-linked galactose, whereas the terminal N-glycan structures of PSA from healthy patients are predominantly α2,6-linked [29] (Fig. 2). Based on this finding, Yoneyama et al. successfully developed a novel assay using a magnetic microbead-based immunoassay to detect α2,3-linked sialylation on free PSA (S2, 3PSA) [30]. The diagnostic accuracy of S2, 3PSA was associated with an AUC of 0.84, and the sensitivity and specificity of the assay was 95.0 and 72.0 %, respectively, a significant increase compared with PSA or %fPSA. Although the study was small and preliminary, the results suggest that assays measuring cancer-associated glycan alterations in serum S2, 3PSA might improve the accuracy of early PC detection and reduce unnecessary prostate biopsies.Fig. 2 Aberrant glycosylation of PSA N-glycans (S2, 3PSA) in PC. In healthy patients, the terminal sialic acid of PSA is predominantly α2,6-linked to galactose residues. In patients with PC, the terminal sialic acid is predominantly α2,3-linked to galactose residues

PC detection and reduce unnecessary prostate biopsies.Fig. 2 Aberrant glycosylation of PSA N-glycans (S2, 3PSA) in PC. In healthy patients, the terminal sialic acid of PSA is predominantly α2,6-linked to galactose residues. In patients with PC, the terminal sialic acid is predominantly α2,3-linked to galactose residues Highly branched (tri- or tetra-antennary) serum N-glycans Although cancer-associated glycan alterations represent potential cancer biomarkers, glycan analysis has not been incorporated into clinical use because the protocols for preparing glycan derivatives vary depending on the analytical method and conducting these protocols requires specialized expertise. Therefore, a practical procedure for analyzing a large number of glycan samples in biological materials such as serum is not currently available.

nto clinical use because the protocols for preparing glycan derivatives vary depending on the analytical method and conducting these protocols requires specialized expertise. Therefore, a practical procedure for analyzing a large number of glycan samples in biological materials such as serum is not currently available. Recently, an approach that combines high-throughput, quantitative N-glycomics with mass spectrometry analysis was developed. The technique is based on a chemoselective glycan enrichment technology that enables the purification of oligosaccharides from 10 μL of crude glycoproteins. Preliminary results indicate that serum N-glycan analysis is a promising approach to screening diagnostic and prognostic markers associated with multiple types of cancer [31, 32]. Ishibashi et al. evaluated the potential predictive value of serum N-glycomics in patients with castration-resistant PC (CRPC) [33]. They used serum N-glycomics with the glycoblotting method to analyze 80 healthy volunteers, 286 patients with benign prostatic hyperplasia, 258 patients with early-stage PC, 46 patients with PC that had been treated with androgen deprivation therapy (ADT), and 68 patients with CRPC. They found that tri- and tetra-antennary N-glycans were significantly enriched in patients with CRPC compared with the other groups. The longitudinal follow-up of highly branched N-glycan levels predicted CRPC despite castrate levels of testosterone. These results suggest that the overexpression of specific N-glycans might be associated with CRPC and that it might represent a predictive CRPC biomarker.

ients with CRPC compared with the other groups. The longitudinal follow-up of highly branched N-glycan levels predicted CRPC despite castrate levels of testosterone. These results suggest that the overexpression of specific N-glycans might be associated with CRPC and that it might represent a predictive CRPC biomarker. Core2 β-1,6-N-acetylglucosaminyltransferase (GCNT1) Core2 β-1,6-N-acetylglucosaminyltransferase-1 (GCNT1) is an enzyme that plays a key role in the formation of core 2-branched O-glycans, and GCNT1 expression is associated with the progression of several types of cancer [34–37]. GCNT1 expression is strongly associated with disease aggressiveness in patients with PC [38–40]. Kojima et al. recently evaluated the utility of GCNT1 for the detection of aggressive PC using immunohistochemistry and immunoblotting assays in post-DRE urine [41]. They reported that over 90 % of GCNT1-positive PC patients with high concentrations of PSA presented with extracapsular extensions, further confirming that GCNT1 expression is strongly associated with aggressive PC. Further research is needed to develop an efficient assay that can detect GCNT1 in post-DRE urine and facilitate the use of GCNT1 as a marker of PC aggressiveness in the clinical setting.

s of PSA presented with extracapsular extensions, further confirming that GCNT1 expression is strongly associated with aggressive PC. Further research is needed to develop an efficient assay that can detect GCNT1 in post-DRE urine and facilitate the use of GCNT1 as a marker of PC aggressiveness in the clinical setting. Circulating tumor cells (CTCs) Circulating tumor cells (CTCs) are tumor cells that have shed into the peripheral circulation from solid malignancies. CTCs have generally been considered surrogates for metastatic cells, and CTCs levels in patients undergoing treatment have proven to be a response marker with a strong independent prognostic value [42]. In addition, CTCs have been characterized as a ‘‘liquid biopsy” that can facilitate the real-time monitoring of therapeutic efficacy, and the identification of therapeutic targets and resistance mechanisms [43].

ts undergoing treatment have proven to be a response marker with a strong independent prognostic value [42]. In addition, CTCs have been characterized as a ‘‘liquid biopsy” that can facilitate the real-time monitoring of therapeutic efficacy, and the identification of therapeutic targets and resistance mechanisms [43]. Recently, the presence of the AR splice variant 7 (AR-V7) in CTCs was shown to be strongly associated with resistance to anti-AR therapy [44]. The presence of AR-V7 in CTCs was associated with poor overall survival, with a hazard ratio (HR) of 6.9 [95 % confidence interval (CI) 1.7–28.1, P = 0.002] in patients receiving enzalutamide cohort and an HR of 12.7 (95 % CI 1.3–125.3, P = 0.006) in patients receiving abiraterone cohort. The same group also reported that the presence of AR-V7 did not significantly influence the effect of taxane treatment [45]. More recently, Scher et al. reported similar results from a prospective study of 191 blood samples from patients with metastatic CRPC. Although the proportion of patients with AR-V7-positive CTCs in this study was relatively low (34/191 samples, 18 %), AR-V7 positive patients were resistant to AR inhibition therapy, were on therapy for less time, and experienced a reduction in radiographic progression-free survival and overall survival compared with patients with AR-V7-negative CTCs [46]. However, there are substantial limitations to the clinical application of AR-V7 monitoring as only half of patients have detectable CTCs, CTSs cannot be stored, and the procedure is costly and can only be conducted in specialized labs. Although AR-V7 appears to be a promising prognostic factor, additional studies and technological innovations are needed to facilitate its widespread clinical use.

7 monitoring as only half of patients have detectable CTCs, CTSs cannot be stored, and the procedure is costly and can only be conducted in specialized labs. Although AR-V7 appears to be a promising prognostic factor, additional studies and technological innovations are needed to facilitate its widespread clinical use. Conclusion and perspectives Although serum PSA has been used as a prostate cancer biomarker for several decades, studies demonstrating its limitations have incited much controversy and debate. The ideal PC biomarker would be capable of distinguishing PC from benign prostate conditions and differentiating between aggressive and indolent tumors. Recent technological innovations have led to substantial improvements in biomarker screening assays. An overview of the emerging prostate cancer biomarkers described in this review are summarized in Fig. 3. Specifically, two new tests designed to help determine the need for prostate biopsy have recently been approved by the US FDA. Currently, the incorporation of new biomarkers appears to be a promising approach for improving the sensitivity and specificity of PSA assays. However, comparative studies evaluating these various biomarker assays are still needed, as are studies with larger sample sizes and improvements in cost effectiveness. Large-scale, prospective trials can help evaluate the utility of these new approaches in multiple clinical contexts. The use of novel biomarkers that can serve as alternatives to PSA appears to be a promising approach to improving risk assessment strategies and has the potential to improve outcomes in patients with PC.Fig. 3 Overview of potential prostate cancer biomarkers. Novel biomarkers were classified according to screening to prognostic phase based on types of biomaterials. Asterisk US FDA approved, N/A not available

approach to improving risk assessment strategies and has the potential to improve outcomes in patients with PC.Fig. 3 Overview of potential prostate cancer biomarkers. Novel biomarkers were classified according to screening to prognostic phase based on types of biomaterials. Asterisk US FDA approved, N/A not available We thank the members of the Department of Urology, Hirosaki University Graduate school of Medicine, and Yukie Nishizawa and Yuki Fujita for their invaluable help with the data and sample collection. This work was supported by a Grant-in-Aid for Scientific Research 23791737, 24659708, 22390301, 15H02563, and 15K15579 from the Japan Society for the Promotion of Science. Compliance with ethical standards Conflict of interest The authors declare no potential conflicts of interest.

Introduction Colorectal cancer (CRC) is one of the most common cancers in Japan, with over 147,000 new cases expected in 2016 [1]. Postoperative adjuvant chemotherapy for patients with stage III CRC is the internationally accepted standard of care to improve patient survival. In the mid-1990s, based on the results of several studies [2, 3], a 6-month course of intravenous 5-fluorouracil (5-FU) plus leucovorin (LV) came to be regarded as the standard regimen of adjuvant chemotherapy for colon cancer. From the following studies investigating oral FUs (such as tegafur-uracil [UFT] plus LV, capecitabine), and oxaliplatin-containing regimens (i.e., FOLFOX and CapeOX) as adjuvant chemotherapy for colon cancer, Western countries selected 6 months as the standard treatment duration [4–7]. Therefore, 6 months of adjuvant chemotherapy has been recognized as the global clinical standard.

ur-uracil [UFT] plus LV, capecitabine), and oxaliplatin-containing regimens (i.e., FOLFOX and CapeOX) as adjuvant chemotherapy for colon cancer, Western countries selected 6 months as the standard treatment duration [4–7]. Therefore, 6 months of adjuvant chemotherapy has been recognized as the global clinical standard. On the other hand, by analyzing the data of >20,800 patients from 18 randomized controlled studies (RCTs) in the Adjuvant Colon Cancer Endpoints (ACCENT) database, Sargent et al. [8] demonstrated that the risk of stage II-III CRC recurrence was highest between 12 and 18 months after surgery and proposed that decreasing the recurrence risk at 12–18 months after surgery might improve survival. The results of a meta-analysis of three studies (JFMC 7-1, 7-2, and 15) by Hamada et al. [9], investigating the risk of recurrence in 2848 patients with curatively resected colon cancer followed by 1-year administration of oral FUs, strongly suggested that 12 months of oral FU drugs might translate the short-term (1–2 years) reduction in the risk of recurrence into a delayed advantage in overall survival (OS).

investigating the risk of recurrence in 2848 patients with curatively resected colon cancer followed by 1-year administration of oral FUs, strongly suggested that 12 months of oral FU drugs might translate the short-term (1–2 years) reduction in the risk of recurrence into a delayed advantage in overall survival (OS). Those findings suggest that extending adjuvant oral FU therapy from 6 to 12 months might be able to improve prognosis. However, whether 12 months of adjuvant chemotherapy can decrease the peak of recurrence risk between 12 and 18 months postoperatively and improve survival has not been investigated by RCT. Therefore, we conducted a phase III study, JFMC37-0801 (UMIN-CTR; UMIN000001367), to compare 6 and 12 months of adjuvant chemotherapy using capecitabine (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan), the most commonly used oral FU for CRC worldwide, in patients with stage III colon cancer. The safety of adjuvant capecitabine for colon cancer has not been studied in a large sample of Japanese patients, even though adjuvant capecitabine is widely used clinically for CRC in Japan. Furthermore, it is unclear whether a longer treatment period might influence the incidence and severity of adverse events (AEs). We therefore report the results of a preplanned safety analysis, to increase the safety of capecitabine use in clinical practice.

vant capecitabine is widely used clinically for CRC in Japan. Furthermore, it is unclear whether a longer treatment period might influence the incidence and severity of adverse events (AEs). We therefore report the results of a preplanned safety analysis, to increase the safety of capecitabine use in clinical practice. Patients and methods Enrollment and assignment This study was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Clinical Research in Japan, and was approved by the Institutional Review Boards of each participating institute. Written informed consent was obtained from all patients before enrollment, and eligible patients were centrally registered. The main eligibility criteria were (1) histologically confirmed stage III colon adenocarcinoma; (2) curatively resected with extended lymph node dissection (D2 or D3 in the Japanese Classification of Colorectal Carcinoma, 8th edition) [10]; (3) aged 20–79 years; (4) Eastern Cooperative Oncology Group performance status (ECOG-PS) of 0 to 1; (5) no prior chemotherapy or radiotherapy for CRC; (6) no other active malignancies; (7) adequate oral intake; (8) preserved major organ functions, and (9) no uncontrollable severe infection.

rcinoma, 8th edition) [10]; (3) aged 20–79 years; (4) Eastern Cooperative Oncology Group performance status (ECOG-PS) of 0 to 1; (5) no prior chemotherapy or radiotherapy for CRC; (6) no other active malignancies; (7) adequate oral intake; (8) preserved major organ functions, and (9) no uncontrollable severe infection. Randomization and masking After confirming eligibility, enrolled patients were randomly assigned to receive either 8 cycles (6 months) or 16 cycles (12 months) of capecitabine at the central registration center, using a minimization method, with stratification by lymph node metastasis (N1 or N2-3 in the Japanese Classification of Colorectal Carcinoma, 8th edition) [10] and institution. The assigned treatment arm was not blinded from both investigators and patients. Protocol treatment Capecitabine was orally given at a dose of 1250 mg/m2 twice daily after meals for 14 consecutive days, followed by a 7-day rest. This 3-week treatment comprised 1 cycle. The control group (6M group) received 8 cycles and the study group (12M group) received 16 cycles. After completing the scheduled treatment, each group was switched to the follow-up schedule defined in the protocol, without any treatment until confirmation of metastasis or recurrence.

-week treatment comprised 1 cycle. The control group (6M group) received 8 cycles and the study group (12M group) received 16 cycles. After completing the scheduled treatment, each group was switched to the follow-up schedule defined in the protocol, without any treatment until confirmation of metastasis or recurrence. The assigned treatment was started within 8 weeks after surgery. During treatment, clinical findings and laboratory values were evaluated at least every 3 weeks. Evaluation at the beginning of each cycle was mandatory. Patients received treatment if they fulfilled the following criteria—leukocytes ≥3000/mm3, neutrophils ≥1500/mm3, platelets ≥75,000/mm3, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5 × upper limit of normal (ULN), total bilirubin ≤1.5 × ULN, creatinine <1.5 × ULN, and no higher than grade 1 non-hematologic toxicities (i.e., anorexia, nausea, vomiting, and diarrhea). If the criteria for starting/continuing treatment were not fulfilled, treatment was postponed or temporarily suspended until AEs had improved sufficiently to meet the criteria. Supportive care including antiemetics, antidiarrheal drugs, liver supporting therapy (e.g., ursodeoxycholic acid), granulocyte colony-stimulating factor, oral vitamin B6, and external use of hydrating cream and steroids were allowed when physicians considered necessary.

s had improved sufficiently to meet the criteria. Supportive care including antiemetics, antidiarrheal drugs, liver supporting therapy (e.g., ursodeoxycholic acid), granulocyte colony-stimulating factor, oral vitamin B6, and external use of hydrating cream and steroids were allowed when physicians considered necessary. Depending upon the severity of the AEs at the time of treatment suspension, the dose of capecitabine was reduced in accordance with the protocol. When a grade 2 AE developed the first time, treatment with capecitabine was suspended until the AE improved to grade ≤1, and then resumed at the same dose. If a grade 2 AE occurred twice or if a grade 3 AE occurred, the dose of capecitabine was reduced by 25%. The minimum dose was 50% of the initial dose recommended in the protocol. The treatment was discontinued if (1) recurrence or other malignancies developed; (2) a grade 4 AE occurred; (3) treatment could not be resumed within 21 days after its postponement or temporary suspension; (4) further dose reduction was necessary even after the specified dose was reduced by two levels (−50%); (5) the physician judged that the protocol treatment was too difficult to continue; (6) the patient requested discontinuation of the treatment, and (7) the patient withdrew their informed consent.

porary suspension; (4) further dose reduction was necessary even after the specified dose was reduced by two levels (−50%); (5) the physician judged that the protocol treatment was too difficult to continue; (6) the patient requested discontinuation of the treatment, and (7) the patient withdrew their informed consent. Data collection Treatment information, such as the daily dose and the number of days of administration in each cycle, was collected from the case report forms of each patient. The relative dose intensity (RDI) for each cycle was defined as the ratio of the actual cumulative dose to the protocol-specified cumulative dose in each cycle. Completion rate of the protocol treatment was defined as the ratio of the number of patients who completed 8 or 16 cycles of capecitabine treatment to the number of patients included the safety analysis set of each treatment group. The type and severity of AEs in each cycle were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. The most severe grade of each AE during each cycle was reported. Statistical analysis All statistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA). Descriptive statistics such as means, standard deviations, and medians were calculated. The chi-squared test was used to compare the incidence of AEs between the treatment groups. A p value <0.05 was considered significant.

atistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA). Descriptive statistics such as means, standard deviations, and medians were calculated. The chi-squared test was used to compare the incidence of AEs between the treatment groups. A p value <0.05 was considered significant. Results Patient characteristics From September 2008 through December 2009, a total of 1304 patients were enrolled from 333 institutes in Japan, and randomized. Of these, 1278 patients (642 in the 6M group and 636 in the 12M group) who received capecitabine treatment were included in the safety analysis set (Fig. 1). All data for the analysis were finalized in March of 2016.Fig. 1 CONSORT diagram Patient characteristics are shown in Table 1. Median age at enrollment was 65 years (range 23–79); 53.7% were male, and 96.1% were PS 0. Baseline characteristics of the two treatment groups were well balanced.Table 1 Patient characteristics 6M group (n = 642) 12M group (n = 636)

Results Patient characteristics From September 2008 through December 2009, a total of 1304 patients were enrolled from 333 institutes in Japan, and randomized. Of these, 1278 patients (642 in the 6M group and 636 in the 12M group) who received capecitabine treatment were included in the safety analysis set (Fig. 1). All data for the analysis were finalized in March of 2016.Fig. 1 CONSORT diagram Patient characteristics are shown in Table 1. Median age at enrollment was 65 years (range 23–79); 53.7% were male, and 96.1% were PS 0. Baseline characteristics of the two treatment groups were well balanced.Table 1 Patient characteristics 6M group (n = 642) 12M group (n = 636) n (%) n (%) Age Median [range] 65 [23–79] 65 [34–79] Gender Male 347 (54.0%) 339 (53.3%) Female 295 (46.0%) 297 (46.7%) ECOG performance status 0 611 (95.2%) 617 (97.0%) 1 31 (4.8%) 19 (3.0%) Creatinine clearance (mL/min) Median [range] 76.6 [35.1–350.4] 78.1 [39.2–176.3] Body surface area (m2) Median [range] 1.59 [1.06–2.42] 1.56 [1.10–2.14] <1.33 (dose 3000 mg/body/day) 41 (6.4%) 48 (7.5%) ≥1.33 to <1.57 (dose 3600 mg/body/day) 262 (40.8%) 272 (42.8%) ≥1.57 to <1.81 (dose 4200 mg/body/day) 274 (42.7%) 261 (41.0%) ≥1.81 (dose 4800 mg/body/day) 65 (10.1%) 55 (8.6%) Tumor location Right-sided colon (C, A, T) 261 (40.6%) 258 (40.6%) Left-sided colon (D, S) 247 (38.5%) 247 (38.8%) Rectosigmoid colon 134 (20.9%) 131 (20.6%) Depth of tumor invasion (TNM 7th) T1 44 (6.9%) 45 (7.1%) T2 54 (8.4%) 52 (8.2%) T3 360 (56.1%) 355 (55.8%) T4 184 (28.7%) 184 (28.9%) LN metastasis (JSCCR classificationa) N1 491 (76.5%) 486 (76.4%) N2 120 (18.7%) 121 (19.0%) N3 31 (4.8%) 29 (4.6%) Stage (TNM 7th) IIIA 91 (14.2%) 93 (14.6%) IIIB 455 (70.9%) 450 (70.8%) IIIC 96 (15.0%) 93 (14.6%) Scope of LN dissection (JSCCR classificationa) D2 129 (20.1%) 133 (20.9%) D3 513 (79.9%) 503 (79.1%) Surgical approach Open (conventional) 371 (57.8%) 388 (61.0%) Laparoscopic 271 (42.2%) 248 (39.0%)

3 31 (4.8%) 29 (4.6%) Stage (TNM 7th) IIIA 91 (14.2%) 93 (14.6%) IIIB 455 (70.9%) 450 (70.8%) IIIC 96 (15.0%) 93 (14.6%) Scope of LN dissection (JSCCR classificationa) D2 129 (20.1%) 133 (20.9%) D3 513 (79.9%) 503 (79.1%) Surgical approach Open (conventional) 371 (57.8%) 388 (61.0%) Laparoscopic 271 (42.2%) 248 (39.0%) ECOG Eastern Cooperative Oncology Group, C cecum, A ascending colon, T transverse colon, D descending colon, S sigmoid colon, LN lymph node N1: Metastasis in 1–3 pericolic/perirectal LNs or intermediate LNs (LNs along the colic artery) N2: Metastasis in ≥4 pericolic/perirectal or intermediate LNs N3: Metastasis in LNs around the origin of the ileocolic, right colic, middle colic, or inferior mesenteric artery D2: Complete dissection of pericolic/perirectal and intermediate LNs D3: Complete dissection of all regional LNs aDefined in the Japanese Classification of Colorectal Carcinoma 8th edition, published by the Japanese Society for Cancer of the Colon and Rectum (JSCCR) [10] Treatment duration The median number of administered cycles was 8 in the 6M group and 15 in the 12M group. The completion rate for 8 cycles of capecitabine was similar in the 6M group (71.5%) and 12M group (71.7%). The final 16-cycle completion rate in the 12M group was 46.1% (Table 2). In both treatment groups, treatment discontinuation occurred at a similar frequency during both the first and last 8 cycles. Among 417 patients in the 12M group who began the 9th cycle, 293 (70.3%) completed 16 cycles (Table 2).Table 2 Treatment discontinuation by cycle 6M group (n = 642) 12M group (n = 636)

Treatment duration The median number of administered cycles was 8 in the 6M group and 15 in the 12M group. The completion rate for 8 cycles of capecitabine was similar in the 6M group (71.5%) and 12M group (71.7%). The final 16-cycle completion rate in the 12M group was 46.1% (Table 2). In both treatment groups, treatment discontinuation occurred at a similar frequency during both the first and last 8 cycles. Among 417 patients in the 12M group who began the 9th cycle, 293 (70.3%) completed 16 cycles (Table 2).Table 2 Treatment discontinuation by cycle 6M group (n = 642) 12M group (n = 636) n (%) n (%) No. of patients discontinued During cycle 1 23 (3.6%) 25 (3.9%) During cycle 2 21 (3.3%) 32 (5.0%) During cycle 3 37 (5.8%) 33 (5.2%) During cycle 4 21 (3.3%) 26 (4.1%) During cycle 5 23 (3.6%) 20 (3.1%) During cycle 6 21 (3.3%) 13 (2.0%) During cycle 7 23 (3.6%) 28 (4.4%) During cycle 8 14 (2.2%) 42 (6.6%) During cycle 9 – – 19 (3.0%) During cycle 10 – – 15 (2.4%) During cycle 11 – – 16 (2.5%) During cycle 12 – – 10 (1.6%) During cycle 13 – – 14 (2.2%) During cycle 14 – – 14 (2.2%) During cycle 15 – – 25 (3.9%) During cycle 16 – – 11 (1.7%) No. of patients completed 8 cycles of capecitabine 459 (71.5%) 456 (71.7%) No. of patients completed 16 cycles of capecitabine – – 293 (46.1%)

ring cycle 11 – – 16 (2.5%) During cycle 12 – – 10 (1.6%) During cycle 13 – – 14 (2.2%) During cycle 14 – – 14 (2.2%) During cycle 15 – – 25 (3.9%) During cycle 16 – – 11 (1.7%) No. of patients completed 8 cycles of capecitabine 459 (71.5%) 456 (71.7%) No. of patients completed 16 cycles of capecitabine – – 293 (46.1%) Reasons for treatment discontinuation In all, 183 patients (28.5%) and 343 patients (53.9%) in the 6M group and 12M group, respectively, dropped out from the protocol treatment. Their reasons for treatment discontinuation are listed in Table 3. The distribution of patients according to reason for discontinuation was similar between the two groups. AEs (listed in the protocol as treatment discontinuation criteria) or physician’s judgment were the most common reasons for discontinuation, and occurred in 50.3, 53.0, and 34.7% of discontinued patients during cycles 1–8 in the 6M group, and cycles 1–8 and 9–16 in the 12M group, respectively. Approximately 14.8, 17.4, and 15.3% of discontinued patients, respectively, requested to discontinue the treatment because of AEs not mentioned in the discontinuation criteria.Table 3 Reasons for treatment discontinuation 6M group (n = 642) 12M group (n = 636) n (%) During cycles 1–8 During cycles 9–16 n (%) n (%) No. of patients with discontinuation 183 (100%) 219 (100%) 124 (100%) Reasons for discontinuation

Reasons for treatment discontinuation In all, 183 patients (28.5%) and 343 patients (53.9%) in the 6M group and 12M group, respectively, dropped out from the protocol treatment. Their reasons for treatment discontinuation are listed in Table 3. The distribution of patients according to reason for discontinuation was similar between the two groups. AEs (listed in the protocol as treatment discontinuation criteria) or physician’s judgment were the most common reasons for discontinuation, and occurred in 50.3, 53.0, and 34.7% of discontinued patients during cycles 1–8 in the 6M group, and cycles 1–8 and 9–16 in the 12M group, respectively. Approximately 14.8, 17.4, and 15.3% of discontinued patients, respectively, requested to discontinue the treatment because of AEs not mentioned in the discontinuation criteria.Table 3 Reasons for treatment discontinuation 6M group (n = 642) 12M group (n = 636) n (%) During cycles 1–8 During cycles 9–16 n (%) n (%) No. of patients with discontinuation 183 (100%) 219 (100%) 124 (100%) Reasons for discontinuation Oncologic events Recurrences 17 (9.3%) 12 (5.5%) 12 (9.7%) Second cancers 3 (1.6%) 2 (0.9%) 1 (0.8%) Adverse events (AEs) AEs (listed on the discontinuation criteria) or physician’s judgement 92 (50.3%) 116 (53.0%) 43 (34.7%) Patient’s request due to AEs not mentioned in the discontinuation criteria 27 (14.8%) 38 (17.4%) 19 (15.3%) Others Aggravation of comorbidities 5 (2.7%) 9 (4.1%) 8 (6.5%) Patient’s request due to non-medical reasons 16 (8.7%) 35 (16.0%) 20 (16.1%) Others 23 (12.6%) 7 (3.2%) 21 (16.9%)

92 (50.3%) 116 (53.0%) 43 (34.7%) Patient’s request due to AEs not mentioned in the discontinuation criteria 27 (14.8%) 38 (17.4%) 19 (15.3%) Others Aggravation of comorbidities 5 (2.7%) 9 (4.1%) 8 (6.5%) Patient’s request due to non-medical reasons 16 (8.7%) 35 (16.0%) 20 (16.1%) Others 23 (12.6%) 7 (3.2%) 21 (16.9%) Types of AEs leading to treatment discontinuation are presented in Table 4. Distribution of patients according to cause of discontinuation was similar between the 6M and 12M group, and between cycles 1–8 and cycles 9–16 in the 12M group. HFS was the most common discontinuation criteria-specified AE leading to discontinuation or the basis for physician's judgment to discontinue treatment. The proportion of patients requesting treatment discontinuation because of HFS was similar to that requesting treatment discontinuation because of other non-hematologic toxicities. Overall, HFS was the leading AE for treatment discontinuation.Table 4 Adverse events causing discontinuation of treatment

ntinue treatment. The proportion of patients requesting treatment discontinuation because of HFS was similar to that requesting treatment discontinuation because of other non-hematologic toxicities. Overall, HFS was the leading AE for treatment discontinuation.Table 4 Adverse events causing discontinuation of treatment 6M group (n = 642) 12M group (n = 636) During cycles 1–8 During cycles 9–16 Discontinuation due to AEs 119 patients 123 AEs (100%) 154 patients 160 AEs (100%) 62 patients 65 AEs (100%) AEs (listed on the discontinuation criteria) or physician’s judgement Hematologic toxicities 18 (14.6%) 25 (15.6%) 9 (13.8%) Abnormal liver function 18 (14.6%) 22 (15.8%) 8 (12.3%) Hand-foot syndrome 38 (30.9%) 60 (37.5%) 19 (29.2%) Non-hematologic toxicitiesa 18 (14.6%) 11 (6.9%) 7 (10.8%) Patient’s request due to AEs not mentioned in the discontinuation criteria Hematologic toxicities 2 (1.6%) 2 (1.3%) 1 (1.5%) Abnormal liver function 0 (0%) 0 (0%) 0 (0%) Hand-foot syndrome 11 (8.9%) 21 (13.1%) 10 (15.4%) Non-hematologic toxicities* 18 (14.6%) 18 (11.3%) 9 (13.8%) Unknown 0 (0%) 1 (0.6%) 2 (3.1%) AEs adverse events aNot including hand-foot syndrome

6M group (n = 642) 12M group (n = 636) During cycles 1–8 During cycles 9–16 Discontinuation due to AEs 119 patients 123 AEs (100%) 154 patients 160 AEs (100%) 62 patients 65 AEs (100%) AEs (listed on the discontinuation criteria) or physician’s judgement Hematologic toxicities 18 (14.6%) 25 (15.6%) 9 (13.8%) Abnormal liver function 18 (14.6%) 22 (15.8%) 8 (12.3%) Hand-foot syndrome 38 (30.9%) 60 (37.5%) 19 (29.2%) Non-hematologic toxicitiesa 18 (14.6%) 11 (6.9%) 7 (10.8%) Patient’s request due to AEs not mentioned in the discontinuation criteria Hematologic toxicities 2 (1.6%) 2 (1.3%) 1 (1.5%) Abnormal liver function 0 (0%) 0 (0%) 0 (0%) Hand-foot syndrome 11 (8.9%) 21 (13.1%) 10 (15.4%) Non-hematologic toxicities* 18 (14.6%) 18 (11.3%) 9 (13.8%) Unknown 0 (0%) 1 (0.6%) 2 (3.1%) AEs adverse events aNot including hand-foot syndrome Dose modification The dose was reduced 314 times in 241 patients (37.5%) in the 6M group and 477 times in 306 patients (48.1%) in the 12M group. In the 12M group, the proportion of patients with dose reduction was lower during cycles 9–16 than during cycles 1–8 (26.1 vs 40.4%) (Table 5). The most common reason for dose reduction was HFS, which occurred 191 times (60.8%) in the 6M group and 290 times (60.8%) in the 12M group.Table 5 Dose reduction Dose reduction 6M group (n = 642) 12M group (n = 636) Overall (n = 636) During cycles 1–8 (n = 636) During cycles 9–16 (n = 417) n (%) n (%) n (%) n (%) (−) 401 (62.5%) 330 (51.9%) 379 (59.6%) 308 (73.9%) (+) 241 (37.5%) 306 (48.1%) 257 (40.4%) 109 (26.1%)

Dose modification The dose was reduced 314 times in 241 patients (37.5%) in the 6M group and 477 times in 306 patients (48.1%) in the 12M group. In the 12M group, the proportion of patients with dose reduction was lower during cycles 9–16 than during cycles 1–8 (26.1 vs 40.4%) (Table 5). The most common reason for dose reduction was HFS, which occurred 191 times (60.8%) in the 6M group and 290 times (60.8%) in the 12M group.Table 5 Dose reduction Dose reduction 6M group (n = 642) 12M group (n = 636) Overall (n = 636) During cycles 1–8 (n = 636) During cycles 9–16 (n = 417) n (%) n (%) n (%) n (%) (−) 401 (62.5%) 330 (51.9%) 379 (59.6%) 308 (73.9%) (+) 241 (37.5%) 306 (48.1%) 257 (40.4%) 109 (26.1%) The RDI for each cycle is shown in Fig. 2. RDI decreased gradually with each successive treatment cycle, and was ≥60% in 424 patients (66.0%) in the 6M group at cycle 8, 397 patients (62.4%) in the 12M group at cycle 8, and 222 patients (34.9%) in the 12M group at cycle 16. The mean RDI for the entire treatment period and all patients, including those who discontinued prematurely, was 79.5% in the 6M group and 61.3% in the 12M group (median was 89.6 and 65.4%, respectively).Fig. 2 Relative dose intensity in each cycle. a 6M group; b 12M group

patients (34.9%) in the 12M group at cycle 16. The mean RDI for the entire treatment period and all patients, including those who discontinued prematurely, was 79.5% in the 6M group and 61.3% in the 12M group (median was 89.6 and 65.4%, respectively).Fig. 2 Relative dose intensity in each cycle. a 6M group; b 12M group Safety profile (6M group vs 12M group) A total of 589 patients (91.7%) in the 6M group and 602 patients (94.7%) in the 12M group experienced AEs (p = 0.051). Moreover, 158 patients (24.6%) in the 6M group and 197 patients (31.0%) in the 12M group experienced grade ≥3 AEs (p = 0.013). The incidence of major AEs (by worst grade throughout the treatment period) is shown in Table 6. The most common AE was HFS; the incidence of grade ≥3 HFS was 16.8 and 22.6% in the 6M group and 12M group, respectively. Other grade ≥3 AEs with ≥1% incidence included neutropenia, diarrhea, fatigue, and anorexia. There was no treatment-related death in the study.Table 6 Incidence of adverse events by the treatment group 6M group (n = 642) 12M group (n = 636) Any grade, p valuea

Safety profile (6M group vs 12M group) A total of 589 patients (91.7%) in the 6M group and 602 patients (94.7%) in the 12M group experienced AEs (p = 0.051). Moreover, 158 patients (24.6%) in the 6M group and 197 patients (31.0%) in the 12M group experienced grade ≥3 AEs (p = 0.013). The incidence of major AEs (by worst grade throughout the treatment period) is shown in Table 6. The most common AE was HFS; the incidence of grade ≥3 HFS was 16.8 and 22.6% in the 6M group and 12M group, respectively. Other grade ≥3 AEs with ≥1% incidence included neutropenia, diarrhea, fatigue, and anorexia. There was no treatment-related death in the study.Table 6 Incidence of adverse events by the treatment group 6M group (n = 642) 12M group (n = 636) Any grade, p valuea Any grade (%) Grade ≥3 (%) Any grade (%) Grade ≥3 (%) Hemoglobin 36.0 0.3 40.6 0.3 0.103 Leukocytes 19.2 0.6 25.6 0.3 0.007 Neutrophils 15.4 2.6 20.6 3.6 0.020 Platelets 13.7 0.5 13.7 0.5 0.839 Total bilirubin 31.2 0.5 39.2 0.8 0.003 AST 18.1 0.2 21.7 0.6 0.120 ALT 17.0 0.3 20.6 0.5 0.113 Creatinine 5.5 0 8.0 0 0.085 Anorexia 20.9 1.2 21.4 0.9 0.877 Nausea 15.6 0.5 13.7 0.6 0.379 Vomiting 6.4 0.2 5.2 0.5 0.425 Stomatitis 17.9 0.8 21.9 0.9 0.090 Diarrhea 18.1 3.0 14.9 2.0 0.152 Fatigue 15.3 1.7 16.0 1.3 0.762 Rash 10.9 0.5 10.1 0.2 0.690 Hyperpigmentation 27.9 0 25.2 0.5 0.299 Alopecia 1.9 0.2 2.5 0.2 0.550 Hand-foot syndrome 72.0 16.8 77.0 22.6 0.043 AST aspartate aminotransferase, ALT alanine aminotransferase achi-squared test

Any grade (%) Grade ≥3 (%) Any grade (%) Grade ≥3 (%) Hemoglobin 36.0 0.3 40.6 0.3 0.103 Leukocytes 19.2 0.6 25.6 0.3 0.007 Neutrophils 15.4 2.6 20.6 3.6 0.020 Platelets 13.7 0.5 13.7 0.5 0.839 Total bilirubin 31.2 0.5 39.2 0.8 0.003 AST 18.1 0.2 21.7 0.6 0.120 ALT 17.0 0.3 20.6 0.5 0.113 Creatinine 5.5 0 8.0 0 0.085 Anorexia 20.9 1.2 21.4 0.9 0.877 Nausea 15.6 0.5 13.7 0.6 0.379 Vomiting 6.4 0.2 5.2 0.5 0.425 Stomatitis 17.9 0.8 21.9 0.9 0.090 Diarrhea 18.1 3.0 14.9 2.0 0.152 Fatigue 15.3 1.7 16.0 1.3 0.762 Rash 10.9 0.5 10.1 0.2 0.690 Hyperpigmentation 27.9 0 25.2 0.5 0.299 Alopecia 1.9 0.2 2.5 0.2 0.550 Hand-foot syndrome 72.0 16.8 77.0 22.6 0.043 AST aspartate aminotransferase, ALT alanine aminotransferase achi-squared test AEs (any grade) with a higher incidence in the 12M group than in the 6M group included leukocytopenia (25.6 vs 19.2%, p = 0.007), neutropenia (20.6 vs 15.4%, p = 0.020), hyperbilirubinemia (39.2 vs 31.2%, p = 0.003), and HFS (77.0 vs 72.0%, p = 0.043). HFS was the only grade ≥3 AE to occur more frequently in the 12M group than in the 6M group (22.6 vs 16.8%, p = 0.011).

roup than in the 6M group included leukocytopenia (25.6 vs 19.2%, p = 0.007), neutropenia (20.6 vs 15.4%, p = 0.020), hyperbilirubinemia (39.2 vs 31.2%, p = 0.003), and HFS (77.0 vs 72.0%, p = 0.043). HFS was the only grade ≥3 AE to occur more frequently in the 12M group than in the 6M group (22.6 vs 16.8%, p = 0.011). Safety profile (cycles 1–8 vs cycles 9–16) A comparison of the incidence of AEs between the first 8 cycles and the second 8 cycles in the 12M group (Table 7) revealed no difference in the incidence of hematologic toxicities. Another non-hematological AEs (any grade) including anorexia (9.1 vs 18.4%, p < 0.001), nausea (4.1 vs 12.4%, p < 0.001), stomatitis (10.6 vs 18.9%, p < 0.001), diarrhea (6.7 vs 11.6%, p = 0.011), fatigue (7.9 vs 13.1%, p = 0.012), and vomiting (1.7 vs 4.2%, p = 0.034) were lower in cycles 9–16 than in cycles 1–8. This shows that the incidence of gastrointestinal toxicities was lower during the later period of treatment while that of hematologic toxicities remained fairly constant over the entire treatment course. HFS was the only grade ≥3 AE with a significantly lower incidence in the later period (8.6 vs 19.0%, p < 0.001). However, the incidence of grade ≥3 HFS (8.6%) was by far the highest of any grade ≥3 AE occurring during cycles 9–16.Table 7 Adverse events in the 12M group during cycles 1–8 and 9–16

ntire treatment course. HFS was the only grade ≥3 AE with a significantly lower incidence in the later period (8.6 vs 19.0%, p < 0.001). However, the incidence of grade ≥3 HFS (8.6%) was by far the highest of any grade ≥3 AE occurring during cycles 9–16.Table 7 Adverse events in the 12M group during cycles 1–8 and 9–16 During cycles 1–8 (n = 636) During cycles 9–16 (n = 417) Any grade (%) Grade ≥3 (%) Any grade (%) Grade ≥3 (%) Hemoglobin 34.9 0 31.7 0.5 Leukocytes 19.3 0.3 21.1 0 Neutrophils 17.6 2.8 12.9 2.2 Platelets 9.6 0.5 12.7 0 Total bilirubin 32.5 0.6 36.0 0.5 AST 16.2 0.3 17.5 0.5 ALT 15.4 0.3 14.1 0.2 Creatinine 5.2 0 7.7 0 Anorexia 18.4 0.9 9.1 0 Nausea 12.4 0.6 4.1 0 Vomiting 4.2 0.5 1.7 0 Stomatitis 18.9 0.9 10.6 0 Diarrhea 11.6 1.7 6.7 0.5 Fatigue 13.1 0.9 7.9 0.5 Rash 7.5 0.2 6.7 0 Hyperpigmentation 20.8 0.5 18.7 0 Alopecia 1.9 0 1.4 0.2 Hand-foot syndrome 71.1 19.0 66.7 8.6 AST aspartate aminotransferase, ALT alanine aminotransferase The cumulative incidence of grade ≥1, grade ≥2, and grade ≥3 HFS by treatment group is shown in Fig. 3. The rise in cumulative onset of HFS during cycles 1–8 was quite similar between the 6M group and 12M group. In the 12M group, the rise in cumulative onset was gradual and constant even during cycles 9–16.Fig. 3 Cumulative incidence of hand-foot syndrome

grade ≥2, and grade ≥3 HFS by treatment group is shown in Fig. 3. The rise in cumulative onset of HFS during cycles 1–8 was quite similar between the 6M group and 12M group. In the 12M group, the rise in cumulative onset was gradual and constant even during cycles 9–16.Fig. 3 Cumulative incidence of hand-foot syndrome Discussion We compared the treatment details and AE profile after 6 and 12 months of adjuvant capecitabine in 1278 Japanese patients with stage III colon cancer. This is the first prospective RCT data demonstrating the safety of adjuvant capecitabine in a large sample of Japanese patients. The most common AE was HFS, a characteristic toxic reaction to capecitabine; 72.0 and 77.0% of patients experienced grade ≥1 HFS, and 16.8 and 22.6% experienced grade ≥3 HFS in the 6M group and 12M group, respectively. The incidences of other AEs were relatively low overall and acceptable.

Discussion We compared the treatment details and AE profile after 6 and 12 months of adjuvant capecitabine in 1278 Japanese patients with stage III colon cancer. This is the first prospective RCT data demonstrating the safety of adjuvant capecitabine in a large sample of Japanese patients. The most common AE was HFS, a characteristic toxic reaction to capecitabine; 72.0 and 77.0% of patients experienced grade ≥1 HFS, and 16.8 and 22.6% experienced grade ≥3 HFS in the 6M group and 12M group, respectively. The incidences of other AEs were relatively low overall and acceptable. A comparison of the AE profile between our study and the X-ACT trial [5], a pivotal study of adjuvant capecitabine for colon cancer patients (Table 8), found no difference in the incidence of grade ≥3 HFS between Japanese and Western patients. It was reported that oral FUs were less frequently associated with gastrointestinal toxicities in Asian patients than in Caucasian patients [11, 12]. Indeed, the incidences of gastrointestinal toxicities (i.e., diarrhea, nausea, vomiting, and stomatitis) were lower in our study than in the X-ACT. When compared to toxicities associated with other oral FUs (such as UFT/LV and S-1) used as adjuvant chemotherapy for CRC in Japan (Table 8), anorexia, nausea, and diarrhea associated with capecitabine were less frequent [11, 12]. From these findings, we suggest that capecitabine might be an easy-to-use oral FU for Japanese patients, when HFS is well-controlled.Table 8 Reported incidence of grade ≥3 adverse events in other studies [5, 11, 12]

apan (Table 8), anorexia, nausea, and diarrhea associated with capecitabine were less frequent [11, 12]. From these findings, we suggest that capecitabine might be an easy-to-use oral FU for Japanese patients, when HFS is well-controlled.Table 8 Reported incidence of grade ≥3 adverse events in other studies [5, 11, 12] HFS was the only grade ≥3 AE whose incidence increased with treatment during the period extending from 6 to 12 months. Although lower in the second 6-month period than in the first 6-month period, the overall incidence of HFS increased gradually and constantly even in the later period and therefore was higher in the 12M group than in the 6M group (Table 6). Although the completion rate for 6-month treatment in our study was similar to that in other studies of adjuvant oral FU therapy for colon cancer [4, 5, 11–13], the completion rate for 12-month treatment was <50%. Approximately half of the discontinuations during cycles 9–16 was due to AEs, most commonly HFS. These findings indicate that prolonging the treatment duration resulted in a higher incidence of HFS with constant cumulative onset, and that HFS led mainly to dose reduction and treatment discontinuation, but was not lethal. Therefore, effective management of HFS could improve the completion rate for 12-month treatment.

ese findings indicate that prolonging the treatment duration resulted in a higher incidence of HFS with constant cumulative onset, and that HFS led mainly to dose reduction and treatment discontinuation, but was not lethal. Therefore, effective management of HFS could improve the completion rate for 12-month treatment. The use of a hydrating cream, external steroid, and oral vitamin B6 is the accepted supportive treatment for HFS [14] and was allowed in our study. However, in this study, 477 (74.3%) and 471 (74.5%) patients in the 6M group and 12M group, respectively, used oral vitamin B6. These recommended supportive measures should be taken in all cases, even at the start of the treatment. The X-ACT trial reported that appropriate dose reduction does not impair the efficacy of adjuvant capecitabine therapy and that the survival rate was better among those who developed HFS than among those who did not [15]. These observations suggest that appropriate dose reduction to manage HFS is important to maintain the treatment duration and to improve patient outcome. The result of the primary object of our study, a comparison of survival rate between the 6M group and 12M group, will be available in late 2016.

hose who did not [15]. These observations suggest that appropriate dose reduction to manage HFS is important to maintain the treatment duration and to improve patient outcome. The result of the primary object of our study, a comparison of survival rate between the 6M group and 12M group, will be available in late 2016. In conclusion, compared to the standard 6 month-treatment, the cumulative incidence of HFS (the most common reason for treatment dropout) increased further after 12 months of adjuvant capecitabine, although overall, the incidence and severity of AEs after 12 months of capecitabine were acceptable. Appropriate dose modification and supportive care could reduce the rate of treatment discontinuation due to HFS and improve treatment compliance. An erratum to this article is available at http://dx.doi.org/10.1007/s10147-017-1146-6. Acknowledgements We are grateful to all patients and co-investigators for their cooperation for the JFMC37-0801 trial. The authors also thank the following additional investigators for their contributions to this trial—Yukari Kawamura (the Japanese Foundation for Multidisciplinary Treatment of Cancer) for data management, and Minako Nakashima (the Japanese Foundation for Multidisciplinary Treatment of Cancer) for data analysis. Funding JFMC37-0801 study is funded and conducted by the Japanese Foundation for Multidisciplinary Treatment of Cancer.

Acknowledgements We are grateful to all patients and co-investigators for their cooperation for the JFMC37-0801 trial. The authors also thank the following additional investigators for their contributions to this trial—Yukari Kawamura (the Japanese Foundation for Multidisciplinary Treatment of Cancer) for data management, and Minako Nakashima (the Japanese Foundation for Multidisciplinary Treatment of Cancer) for data analysis. Funding JFMC37-0801 study is funded and conducted by the Japanese Foundation for Multidisciplinary Treatment of Cancer. Compliance with ethical standards Conflict of interest MI has received consulting fees from Taiho Pharmaceutical; honoraria from Taiho, Yakult Honsha, and Merck Serono; research funding from Taiho Pharmaceutical and Yakult Honsha. SH has received research funding from Toyo-Kohan and NEC Corporation. HMi has received honoraria from Chugai and Merck Serono; research funding from Chugai, Taiho, Yakult, and Daiichi-Sankyo. KSu has received honoraria from Chugai Pharmaceutical Co., Taiho Pharmaceutical Co., Merk Serono Co., Bayer Yakuhin Ltd., and Eli Lilly Co. as well as research funding from Chugai Pharmaceutical Co. and Taiho Pharmaceutical Co. JS has received honoraria from Yakult Honsha Co. Ltd, and consultancy fee from Takeda Co. Ltd. All remaining authors have declared no conflict of interest.

Introduction A greater understanding of molecular biology has led to major breakthroughs in medical treatment for patients with renal cell cancer (RCC). Vascular endothelial growth factor (VEGF), platelet-derived growth factor, and mammalian target of rapamycin (mTOR) signaling pathways have become recognized as rational targets for targeted therapy [1]. Angiogenesis inhibitors, which include sorafenib (Nexavar®, Bayer), sunitinib (Sutent®, Pfizer), bevacizumab (Avastin®, Genentech/Roche), pazopanib (Votrient®, Novartis), and axitinib (Inlyta®, Pfizer) [2–6]; and two mTOR inhibitors, temsirolimus (Torisel®, Pfizer) and everolimus (Afinitor®, Novartis) [7, 8], are all currently available as a result of the first breakthrough in metastatic RCC therapy, although bevacizumab has not been approved in Japan.

, pazopanib (Votrient®, Novartis), and axitinib (Inlyta®, Pfizer) [2–6]; and two mTOR inhibitors, temsirolimus (Torisel®, Pfizer) and everolimus (Afinitor®, Novartis) [7, 8], are all currently available as a result of the first breakthrough in metastatic RCC therapy, although bevacizumab has not been approved in Japan. We are currently on the verge of the second breakthrough. Nivolumab (Opdivo®, Ono/Bristol-Myers Squibb) is a novel targeted agent that has just been launched for clinical practice in the treatment of metastatic RCC [9]. Nivolumab, which is a fully human immunoglobulin (Ig) G4 programmed death 1 (PD-1) antibody, selectively inhibits the interaction between PD-1 (which is expressed on activated T cells) and PD-1 ligand 1 (PD-L1) and 2 (PD-L2) (which are expressed on antigen-presenting cells [APCs] and cancer cells) [9]. Its promising anti-tumor efficacy and manageable safety profile were demonstrated in the phase III Checkmate 025 trial. Nivolumab therapy is thus being rapidly introduced in metastatic RCC clinical practice in Japan. Recently, excellent treatment results for the phase Ia study of atezolizumab (Roche/Genentech), which is a humanized anti-PD-L1 monoclonal IgG1 antibody, were also demonstrated [10]. The identification of biomarkers to predict the response and side-effects for checkpoint inhibitor therapy is urgently needed.

in Japan. Recently, excellent treatment results for the phase Ia study of atezolizumab (Roche/Genentech), which is a humanized anti-PD-L1 monoclonal IgG1 antibody, were also demonstrated [10]. The identification of biomarkers to predict the response and side-effects for checkpoint inhibitor therapy is urgently needed. Previously, we reviewed the candidate biomarkers of angiogenesis inhibitor therapy in terms of clinical variables, genetic factors, and circulating proteins and endothelial cells [11]. Regarding biomarkers of RCC patients treated with checkpoint inhibitors, however, the role of potential predictive biomarkers to benefit the PD-1/PD-L1 blockade remains controversial and is still under investigation. Most of the ongoing clinical trials have established exploratory biomarker sub-analyses to attempt to identify predictive biomarkers of response to PD-1/PD-L1 inhibition, including PD-L1 expression. Rodriguez-Vida et al. reviewed them comprehensively [12]. Research on other malignancies may also shed light on biomarker analyses in metastatic RCC therapy. Here, we provide a brief overview of biomarkers in terms of the tumor immune microenvironment and clinical factors of RCC and other malignant tumor studies.

sion. Rodriguez-Vida et al. reviewed them comprehensively [12]. Research on other malignancies may also shed light on biomarker analyses in metastatic RCC therapy. Here, we provide a brief overview of biomarkers in terms of the tumor immune microenvironment and clinical factors of RCC and other malignant tumor studies. Tumor immune microenvironment Cancer cells are recognized by APCs in which cancer cells are processed to peptide antigens; cancer cells are then presented on major histocompatibility class I (MHC-I) or class II (MHC-II) molecules as cancer-specific neoantigens [13, 14]. When CD8-positive cytotoxic T lymphocytes (CTLs) recognize these neoantigens presented on the MHC molecules, the CTLs are activated and proliferate, leading to an antigen-specific immune response that kills neoantigen-bearing cancer cells [13, 14]. However, negative regulators exist, namely, the complex of PD-1 and PD-L1/PD-L2. PD-L1 and PD-L2, which are known to be expressed on the surface of APCs and cancer cells, engage PD-1, which expresses on CD8-positive CTLs [13, 14]. When these PD-1 and PD-L1/L2 complexes are complete, they trigger an inhibitory signal to the downstream of the T-cell receptor (TCR), and block effector and CTL functions [13, 14]. Here, immune tolerance is achieved. Destruction of this immune tolerance using immune checkpoint inhibitors forms the basis for the current novel immune therapy (Fig. 1a). In this scenario, the number of neoantigens and the expression of MHC molecules (Fig. 1b) and PD-1/PD-L1 expression (Fig. 1c) can be considered potential biomarkers for immune checkpoint inhibitor therapy.Fig. 1 Tumor microenvironment and immune checkpoint inhibitors (Fig. 1a). Cancer cells are recognized by APCs in which cancer cells are processed to peptide antigen; cancer cells are then presented on MHC-I/II as cancer-specific neoantigens. After recognizing these neoantigens, the CTLs are activated and proliferate, and kill neoantigen-bearing cancer cells (Fig. 1b). When a complex of PD-L1 expressed by APCs and cancer cells engage PD-1 expressed on CD8-positive CTLs is complete, immune tolerance is achieved. Destruction of this immune tolerance using immune checkpoint inhibitors is the current novel immune therapy (Fig. 1c). MHC major histocompatibility, CTL cytotoxic T lymphocytes, PD-1 programmed death-1, PD-L1 programmed death-ligand 1, APC antigen-presenting cell, TCR T cell receptor

lete, immune tolerance is achieved. Destruction of this immune tolerance using immune checkpoint inhibitors is the current novel immune therapy (Fig. 1c). MHC major histocompatibility, CTL cytotoxic T lymphocytes, PD-1 programmed death-1, PD-L1 programmed death-ligand 1, APC antigen-presenting cell, TCR T cell receptor Neoantigens and MHC antigens Neoantigens, which constitute between 8 and 10 peptides, are generally established from tumor-specific mutations, presented by MHC class I or MHC class II molecules on the surface of APCs, and recognized by CD8-positive CTLs that may be able to destroy cancer cells (Fig. 1b) [13, 14]. Although all of the non-synonymous mutations do not always constitute neoantigens, it is probable that the more non-synonymous mutations are affected, the more neoantigens develop. Lawrence et al. investigated the heterogeneity across patients with 27 cancer types, and revealed that the median frequency of non-synonymous mutations varied by >1000-fold across cancer types [15]. Melanoma and lung cancer showed the highest mutation frequencies, exceeding 100/Mb [15]. These may be attributable to extensive exposure to well-known carcinogens, such as ultraviolet radiation in the case of melanoma and tobacco smoke in the case of lung cancers [15]. Among the cancers, RCC (including clear cell cancer and papillary cancer) were in the middle position and demonstrated a low frequency of mutation burden compared with lung cancer and melanoma [15]. Mutation frequencies, however, varied markedly across patients within a cancer type. In clear cell renal cancer, the frequency ranged from 0.1−10/Mb [15]. Rizvi et al. examined the association between the mutation burden and the response of the immune checkpoint inhibitor in non-small cell lung cancer (NSCLC) patients treated with pembrolizumab (Keytruda®, MSD), which is a humanized antibody for PD-1 [16]. In this study, they used whole-exome sequencing and reported that higher non-synonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and longer progression-free survival (PFS) [16]. In addition, the efficacy was also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations [16]. Interestingly, although the efficacy was significantly correlated with the molecular smoking signature, self-reported smoking history did not significantly discriminate those most likely to benefit from pembrolizumab [16].

the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations [16]. Interestingly, although the efficacy was significantly correlated with the molecular smoking signature, self-reported smoking history did not significantly discriminate those most likely to benefit from pembrolizumab [16]. A small fraction of advanced colorectal cancer occurs as a result of mismatch-repair (MMR) deficiency. Uram et al. investigated the efficacy of immune checkpoint inhibitors for colorectal and non-colorectal gastrointestinal cancer patients who have MMR deficiency treated with pembrolizumab [17]. In this study, whole-exome sequencing revealed that a mean of 1782 somatic mutations per tumor in MMR-deficient tumors was much greater than in MMR-proficient tumors, which had a mean of only 73 per tumor (p = 0.007) [17]. The median PFS and overall survival (OS) periods, and objective response rate of patients with MMR-deficient colorectal cancer were significantly superior to those of patients with MMR-proficient colorectal cancer [HR for PFS and OS, 0.10 (p < 0.001) and 0.22 (p = 0.05), respectively] [17]. In addition, patients with MMR-deficient non-colorectal gastrointestinal cancer had responses similar to those of patients with MMR-deficient colorectal cancer [17].

ficantly superior to those of patients with MMR-proficient colorectal cancer [HR for PFS and OS, 0.10 (p < 0.001) and 0.22 (p = 0.05), respectively] [17]. In addition, patients with MMR-deficient non-colorectal gastrointestinal cancer had responses similar to those of patients with MMR-deficient colorectal cancer [17]. Regarding the receiver of the neoantigen, the expression of MHC antigen might play a role in the efficacy of immune checkpoint inhibitors (Fig. 1b). Using two independent cohorts of anti-PD-1-treated melanoma patients, Johnson et al. reported that MHC-II positivity on cancer cells is associated with therapeutic response, PFS, and OS, as well as CD4 and CD8 tumor infiltration [18]. They concluded that MHC-II expression on cancer cells can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR in order to improve anti-PD-1 patient selection [18]. In addition, in an in vivo study using murine lung cancer cells and anti-mouse PD-1 antibodies, Wang et al. reported that MHC class I and II were significantly downregulated in anti-PD1-resistant tumors compared with anti-PD1-sensitive tumors [19].

bodies for HLA-DR in order to improve anti-PD-1 patient selection [18]. In addition, in an in vivo study using murine lung cancer cells and anti-mouse PD-1 antibodies, Wang et al. reported that MHC class I and II were significantly downregulated in anti-PD1-resistant tumors compared with anti-PD1-sensitive tumors [19]. PD-L1 expression Before describing PD-L1 expression, we must note that there are various factors that influence the PD-L1 expression and clinical efficacy of immune checkpoint inhibitors (Table 1). There are also various assays, including antibodies and cut-off points. There might be a difference between newly collected specimens and archival tumor samples. Furthermore, PD-L1 expression is dynamic and is affected by many factors, including prior therapy and the presence of tumor-infiltrating immune cells, which lead to intra-tumor differences of PD-L1 expression among primary tumors and individual metastatic sites.Table 1 Various interference factors for programmed death-ligand 1 (PD-L1) expression Used antibody Immunohistochemistry procedure Cut-off point of stained sample Newly corrected specimen or archival tumor sample Heterogeneity between primary and metastatic sites Heterogeneity among metastatic sites Past treatment history

PD-L1 expression Before describing PD-L1 expression, we must note that there are various factors that influence the PD-L1 expression and clinical efficacy of immune checkpoint inhibitors (Table 1). There are also various assays, including antibodies and cut-off points. There might be a difference between newly collected specimens and archival tumor samples. Furthermore, PD-L1 expression is dynamic and is affected by many factors, including prior therapy and the presence of tumor-infiltrating immune cells, which lead to intra-tumor differences of PD-L1 expression among primary tumors and individual metastatic sites.Table 1 Various interference factors for programmed death-ligand 1 (PD-L1) expression Used antibody Immunohistochemistry procedure Cut-off point of stained sample Newly corrected specimen or archival tumor sample Heterogeneity between primary and metastatic sites Heterogeneity among metastatic sites Past treatment history Regarding the difference between PD-L1 expression and the characteristics of RCC, the Mayo Clinic published interesting reports. Thompson et al. reported that PD-L1 expression was demonstrated in both clear cell RCC tumor cells (present in 66% of specimens) and tumor-infiltrating mononuclear cells (present in 59% of specimens) by immunohistochemical analysis [20, 21]. High levels of PD-L1 within the tumors were significantly more likely to exhibit aggressive pathologic features, including higher nuclear grade (p < 0.001), positive lymph node metastases (p < 0.001), and distant metastases (p = 0.022) [20, 21]. In addition, they reported that both metastatic RCC cells and infiltrating lymphocytes express PD-L1 at rates similar to those observed in primary clear cell RCC tumor lesions [20, 21].

, including higher nuclear grade (p < 0.001), positive lymph node metastases (p < 0.001), and distant metastases (p = 0.022) [20, 21]. In addition, they reported that both metastatic RCC cells and infiltrating lymphocytes express PD-L1 at rates similar to those observed in primary clear cell RCC tumor lesions [20, 21]. Taube et al. investigated PD-L1 and PD-L2 expression of cancer cells and infiltrating immune cells in various cancer types. Cell surface PD-L1 expression by cancer cells and immune-infiltrating cells varied significantly by tumor type, and the most abundant expression was demonstrated in melanoma, NSCLC, and RCC [22]. Expression of PD-L1 by cancer cells and infiltrating immune cells was significantly associated with expression of PD-1 on lymphocytes [22]. PD-L2 expression was also associated with PD-L1 expression [22]. In addition, PD-L1 expression on cancer cells demonstrated a significant correlation with an objective response to anti-PD-1 therapy [22]. Daud et al. also investigated the relationship between anti-PD-1 activity and PD-L1 expression in patients with advanced melanoma who were treated with pembrolizumab in the phase Ib KEYNOTE-001 study [23]. In this study of 451 patients with evaluable PD-L1 expression, 344 (76%) demonstrated PD-L1 expression. High PD-L1 expression demonstrated a significant correlation with a high response rate and long PFS (HR 0.76; 95% confidence interval [CI] 0.71–0.82, p < 0.001) and long OS (HR 0.76; 95% CI 0.69–0.83, p < 0.001) [23]. Expression of PD-L1 is thus a potential predictive biomarker for response and outcome following treatment with PD-L1/PD-1 immune checkpoint inhibitor therapy.

tion with a high response rate and long PFS (HR 0.76; 95% confidence interval [CI] 0.71–0.82, p < 0.001) and long OS (HR 0.76; 95% CI 0.69–0.83, p < 0.001) [23]. Expression of PD-L1 is thus a potential predictive biomarker for response and outcome following treatment with PD-L1/PD-1 immune checkpoint inhibitor therapy. From the viewpoint of immune checkpoint inhibitor therapy, Hodgkin’s lymphoma is very interesting. In all cases of classical Hodgkin’s lymphoma, Hodgkin Reed−Sternberg cells have copy number alterations of 9p24.1, a region that includes PD-L1 and PD-L2, and contributes to robust expression of these PD-1 ligands [24]. Amplification of 9p24.1 is more common in patients with advanced-stage Hodgkin’s lymphoma. Like a driver gene mutation, amplification of PD-L1 and PD-L2 plays an important role in pathogenesis and treatment resistance in this disease. Before the checkpoint inhibitor therapy era, PD-L1 and PD-L2 amplification was associated with poor prognosis [24]. Therefore, checkpoint inhibitor therapy was warranted. In fact, in the phase I study of nivolumab, 23 patients with relapsed or refractory Hodgkin’s lymphoma, who had already been heavily treated, received nivolumab [25]. An excellent objective response rate of 87% was obtained, including 17% with a complete response and 70% with a partial response [25]. In December 2016 in Japan, nivolumab received approval for treatment of patients with classical Hodgkin’s lymphoma that had relapsed or progressed after initial treatment.

volumab [25]. An excellent objective response rate of 87% was obtained, including 17% with a complete response and 70% with a partial response [25]. In December 2016 in Japan, nivolumab received approval for treatment of patients with classical Hodgkin’s lymphoma that had relapsed or progressed after initial treatment. Clinical factors In the cytokine era, prognostic factors that could predict outcome in patients with metastatic RCC treated with interferon (IFN)-α as initial systemic therapy were defined by the Memorial Sloan Kettering Cancer Center (MSKCC) study group [26]. The MSKCC group extracted five variable risk factors for short survival—low Karnofsky performance status (PS), high serum lactate dehydrogenase (LDH), low blood hemoglobin (Hb), high corrected serum calcium (Ca), and time from initial RCC diagnosis to start of IFN-α therapy of <1 year (26). Later, the MSKCC group reported the prognostic factors of previously treated RCC patients who had received new agents as salvage therapy [27]. Three factors, including low Karnofsky PS, low Hb level, and high corrected serum Ca level, were extracted as the MSKCC prognostic factors for patients treated by the second-line therapy [27]. In the molecular targeted therapy era, Heng et al. first reported results from a large, multicenter study of 645 patients with anti-VEGF therapy-naive metastatic RCC [28]. In this study, four of the five adverse prognostic factors according to MSKCC score (low Hb, high corrected Ca level, low PS, and time from diagnosis to treatment of <1 year) emerged as independent predictors of poor OS [28]. In addition, high levels of neutrophils and platelets emerged as independent adverse prognostic factors [28]. Later, these prognostic factors were applied to patients previously treated with targeted therapy, in addition to previously validated populations in first-line targeted therapy [29]. These six risk factors are now widely used and are known as the International Metastatic RCC Database Consortium (IMDC) criteria. In the immune checkpoint inhibitor era, these known and widely used criteria must be re-evaluated.

erapy, in addition to previously validated populations in first-line targeted therapy [29]. These six risk factors are now widely used and are known as the International Metastatic RCC Database Consortium (IMDC) criteria. In the immune checkpoint inhibitor era, these known and widely used criteria must be re-evaluated. Baseline clinical factors associated with OS after immune checkpoint blockade for melanoma patients treated by pembrolizumab have been reported [30]. Relative eosinophil count ≥1.5%, relative lymphocyte count ≥17.5%, ≤2.5-fold elevation of LDH, and absence of metastasis other than soft tissue/lung were extracted as independent favorable prognostic factors (all p < 0.001). In terms of eosinophil count, however, another group reported that eosinophilia was a favorable prognostic factor independent of therapeutic agents [31].

≥17.5%, ≤2.5-fold elevation of LDH, and absence of metastasis other than soft tissue/lung were extracted as independent favorable prognostic factors (all p < 0.001). In terms of eosinophil count, however, another group reported that eosinophilia was a favorable prognostic factor independent of therapeutic agents [31]. Other groups also reported serum LDH level as a prognostic factor for advanced/metastatic melanoma patients treated with nivolumab or pembrolizumab [32]. After a median follow-up of 9 months, patients with an elevated baseline LDH had a significantly shorter OS compared to patients with normal LDH (6 month OS 60.8 vs 81.6% and 12 month OS 44.2 vs 71.5% (p = 0.0292) [32]. In addition, patients with a relative increase of >10% from elevated baseline LDH had a significantly shorter OS compared to patients with a decrease or <10% increase (4.3 vs 15.7 months, p = 0.00623) [32]. They concluded that LDH could be a useful marker at baseline as well as during treatment to predict early response or progression in patients with advanced melanoma who received immune checkpoint inhibitor therapy [32]. Similarly, Nakayama et al. reported pretreatment as well as on-treatment prognostic factors for patients with melanoma treated with nivolumab [33]. The Eastern Cooperative Oncology Group (ECOG) PS ≥1, maximum tumor diameter of ≥30 mm, elevated LDH, and elevated C-reactive protein (CRP) were significantly associated with poor OS [HR 0.29 (p < 0.001), HR 0.40 (p = 0.003), HR 0.29 (p < 0.001), HR 0.42 (p = 0.004), respectively] on univariate analysis [33]. Among these factors, PS and LDH were identified as independent variables by multivariate analysis [33]. In addition, for early treatment responding markers, patients with absolute lymphocyte count ≥1000/μl [week 3, HR 0.40 (p = 0.004); week 6, HR 0.33 (p = 0.001)] and absolute neutrophil count <4000/μl [week 3, HR 0.46 (p = 0.014); week 6, HR 0.51 (p = 0.046)] had significantly better OS [33].

multivariate analysis [33]. In addition, for early treatment responding markers, patients with absolute lymphocyte count ≥1000/μl [week 3, HR 0.40 (p = 0.004); week 6, HR 0.33 (p = 0.001)] and absolute neutrophil count <4000/μl [week 3, HR 0.46 (p = 0.014); week 6, HR 0.51 (p = 0.046)] had significantly better OS [33]. The final topic in terms of clinical factors is adverse events. Are adverse events associated with the efficacy of immune checkpoint inhibitors? In melanoma patients treated with nivolumab, immune-related adverse events (irAEs) are reported to be associated with improved survival [34]. In this study, irAEs of any grade were observed in 68.2% of patients (101 of 148). A statistically significant OS difference was noted among patients with any grade of irAE versus those without (p < 0.001), and OS benefit was noted in patients who reported ≥3 irAE events (p < 0.001) [34]. In addition, rash and vitiligo correlated with statistically significant OS differences in patients with metastatic disease (p = 0.004 and p = 0.028, respectively) [34].

g patients with any grade of irAE versus those without (p < 0.001), and OS benefit was noted in patients who reported ≥3 irAE events (p < 0.001) [34]. In addition, rash and vitiligo correlated with statistically significant OS differences in patients with metastatic disease (p = 0.004 and p = 0.028, respectively) [34]. Conclusion In this review, we introduced the current candidate biomarkers of immune checkpoint inhibitor therapy. Based on the mechanism of efficacy, the number of neoantigens and expression of MHC molecules are strong candidate biomarkers (Fig. 1b). Despite the various interference factors (Table 1), PD-1/PD-L1 expression can be considered a potential biomarker (Fig. 1c). Regarding clinical factors in metastatic RCC patients, we already have two well-known criteria, including MSKCC and IMDC; however, these widely used criteria must be re-evaluated. Finally, we introduced serum clinical factors and severity of adverse effects as candidate biomarkers of favorable efficacy (Fig. 2). Although further implementation in prospective studies is necessary, if validated, these biomarkers can be utilized to measure therapeutic response and design treatment strategies for metastatic RCC.Fig. 2 Clinical factors as candidate biomarkers. In addition to various baseline factors, correlations between immune checkpoint inhibitor efficacy and adverse effects have been reported. PS performance status, HB hemoglobin, LDH lactate dehydrogenase, Ca calcium, Dx diagnosis, LLN lower limit of normal range, ULN upper limit of normal range, CRP C-reactive protein

. In addition to various baseline factors, correlations between immune checkpoint inhibitor efficacy and adverse effects have been reported. PS performance status, HB hemoglobin, LDH lactate dehydrogenase, Ca calcium, Dx diagnosis, LLN lower limit of normal range, ULN upper limit of normal range, CRP C-reactive protein Acknowledgements This work was partly supported by the Smoking Research Foundation and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Compliance with ethical standards Conflict of interest TY received remuneration for a lecture from Astellas (Tokyo, Japan), Sanofi Japan (Tokyo, Japan), Pfizer Japan (Tokyo, Japan), Novartis Pharma Japan (Tokyo, Japan), Ono Pharma (Osaka, Japan), and Daiichi-Sankyo (Tokyo, Japan). The other authors have declared no conflict of interest.

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a poor prognosis; it is the fourth leading cause of cancer-related deaths in Japan and the fourth most common malignancy in the USA [1, 2]. At the time of PDAC diagnosis, less than 20% of the tumors are resectable, and the actual 5-year survival rate is reported to range from 15 to 25% [3]. Current clinical treatments for PDAC have limited efficacy; thus, improved treatment strategies are required to prolong patient survival. Expression of programmed death-1 (PD-1) is significantly upregulated on activated cancer-specific T cells. The PD-1 receptor attaches to its ligand PD-L1, which is expressed by tumor cells and infiltrating immune cells. The interaction of PD-1 and PD-L1 inhibits T-cell activation and promotes tumor immune escape [4–7]. The escape mechanism acquired by tumor cells to avoid immune recognition and destruction is a major contributor to the limitations of the therapeutic efficacy. However, recently developed therapeutic antibodies against PD1/PD-L1 show promising clinical results for several tumors, such as melanoma, renal cancer, and non-small cell lung cancer [8].

d by tumor cells to avoid immune recognition and destruction is a major contributor to the limitations of the therapeutic efficacy. However, recently developed therapeutic antibodies against PD1/PD-L1 show promising clinical results for several tumors, such as melanoma, renal cancer, and non-small cell lung cancer [8]. Although the efficacy of PD-1/PD-L1 antibody therapy should be correlated with PD-L1 protein expression in tumor cells, approximately 10–40% of PD-L1 immuno-negative cases also respond to anti-PD-1/PD-L1 therapy [9–11]. This contradiction may be caused by the performance of the PD-L1 immunostaining assay which is based on the color intensity visualized using the chromogen dye diaminobenzidine (DAB). We recently developed an immunohistochemistry method using fluorescence-emitting phosphor-integrated dot (PID) nanoparticles as a fluorescent dye. PID shows a higher luminance and dynamic range than those of conventional fluorescent dyes and DAB [12]. Specifically, the fluorescence intensity of the PID particles was found to be approximately 100-fold higher than that of a conventional fluorescent dye. Also, the ratio of particle to antibody binding is 1:1. Thus, this technique is highly sensitive as well as quantitative as compared to the conventional DAB-based method. The aim of the study reported here was to evaluate the expression of PD-L1 in patients with PDAC by immunostaining using PID technology and to compare the results with those obtained after conventional DAB staining.

Although the efficacy of PD-1/PD-L1 antibody therapy should be correlated with PD-L1 protein expression in tumor cells, approximately 10–40% of PD-L1 immuno-negative cases also respond to anti-PD-1/PD-L1 therapy [9–11]. This contradiction may be caused by the performance of the PD-L1 immunostaining assay which is based on the color intensity visualized using the chromogen dye diaminobenzidine (DAB). We recently developed an immunohistochemistry method using fluorescence-emitting phosphor-integrated dot (PID) nanoparticles as a fluorescent dye. PID shows a higher luminance and dynamic range than those of conventional fluorescent dyes and DAB [12]. Specifically, the fluorescence intensity of the PID particles was found to be approximately 100-fold higher than that of a conventional fluorescent dye. Also, the ratio of particle to antibody binding is 1:1. Thus, this technique is highly sensitive as well as quantitative as compared to the conventional DAB-based method. The aim of the study reported here was to evaluate the expression of PD-L1 in patients with PDAC by immunostaining using PID technology and to compare the results with those obtained after conventional DAB staining. Materials and methods Patients and samples This study included 42 patients with PDAC, of whom 31 underwent pancreaticoduodenectomy and 11 underwent distal pancreatectomy at the Department of Surgery at Kansai Medical University Hospital (Osaka, Japan) between May 2001 and December 2007. All patients had histologically confirmed PDAC. The tumors were classified according to the TNM classification [13]. The clinical parameters of all patients were collected from a prospectively maintained institutional PDAC database.

t Kansai Medical University Hospital (Osaka, Japan) between May 2001 and December 2007. All patients had histologically confirmed PDAC. The tumors were classified according to the TNM classification [13]. The clinical parameters of all patients were collected from a prospectively maintained institutional PDAC database. Surgically resected specimens were fixed in formalin and embedded in paraffin; and serial sections cut from the embedded specimens were stained with hematoxylin and eosin for histological evaluation. The most representative tumor areas were sampled for the tissue microarray using 2-mm-diameter samples (Azumaya, Tokyo, Japan). This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the institutional review board of our hospital (Protocol no. H151043 and 27-14). Immunohistochemistry Four-micrometer-thick sections were deparaffinized using routine procedures. Endogenous peroxidase activity in the deparaffinized sections was blocked by treating the sections with 3% hydrogen peroxide treatment for 15 min, followed by washing in deionized water for 2–3 min. The sections were subsequently boiled in 10 mM sodium citrate buffer for 10 min at 121 °C, then allowed to cool at room temperature for 40 min, followed by rinsing with deionized water and washing with phosphate-buffered saline for 5 min.

eroxide treatment for 15 min, followed by washing in deionized water for 2–3 min. The sections were subsequently boiled in 10 mM sodium citrate buffer for 10 min at 121 °C, then allowed to cool at room temperature for 40 min, followed by rinsing with deionized water and washing with phosphate-buffered saline for 5 min. Measurement of the fluorescence properties of PID The sections were incubated with the primary antibody toward PD-L1 (E1L3N, 1:1000, Cell Signaling Technology, Danvers, MA). Sections were incubated with 2 μg/mL biotinylated anti-rabbit antibody (LO-RG-1) for 30 min and then with PID-conjugated streptavidin (0.06 nM) for 2 h, both at room temperature. The sections were then irradiated at 580 nm, and the fluorescence intensities were measured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan); images were acquired with a DP73 CCD camera (Olympus) (Fig. 1). The number of PID particles per cell was measured with an automated PID Analyzer (Konica Minolta, Tokyo, Japan). The number of PD-L1 particles was evaluated only on the tumor cells. Five fields at 400× magnification were selected randomly, and the number of PD-L1 particles on each tumor cell was counted and the average number of particles per cell then calculated for each field. The highest value among the five fields was determined to be the PID staining value. The negative control was prepared with PID staining but without the primary antibody.Fig. 1 Immunohistochemistry of pancreatic ductal adenocarcinoma tissue using phosphor-integrated dot (PID) staining. Red spots on tumor cells indicate PID particles

lue among the five fields was determined to be the PID staining value. The negative control was prepared with PID staining but without the primary antibody.Fig. 1 Immunohistochemistry of pancreatic ductal adenocarcinoma tissue using phosphor-integrated dot (PID) staining. Red spots on tumor cells indicate PID particles Measurement of DAB intensity The sections were incubated with the primary antibody toward PD-L1 (E1L3N, 1:200, Cell Signaling Technology) diluted in antibody diluent (Signal Stain Antibody Diluent #8112; Cell Signaling Technology). The sections were treated with peroxidase-labeled secondary antibody (EnVision/HRP system; DAKO, Carpinteria, CA) after linker reagent treatment for 15 min. The sections were then rinsed in the buffer and immersed in DAB to observe color development. For the DAB-naked eye evaluation, three observers (S.Y., H.R., K.T.) assessed the immunostaining results in a blinded manner without knowledge of the clinical or histopathological diagnoses. Intensity was graded on a 3-tier scale (1+, negative to weak; 2+, moderate; 3+, strong). The percentage of staining was recorded, and a semi-quantitative (H-score) approach [14] was used for analysis. PD-L1 expression scores were calculated (from 0 to 300) by multiplying the percentage of the stained tumor area by the staining intensity score.

3-tier scale (1+, negative to weak; 2+, moderate; 3+, strong). The percentage of staining was recorded, and a semi-quantitative (H-score) approach [14] was used for analysis. PD-L1 expression scores were calculated (from 0 to 300) by multiplying the percentage of the stained tumor area by the staining intensity score. Double staining of PD-L1 using the PID method and of CD8+ lymphocytes using the DAB method Double staining, i.e., DAB staining for CD8+ lymphocytes and PD-L1 staining with the PID method, was performed to count the tumor infiltrating lymphocytes (TILs) (primary antibody for PD-L1: E1L3N, 1:100; primary antibody for CD8: C8/114B, 1:250; DAKO; secondary antibody: EnVision/HRP system) (Fig. 2).Fig. 2 Immunohistochemical double staining for programmed death ligand 1 (PD-L1) on tumor cells and CD8+ lymphocytes. PID staining was used for PD-L1 detection, and diaminobenzidine (DAB) staining was used for CD8+ lymphocytes. The number of PID particles measured by the automated PID analyzer is indicated for each tumor cell (white numbers) and lymphocyte (yellow numbers) The correlations between PD-L1 expression, overall survival, and clinico-pathological data were evaluated.

Double staining of PD-L1 using the PID method and of CD8+ lymphocytes using the DAB method Double staining, i.e., DAB staining for CD8+ lymphocytes and PD-L1 staining with the PID method, was performed to count the tumor infiltrating lymphocytes (TILs) (primary antibody for PD-L1: E1L3N, 1:100; primary antibody for CD8: C8/114B, 1:250; DAKO; secondary antibody: EnVision/HRP system) (Fig. 2).Fig. 2 Immunohistochemical double staining for programmed death ligand 1 (PD-L1) on tumor cells and CD8+ lymphocytes. PID staining was used for PD-L1 detection, and diaminobenzidine (DAB) staining was used for CD8+ lymphocytes. The number of PID particles measured by the automated PID analyzer is indicated for each tumor cell (white numbers) and lymphocyte (yellow numbers) The correlations between PD-L1 expression, overall survival, and clinico-pathological data were evaluated. Statistical analysis Statistical analysis was performed using JMP® 10 software (SAS institute Inc., Cary, NC). Student’s t test was used to analyze continuous variables, and the χ2 test was used to analyze categorical variables. Cumulative survival rates were calculated by the Kaplan–Meier method. Significant differences in survival status were evaluated using the log-rank test. The Cox proportional hazards model was used in the multivariate analysis, and values are expressed using the hazard ratio (HR) with a 95% confidence interval (CI). P < 0.05 was considered to be statistically significant.

an–Meier method. Significant differences in survival status were evaluated using the log-rank test. The Cox proportional hazards model was used in the multivariate analysis, and values are expressed using the hazard ratio (HR) with a 95% confidence interval (CI). P < 0.05 was considered to be statistically significant. Results Patient characteristics The patient background and clinico-pathological parameters are shown in Table 1. Of the 42 patients in the study, 16 were women and 26 were men, with a median age at the time of diagnosis of 65s (range 50–83) years. In terms of TMN stage at diagnosis, three, six, 33, and zero patients were diagnosed at the T1, T2, T3, and T4 stages, respectively. Lymph node metastases were detected in 26 of the patients (61.9%). Seven patients were identified as category M1 because of metastasis of the number 16 lymph node without other organ metastasis. In terms of the pathological stage (pStage), as defined in the Union for International Cancer Control classification, three, four, nine, 19, and seven cases were at pStage Ia, Ib, IIa, IIb, and IV, respectively. The median survival time (MST) of the 42 patients was 26 months (Fig. 3).Table 1 Patient characteristics and clinico-pathological data

al stage (pStage), as defined in the Union for International Cancer Control classification, three, four, nine, 19, and seven cases were at pStage Ia, Ib, IIa, IIb, and IV, respectively. The median survival time (MST) of the 42 patients was 26 months (Fig. 3).Table 1 Patient characteristics and clinico-pathological data Patient characteristics Values (n = 42 patients) Age (years) 65 (50–83) Male/female 26/16 Tumor location (Ph/Pbt) 31/11 R0/R1a 27/15 Neo-adjuvant therapy (+/−) 8/34 Adjuvant therapy (+/−) 23/19 Pre-operative tumor marker CA19-9 134.2 (1.6–8116) T stage (½/3)b 3/6/33 N stage (0/1)b 16/26 M stage (0/1)c 35/7 Tumor diameter (mm) 30 (16–75) The data in table are expressed as the median with the range in parenthesis or as the number of patients Ph Pancreatic head, Pbt pancreatic body or tail aR0 corresponds to curative resection or complete remission; R1 corresponds to microscopic residual tumor bT and N stage were based on the TNM classification of malignant tumors, sixth edition cAll M1 cases had No.16 lymph node metastasis without other organ metastasis Fig. 3 Overall survival of 42 patients with pancreatic ductal adenocarcinoma (PDAC) Of the 42 patients, 27 (64.3%) underwent pathological curative resection, eight (19.0%) received neo-adjuvant chemo-radiation therapy, and 23 (54.8%) received adjuvant chemotherapy.

cAll M1 cases had No.16 lymph node metastasis without other organ metastasis Fig. 3 Overall survival of 42 patients with pancreatic ductal adenocarcinoma (PDAC) Of the 42 patients, 27 (64.3%) underwent pathological curative resection, eight (19.0%) received neo-adjuvant chemo-radiation therapy, and 23 (54.8%) received adjuvant chemotherapy. PD-L1 expression in the 42 PDAC patients by DAB and PID staining To judge the rate of positive PID staining, we first established the threshold of the PID staining value. The average of the highest value of the negative control of PID staining was 3.01; therefore, the threshold value for judging positive PID staining was set to 3.0. Using this threshold, we detected PD-L1 expression in 26 of the 42 patients (61.9%). By contrast, PD-L1 expression measured by the DAB-naked eye evaluation was detected in only six of the 42 patients (14.3%). This expression rate was measured to be between the PID positive staining value derived from the threshold set at 4.0 (28.6%, 12/42 patients) and 5.0 (11.9%, 5/42 patients). Relationship between PD-L1 expression and clinico-pathological features The correlations between pathological features and PD-L1 expression are shown in Table 2.Table 2 Relationships between programmed death ligand 1 expression and pathological features Parametera PD-L1 expression (+) (n = 26) PD-L1 expression (−) (n = 16) P

Relationship between PD-L1 expression and clinico-pathological features The correlations between pathological features and PD-L1 expression are shown in Table 2.Table 2 Relationships between programmed death ligand 1 expression and pathological features Parametera PD-L1 expression (+) (n = 26) PD-L1 expression (−) (n = 16) P Age (years) 64 (51–82) 66 (50–78) 0.385 Male/female 20/6 6/10 0.021 Tumor location (Ph/Pbt) 19/7 12/4 0.891 R0/R1 15/11 12/4 0.256 Neo-adjuvant therapy (+/−) 5/21 3/13 0.969 Adjuvant therapy (+/−) 15/11 8/8 0.627 Pre-operative tumor marker CA19-9 143.0 (1.6–8116) 113.0 (20.3–1712) 0.421 T stage (1, 2/3) 5/21 4/12 0.711 N stage (0/1) 10/16 6/10 0.950 M stage (0/1) 21/5 14/2 0.570 Tumor diameter (mm) 29.5 (18–75) 32.5 (16–45) 0.583 The data in table are expressed as the median with the range in parenthesis or as the number of patients PD-L1 Programmed death ligand 1 aSee footnotes to Table 1 for explanations of staging The ratio of males was significantly higher in the PD-L1-positive group (male/female 20/6) than in the PD-L1-negative group (male/female: 6/10 (P = 0.021). There was no significant correlation between PD-L1 expression and tumor size, lymph node metastasis [including distant lymph node metastasis (M1)], pre-operative tumor marker CA19-9 level, and R0/R1 (microscopic residual tumor) status. CD8+ TIL counts were not significantly correlated to PD-L1 expression.

(P = 0.021). There was no significant correlation between PD-L1 expression and tumor size, lymph node metastasis [including distant lymph node metastasis (M1)], pre-operative tumor marker CA19-9 level, and R0/R1 (microscopic residual tumor) status. CD8+ TIL counts were not significantly correlated to PD-L1 expression. Survival analysis Survival curves obtained using the Kaplan–Meier method are shown in Fig. 4. Among the 42 patients, there was a significant difference in the overall survival rate between patients with PD-L1-positive disease and those with PD-L1-negative disease based on PID staining (Fig. 4a. HR 2.07, 95% CI 1.00–4.54; P = 0.049). The MST was 23.5 months in the PD-L1-positive group and 51.6 months in the PDL-1-negative group. Among the 29 patients who had positive CD8+ TILs (>3 cells per 400× field), those in the PD-L1-positive group showed a significantly poorer overall survival rate than those in the PD-L1-negative group based on detection with the PID method (Fig. 4b. HR 3.84, 95% CI 1.59–10.35; P = 0.003).Fig. 4 a Overall survival rates of 42 patients with PDAC correlated to PD-L1 expression. Overall survival in patients with PD-L1-positive disease was significantly poorer than that in patients with PD-L1-negative disease (P = 0.049). b Overall survival rate of 29 patients showing positive CD8+ tumor-infiltrating lymphocytes (TILs) correlated to PD-L1 expression; there was a significant difference in prognosis between patients with PD-L1-positive and PD-L1-negative disease (P = 0.003). HR Hazard ratio, CI confidence interval

ive disease (P = 0.049). b Overall survival rate of 29 patients showing positive CD8+ tumor-infiltrating lymphocytes (TILs) correlated to PD-L1 expression; there was a significant difference in prognosis between patients with PD-L1-positive and PD-L1-negative disease (P = 0.003). HR Hazard ratio, CI confidence interval The results of the univariate and multivariate analyses in the 42 patients are shown in Table 3. Univariate and multivariate analyses revealed that PD-L1 expression determined by the PID method (PID staining value >3.0) was an independent prognostic factor (HR 2.34, 95% CI 1.02–5.74; P = 0.045); specifically, in the 29 TIL-positive patients, PD-L1 expression determined by the PID method was an independent predictive poor prognostic factor (Table 4). Moreover, there was a stronger prognostic effect of PD-L1 expression among these 29 TIL-positive cases compared to the analysis including all 42 patients (HR 4.39, 95% CI 1.64–13.34; P = 0.003).Table 3 Results of the univariate and multivariate analyses for overall survival in all patients (n = 42) Variablesa Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P

The results of the univariate and multivariate analyses in the 42 patients are shown in Table 3. Univariate and multivariate analyses revealed that PD-L1 expression determined by the PID method (PID staining value >3.0) was an independent prognostic factor (HR 2.34, 95% CI 1.02–5.74; P = 0.045); specifically, in the 29 TIL-positive patients, PD-L1 expression determined by the PID method was an independent predictive poor prognostic factor (Table 4). Moreover, there was a stronger prognostic effect of PD-L1 expression among these 29 TIL-positive cases compared to the analysis including all 42 patients (HR 4.39, 95% CI 1.64–13.34; P = 0.003).Table 3 Results of the univariate and multivariate analyses for overall survival in all patients (n = 42) Variablesa Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P R0/1 1.81 (0.87–3.64) 0.108 1.16 (0.51–2.59) 0.710 Neo-adjuvant therapy (+/−) 1.75 (0.74–5.18) 0.220 Adjuvant chemotherapy (+/−) 1.28 (0.64–2.61) 0.480 Tumor marker CA19-9 > 138 1.57 (0.76–3.22) 0.216 T stage (½, 3) 1.28 (0.58–3.21) 0.555 N stage (0/1) 2.06 (1.01–4.44) 0.046 2.21 (0.91–5.62) 0.081 M stage (0/1) 2.25 (0.88–5.08) 0.087 1.09 (0.38–2.86) 0.863 ly (0–1/2–3) 1.23 (0.60–2.65) 0.573 v (0–1/2–3) 1.15 (0.57–2.50) 0.697 PD-L1 expression (+) (PID) 2.07 (1.00–4.54) 0.049 2.34 (1.02–5.74) 0.045 PD-L1 expression (+) (DAB) 1.28 (0.50–4.33) 0.633 CD8+ TILs >3.0 1.31 (0.62–3.15) 0.490 HR Hazard ratio, CI confidence interval, PID phosphor-integrated dots, DAB diaminobenzidine, TILs +tumor infiltrating lymphocytes, ly lymphatic invasion, v blood vessel invasion

R0/1 1.81 (0.87–3.64) 0.108 1.16 (0.51–2.59) 0.710 Neo-adjuvant therapy (+/−) 1.75 (0.74–5.18) 0.220 Adjuvant chemotherapy (+/−) 1.28 (0.64–2.61) 0.480 Tumor marker CA19-9 > 138 1.57 (0.76–3.22) 0.216 T stage (½, 3) 1.28 (0.58–3.21) 0.555 N stage (0/1) 2.06 (1.01–4.44) 0.046 2.21 (0.91–5.62) 0.081 M stage (0/1) 2.25 (0.88–5.08) 0.087 1.09 (0.38–2.86) 0.863 ly (0–1/2–3) 1.23 (0.60–2.65) 0.573 v (0–1/2–3) 1.15 (0.57–2.50) 0.697 PD-L1 expression (+) (PID) 2.07 (1.00–4.54) 0.049 2.34 (1.02–5.74) 0.045 PD-L1 expression (+) (DAB) 1.28 (0.50–4.33) 0.633 CD8+ TILs >3.0 1.31 (0.62–3.15) 0.490 HR Hazard ratio, CI confidence interval, PID phosphor-integrated dots, DAB diaminobenzidine, TILs +tumor infiltrating lymphocytes, ly lymphatic invasion, v blood vessel invasion aSee footnotes to Table 1 for explanations of staging Table 4 The results of the univariate and multivariate analyses for overall survival in patients with positive CD8+ tumor infiltrating lymphocytes (n = 29) Variablesa Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P R0/1 1.90 (0.82–4.24) 0.131 1.55 (0.63–3.77) 0.334 Neo-adjuvant therapy (+/−) 1.42 (0.53–4.88) 0.512 Adjuvant chemotherapy (+/−) 1.00 (0.45–2.21) 0.999 Tumor marker CA19-9 > 138 1.14 (050–2.60) 0.751 T stage (1–2/3) 1.08 (0.39–2.60) 0.864 N stage (0/1) 1.64 (0.73–3.94) 0.233 1.80 (0.66–5.12) 0.250 M stage (0/1) 3.09 (0.97–8.58) 0.056 1.63 (0.47–5.05) 0.420 ly (0–1/2–3) 1.18 (0.48–2.69) 0.710 v (0–1/2–3) 1.05 (0.43–2.43) 0.904 PD-L1 expression (+) (PID) 3.84 (1.59–10.35) 0.003 4.39(1.64-13.34) 0.003 PD-L1 expression (+) (DAB) 1.89 (0.44–5.84) 0.349

0.864 N stage (0/1) 1.64 (0.73–3.94) 0.233 1.80 (0.66–5.12) 0.250 M stage (0/1) 3.09 (0.97–8.58) 0.056 1.63 (0.47–5.05) 0.420 ly (0–1/2–3) 1.18 (0.48–2.69) 0.710 v (0–1/2–3) 1.05 (0.43–2.43) 0.904 PD-L1 expression (+) (PID) 3.84 (1.59–10.35) 0.003 4.39(1.64-13.34) 0.003 PD-L1 expression (+) (DAB) 1.89 (0.44–5.84) 0.349 ly lymphatic invasion, v blood vessel invasion aSee footnotes to Table 1 for explanations of staging Discussion We report here the results of our investigation on PD-L1 expression in patients with PDAC. The PD-L1-positive detection rate in PID staining was higher than that in DAB staining, possibly due to the fact that digital immunostaining can detect proteins at lower concentrations. The higher fluorescence intensity of the nanoparticles contributes to the higher sensitivity of PID compared to DAB. In addition, quantitative analysis was possible even in cells with strong positive expression, without saturation.

due to the fact that digital immunostaining can detect proteins at lower concentrations. The higher fluorescence intensity of the nanoparticles contributes to the higher sensitivity of PID compared to DAB. In addition, quantitative analysis was possible even in cells with strong positive expression, without saturation. In this study, the PD-L1 positive expression rate detected using the PID method in PDAC cases was higher than that reported in previous studies (4–49%) [15–17] using the DAB method. Among the cases in our study, no case was found to be negative for PID and positive for DAB. The PD-L1 positive expression rate was 14.3% using the DAB method and 61.9% using the PID method when the PID staining threshold value was set to 3.0. This result supported the current PD-L1 protein expression data using PID, which was found to be more sensitive than DAB. However, the DAB positive staining rate was similar to the PID positive staining rate when the threshold was set to 4.0 or 5.0; in other words, cells with slightly positive expression showing three to five PID particles could not be detected by conventional methods.

g PID, which was found to be more sensitive than DAB. However, the DAB positive staining rate was similar to the PID positive staining rate when the threshold was set to 4.0 or 5.0; in other words, cells with slightly positive expression showing three to five PID particles could not be detected by conventional methods. Immunotherapeutic approaches, most notably immune checkpoint inhibitors epitomized by antibodies directed against T-lymphocyte regulators, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and PD-1, have demonstrated efficacy in a variety of solid tumors, including metastatic melanoma and lung cancer, and have already received U.S. Food and Drug Administration approval. PD-1/PD-Ll pathway blockade has resulted in significant and durable clinical responses in patients with several malignancies, such as melanoma, renal cell carcinoma, lung cancer, mismatch repair-deficient colorectal cancer, and bladder cancer [18]. However, PDAC has generally been considered to be a non-immunogenic malignancy, given that tumor-infiltrating effector T lymphocytes do not represent a histopathological hallmark of this disease [19, 20]. Investigators have been actively exploring the mechanisms underlying the evasion of immune surveillance by pancreatic cancer cells, and several potential strategies have been proposed to overcome resistance to immune checkpoint inhibitors. In this study, TILs reached tumor cells in 29 of 42 surgical PDAC specimens.

0]. Investigators have been actively exploring the mechanisms underlying the evasion of immune surveillance by pancreatic cancer cells, and several potential strategies have been proposed to overcome resistance to immune checkpoint inhibitors. In this study, TILs reached tumor cells in 29 of 42 surgical PDAC specimens. In a clinical setting, patients with positive PD-L1 expression tend to show significantly unfavorable outcomes. Previous studies using DAB methods have also demonstrated that patients with PD-L1-positive PDAC showed unfavorable outcomes. Wang et al. reported a correlation between B7-H1 (PD-L1) expression and pathological grade and TNM stage [16]. Nomi et al. reported that PD-L1-positive PDAC patients had a significantly poorer prognosis than PD-L1-negative patients [17]. Their data were similar to our results in that they found was no significant correlation between tumor PD-L1 status and clinical indicators, including tumor status, nodal status, metastatic status, and pathological stage. In the current study, among the TIL-positive patients there was PD-L1 expression which showed a stronger prognostic correlation than that observed in the analysis including all patients. Univariate and multivariate analyses showed that positive PD-L1 expression (PID staining value >3.0) was indeed an independent poor prognostic factor in PDAC patients with positive CD8+ TILs. This result suggests that an immunosuppressive tumor microenvironment with high PD-L1 expression can interfere with the attack by TILs on the tumor cells. In these cases, there is a possibility that blockage of the PD-1/PD-L1 signaling pathway can result in the attack of TILs being dramatically more effective. Although there have been no objective responses observed in patients with PDAC who received anti-PD-L1 antibody therapy [21], our results suggest that PD-L1-positive patients with TILs may be good candidates for anti-PD-L1 antibody therapy.

ing pathway can result in the attack of TILs being dramatically more effective. Although there have been no objective responses observed in patients with PDAC who received anti-PD-L1 antibody therapy [21], our results suggest that PD-L1-positive patients with TILs may be good candidates for anti-PD-L1 antibody therapy. Conclusions Phosphor-integrated dot staining provided superior results compared to those obtained by the canonical DAB staining method. We have shown for the first time that PD-L1, detected using PID staining, is a novel prognostic marker for human PDAC. Digital immunostaining is a promising tool for companion diagnostics to evaluate the therapeutic effects of molecular-targeted drugs and immunotherapy. Acknowledgements The authors would like to thank Aoi Nozawa (Konica Minolta, Inc.) for helpful suggestions. We would also like to thank Editage (www.editage.jp) for English language editing. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest to declare.

Introduction Phase I clinical trials of oncologic drugs are conducted to determine the proper dosage of both single-agent and combination therapies. If the therapeutic index is very narrow, it is critical to evaluate the study drug toxicity in dose-escalation studies. Dose-limiting toxicity (DLT) is evaluated during the first cycle of each new dosage level for every trial participant to determine the maximum tolerated dose (MTD). Close monitoring for symptoms as well as physiological and laboratory data are required to determine if DLT is observed. Clinical trials in Japan and Western countries are carried out under the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines [1]. These guidelines do not require hospitalization as part of phase I clinical trials. However, according to Japanese guidelines, participants in phase I oncology clinical trials are required to be hospitalized during the first cycle of treatment to observe toxicity closely for safety reasons [2].

se (ICH) guidelines [1]. These guidelines do not require hospitalization as part of phase I clinical trials. However, according to Japanese guidelines, participants in phase I oncology clinical trials are required to be hospitalized during the first cycle of treatment to observe toxicity closely for safety reasons [2]. Because the assessment period for DLT is approximately 1 month in most phase I oncology clinical trials, in Japan study participants are required to be hospitalized for 1 month. Phase I clinical oncology trial participants are generally refractory to standard treatments and usually have very limited options. Even though these patients have a good performance status and generally good health, their life expectancy is limited, and consequently, the phase I hospitalization requirement is sometimes in conflict with ongoing end-of-life care. Additionally, the patient is usually charged with the cost of hospitalization, making a patient’s participation in the phase I trial burdensome with respect to time and cost. For all these reasons, it is very difficult to recruit patients to participate in phase I clinical trials. Furthermore, Japan’s hospitalization practice makes it an outlier from standard ICH guidelines, limiting the globalization of Japanese oncology drug development.

e I trial burdensome with respect to time and cost. For all these reasons, it is very difficult to recruit patients to participate in phase I clinical trials. Furthermore, Japan’s hospitalization practice makes it an outlier from standard ICH guidelines, limiting the globalization of Japanese oncology drug development. Toxicity assessment is an essential objective of a phase I trial. Hyman et al. have developed a nomogram-based model to predict the risk of a patient developing a cycle-one serious drug-related toxicity (SDRT), but its practical use remains very limited [3]. Additionally, the type of investigational drugs upon which this model was largely derived has changed from cytotoxic drugs to molecularly targeted drugs and immune checkpoint inhibitors during the past several decades. Consequently, as the mechanism of action of new cancer drugs has changed, the toxicity profiles and timing of toxicity events have also changed. For example, Postel-Vinay et al. reported that in phase I oncology trials of molecularly targeted drugs, treatment interruption or discontinuation occurs more frequently in the second cycle rather than the first cycle [4].

cancer drugs has changed, the toxicity profiles and timing of toxicity events have also changed. For example, Postel-Vinay et al. reported that in phase I oncology trials of molecularly targeted drugs, treatment interruption or discontinuation occurs more frequently in the second cycle rather than the first cycle [4]. If the frequency of severe toxicity during first cycle in phase I trials is low, it may be more practical to conduct trials in an outpatient setting or with only minimal hospitalization. Furthermore, minimizing or avoiding hospitalization altogether may facilitate recruitment of participants and accelerate clinical drug development. However, to date, there has been no evaluation of the incidence and risk of severe toxicity in hospitalized patients participating in phase I clinical oncology trials in Japan. In this study, we surveyed severe toxicity observed in the phase I single-agent clinical trials conducted in our institution and investigated the frequency of toxicity that did or did not require hospitalization.

If the frequency of severe toxicity during first cycle in phase I trials is low, it may be more practical to conduct trials in an outpatient setting or with only minimal hospitalization. Furthermore, minimizing or avoiding hospitalization altogether may facilitate recruitment of participants and accelerate clinical drug development. However, to date, there has been no evaluation of the incidence and risk of severe toxicity in hospitalized patients participating in phase I clinical oncology trials in Japan. In this study, we surveyed severe toxicity observed in the phase I single-agent clinical trials conducted in our institution and investigated the frequency of toxicity that did or did not require hospitalization. Patients and methods Patients Patients who participated in single-agent phase I clinical trials at National Cancer Center Hospital between December 1996 and August 2014 were included in this study. Drug type, treatment course, and toxicity were retrieved from medical records and databases. Severity of toxicity was assessed by Common Terminology Criteria for Adverse Events v. 4.0 [5]. Severe toxicity was classified as requiring hospitalization. Such toxicity requiring hospitalization is defined as follows: toxicity that requires intensive treatment (e.g., continuous oxygen administration, invasive procedure) and/or toxicity that requires intravenous intervention (e.g., fluid therapy, intravenous antibiotics, blood transfusion). Study designs were classified into three types: (1) dose-escalation (conventional dose-escalation study to determine MTD in Japanese patients; FIH study was excluded); (2) first-in-human (FIH); and (3) dose-finding (to assess drug safety and pharmacokinetic profiles up to the MTD previously determined in Western studies).

dy designs were classified into three types: (1) dose-escalation (conventional dose-escalation study to determine MTD in Japanese patients; FIH study was excluded); (2) first-in-human (FIH); and (3) dose-finding (to assess drug safety and pharmacokinetic profiles up to the MTD previously determined in Western studies). Ethical considerations The present study involving human subjects was approved by the National Cancer Center Institutional Review Board (2014-148). A copy of the letter from the Institutional Review Board is available for review by the Editor of this journal. Results A total of 945 patients participated in the phase I trial from December 1996 to August 2014 at the National Cancer Center Hospital (Tokyo, Japan). Median patient age was 58 years (range, 18–76 years); 537 patients (57%) were men and 408 (43%) were women. Patients were assigned to receive cytotoxic drugs (n = 207, 22%), molecularly targeted drugs (n = 690, 73%), or immune checkpoint inhibitors (n = 48, 5%). Patients participated in one of three study types: dose-escalation (n = 582, 61%), first-in-human (n = 129, 14%), or dose-finding (n = 234, 25%). Tumor types were non-small cell lung cancer (n = 248, 28%), colorectal cancer (n = 175, 19%), sarcoma (n = 115, 12%), esophageal cancer (n = 49, 5%), pancreatic cancer (n = 45, 5%), bile duct cancer (n = 36, 4%), breast cancer (n = 35, 4%), and gastric cancer (n = 26, 3%) (Table 1).Table 1 Patient characteristics

25%). Tumor types were non-small cell lung cancer (n = 248, 28%), colorectal cancer (n = 175, 19%), sarcoma (n = 115, 12%), esophageal cancer (n = 49, 5%), pancreatic cancer (n = 45, 5%), bile duct cancer (n = 36, 4%), breast cancer (n = 35, 4%), and gastric cancer (n = 26, 3%) (Table 1).Table 1 Patient characteristics Patients, n 945 Age, years 58 (median), 18–76 (range) Sex, n (%) Male 537 (57) Female 408 (43) Drug type, n (%) Cytotoxic 207 (22) Molecularly targeted 690 (73) Checkpoint inhibitor 48 (5) Study type, n (%) Dose escalation 582 (61) First in human (FIH) 129 (14) Dose finding 234 (25) Disease, n (%) NSCLC 248 (26) Colorectal 175 (19) Sarcoma 115 (12) Esophagus 49 (5) Pancreas 45 (5) Biliary 36 (4) Breast 35 (4) Gastric 26 (3) Melanoma 23 (2) Ovarian 23 (2) SCLC 20 (2) HN 18 (2) Prostate 18 (2) Uterine 17 (2) CUP 10 (1) Other 87 (9) NSCLC non-small cell lung cancer, SCLC small cell lung cancer, HN head and neck, CUP carcinoma of unknown primary A total of 76 study drugs were evaluated as part of this pool of phase I studies. Subdivided by mechanism of action, 20 (26%) were cytotoxic, 50 (66%) were molecularly targeted, and 6 (8%) were immune checkpoint inhibitor (Table 2).Table 2 Characteristics of study drugs and clinical studies Study drug, n 76 Study drug, n (%) Cytotoxic 20 (26) Molecularly targeted 50 (66) Checkpoint inhibitor 6 (8) Study type, n (%) Dose escalation 44 (57) First in human 8 (11) Dose findings 24 (32)

A total of 76 study drugs were evaluated as part of this pool of phase I studies. Subdivided by mechanism of action, 20 (26%) were cytotoxic, 50 (66%) were molecularly targeted, and 6 (8%) were immune checkpoint inhibitor (Table 2).Table 2 Characteristics of study drugs and clinical studies Study drug, n 76 Study drug, n (%) Cytotoxic 20 (26) Molecularly targeted 50 (66) Checkpoint inhibitor 6 (8) Study type, n (%) Dose escalation 44 (57) First in human 8 (11) Dose findings 24 (32) Ninety-eight patients (10.4%) developed severe toxicities during the first cycle; a total of 126 patients (13.3%) developed severe toxicities during any cycle. Thirty-six patients (3.8%) had severe toxicities requiring hospitalization during the first cycle (Fig. 1). The overall number of toxicities requiring hospitalization and/or grade 4 toxicities during any cycle was 5.0%. In the first cycle, 31 patients (15.0%) who received cytotoxic drugs and 67 patients (9.7%) who received molecularly targeted drugs developed severe toxicity. However, patients who received immune checkpoint inhibitors did not develop any severe toxicity. Fourteen patients (6.8%) receiving cytotoxic drugs and 22 patients (3.2%) receiving molecularly targeted drugs needed to be hospitalized because of toxicity. For patients receiving cytotoxic agents, 4 hematological (1.9%) and 10 nonhematological (4.8%) toxicity events required hospitalization (Table 3). For patients receiving molecularly targeted drugs, 3 hematological (0.4%) and 19 nonhematological (2.8%) toxicity events requiring hospitalization (Table 3).Table 3 Toxicity by drug type

receiving cytotoxic agents, 4 hematological (1.9%) and 10 nonhematological (4.8%) toxicity events required hospitalization (Table 3). For patients receiving molecularly targeted drugs, 3 hematological (0.4%) and 19 nonhematological (2.8%) toxicity events requiring hospitalization (Table 3).Table 3 Toxicity by drug type Drug type Cytotoxic, n = 207 Molecularly targeted, n = 690 Checkpoint inhibitor, n = 48 Number of severe toxicities in cycle 1, n (%) 31 (15) 67 (9.7) 0 (0) Hospitalization required, n (%) Hematological 14 (6.8) 4 (1.9) 22 (3.2) 3 (0.4) 0 (0) Nonhematological 10 (4.8) 19 (2.8) No hospitalization required, n (%) Hematological 17 (8.2) 8 (3.9) 45 (6.5) 6 (0.9) 0 (0) Nonhematological 9 (4.3) 39 (5.7) Fig. 1 Patient outcomes Seventy-three patients (12.5%) in dose-escalation studies, 14 patients (10.9%) in FIH studies, and 11 patients (4.7%) in dose-finding studies developed severe toxicity, including DLT. Twenty-seven (4.6%) dose-escalation study participants, 4 (3.1%) FIH study participants, and 5 (2.1%) dose-finding study participants needed hospitalization for toxicity (Table 4).Table 4 Toxicity by study type Dose escalation, n = 582 FIH, n = 129 Dose finding, n = 234 Number of severe toxicities in cycle 1, n (%) 73 (12.5) 14 (10.9) 11 (4.7) Required hospitalization, n (%) 27 (4.6) 4 (3.1) 5 (2.1) Required hospitalization and/or G4, n (%) 34 (5.8) 7 (5.4) 6 (2.6) FIH first-in-human

Seventy-three patients (12.5%) in dose-escalation studies, 14 patients (10.9%) in FIH studies, and 11 patients (4.7%) in dose-finding studies developed severe toxicity, including DLT. Twenty-seven (4.6%) dose-escalation study participants, 4 (3.1%) FIH study participants, and 5 (2.1%) dose-finding study participants needed hospitalization for toxicity (Table 4).Table 4 Toxicity by study type Dose escalation, n = 582 FIH, n = 129 Dose finding, n = 234 Number of severe toxicities in cycle 1, n (%) 73 (12.5) 14 (10.9) 11 (4.7) Required hospitalization, n (%) 27 (4.6) 4 (3.1) 5 (2.1) Required hospitalization and/or G4, n (%) 34 (5.8) 7 (5.4) 6 (2.6) FIH first-in-human The observed toxicity profile included increase in aminotransferase (n = 39, 17.3%), neutropenia (n = 22, 9.7%), thrombocytopenia (n = 20, 8.8%), anorexia (n = 13, 5.8%), and proteinuria (n = 13, 5.8%). During the first cycle, toxicities requiring hospitalization included thrombocytopenia (n = 11, 19.0%), febrile neutropenia (n = 9, 15.5%), ileus/bowel obstruction (n = 5, 8.6%), arrhythmia (n = 3, 5.2%), and pneumonia (n = 3, 5.2%) (Table 5).Table 5 Adverse events (AEs) observed during trial Any AEs AEs requiring hospitalization in cycle 1

The observed toxicity profile included increase in aminotransferase (n = 39, 17.3%), neutropenia (n = 22, 9.7%), thrombocytopenia (n = 20, 8.8%), anorexia (n = 13, 5.8%), and proteinuria (n = 13, 5.8%). During the first cycle, toxicities requiring hospitalization included thrombocytopenia (n = 11, 19.0%), febrile neutropenia (n = 9, 15.5%), ileus/bowel obstruction (n = 5, 8.6%), arrhythmia (n = 3, 5.2%), and pneumonia (n = 3, 5.2%) (Table 5).Table 5 Adverse events (AEs) observed during trial Any AEs AEs requiring hospitalization in cycle 1 n % n % Aminotransferase increased 39 17.3 4 6.9 Neutropenia 22 9.7 0 0.0 Thrombocytopenia 20 8.8 11 19.0 Anorexia 13 5.8 1 1.7 Proteinuria 13 5.8 0 0.0 Nausea 10 4.4 0 0.0 Febrile neutropenia 9 4.0 9 15.5 Anemia 7 3.1 2 3.4 Malaise 7 3.1 1 1.7 CPK increased 5 2.2 0 0.0 Diarrhea 5 2.2 0 0.0 Ileus 5 2.2 5 8.6 Rash 5 2.2 1 1.7 T-Bil increased 5 2.2 0 0.0 WBC decreased 5 2.2 0 0.0 Hypertension 4 1.8 0 0.0 GGT increased 4 1.8 0 0.0 Mucositis 4 1.8 1 1.7 Vomiting 4 1.8 2 3.4 Arrhythmia 3 1.4 3 5.2 Pneumonitis 3 1.4 3 5.2 Colitis 2 0.9 2 3.4 Hypoxia 2 0.9 2 3.4 Infection 2 0.9 2 3.4 CIPN 2 0.9 0 0.0 QTc prolonged 2 0.9 0 0.0 Unacceptable by patients 2 0.9 0 0.0 AMY increased 1 0.4 0 0.0 Constipation 1 0.4 1 1.7 Creatinine increased 1 0.4 0 0.0 Dehydration 1 0.4 1 1.7 Drug fever 1 0.4 0 0.0 Dry skin 1 0.4 0 0.0 DVT 1 0.4 0 0.0 Dyspnea 1 0.4 1 1.7 ECG abnormality 1 0.4 0 0.0 Edema 1 0.4 0 0.0 Hearing disorder 1 0.4 0 0.0 Hyperglycemia 1 0.4 1 1.7 Hypoalbuminemia 1 0.4 0 0.0 Hypotension 1 0.4 1 1.7 Lipase increased 1 0.4 0 0.0 Muscle weakness, extremity lower 1 0.4 0 0.0 Perforation 1 0.4 1 1.7 Pericardial effusion 1 0.4 1 1.7 Pleural effusion 1 0.4 1 1.7 Pneumonia 1 0.4 1 1.7 Pyrexia 1 0.4 0 0.0 Uric acid increased 1 0.4 0 0.0 Total 226 100 58 100

a 1 0.4 1 1.7 Hypoalbuminemia 1 0.4 0 0.0 Hypotension 1 0.4 1 1.7 Lipase increased 1 0.4 0 0.0 Muscle weakness, extremity lower 1 0.4 0 0.0 Perforation 1 0.4 1 1.7 Pericardial effusion 1 0.4 1 1.7 Pleural effusion 1 0.4 1 1.7 Pneumonia 1 0.4 1 1.7 Pyrexia 1 0.4 0 0.0 Uric acid increased 1 0.4 0 0.0 Total 226 100 58 100 AEs adverse events, CPK creatinine phosphokinase, T-Bil total bilirubin, WBC white blood cell, GGT gamma-glutamyltransferase, CIPN chemotherapy-induced peripheral neuropathy, AMY amylase, ECG electric cardiogram, DVT deep vein thrombosis Discussion Toxicity evaluation is one of the primary objectives of phase I oncology trials because such studies help define the recommended dose for further studies. The type and timing of toxicity can differ depending on a variety of factors including different dose levels, the class and mechanism of action of the study drug, and the route of administration [6, 7]. As new cancer targets have emerged over the past several decades, there is now a much wider array of investigational drugs with new mechanisms of action. As such, classical a “3 + 3 design” may be not sufficient to evaluate toxicities in the current era [7–9].

of the study drug, and the route of administration [6, 7]. As new cancer targets have emerged over the past several decades, there is now a much wider array of investigational drugs with new mechanisms of action. As such, classical a “3 + 3 design” may be not sufficient to evaluate toxicities in the current era [7–9]. A modeling approach using toxicity data from 3104 participants in 127 phase I trials between 2000 and 2010 to estimate the risk of SDRT has been reported [3]. In this study, SDRT was defined as grade ≥4 hematological toxicity or grade ≥3 nonhematological toxicity attributed, at least possibly, to study drug(s). The parameters used in this empirical model to predict SDRT risk included performance status, white blood cell count, creatinine clearance, serum albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, number of study drug(s), class of study drug, dose level, and constitutional symptoms. Although the resulting nomogram can be useful in predicting a patient’s risk for SDRT at the time of enrollment, its usage remains limited. In our study, we assessed every toxicity observed after study enrollment regardless of study drug causality. Frequent severe toxicities were increased aminotransferase, neutropenia, and thrombocytopenia. Toxicities requiring hospitalization were hematological, such as thrombocytopenia and febrile neutropenia, a tendency similar to that previously observed [3]. Toxicity requiring hospitalization was more frequent in studies involving cytotoxic drugs compared to those using molecularly targeted drugs (15.9% vs. 9.4%, respectively) (Table 3). This result is consistent with other reported observations that molecularly targeted drugs are negatively correlated with SDRT. In this study, we did not observe severe toxicity in patients who received immune checkpoint drugs. However, there are some reports of severe toxicity associated with this class of drugs [10], and the lack of observed severe toxicity in our study may be because only a small number of patients received immune checkpoint drugs compared to the other drug classes.

oxicity in patients who received immune checkpoint drugs. However, there are some reports of severe toxicity associated with this class of drugs [10], and the lack of observed severe toxicity in our study may be because only a small number of patients received immune checkpoint drugs compared to the other drug classes. Severe toxicity was observed in 10.4% of all participants during the first cycle and in 3.2% of all participants during the second or later cycles, a result similar to a previous report. Thus, it is important to observe toxicity carefully not only during the first cycle but in later cycles as well. We observed that 3.8% of participants experienced severe toxicity requiring hospitalization, and that 5.0% of all participants experienced either grade 4 toxicity or severe toxicity requiring hospitalization. This frequency was unexpectedly low and was independent of drug class. This overall low severe toxicity rate suggests that toxicity can be safely monitored in an outpatient setting and that hospitalization during the entire clinical study period is not necessary in most studies. Nonetheless, toxicity observation is still important after the DLT evaluation period. Moreover, it is important to continue careful monitoring for toxicity throughout the course of a phase I trial of immune checkpoint inhibitors because such drugs may have delayed adverse effects [10].

is not necessary in most studies. Nonetheless, toxicity observation is still important after the DLT evaluation period. Moreover, it is important to continue careful monitoring for toxicity throughout the course of a phase I trial of immune checkpoint inhibitors because such drugs may have delayed adverse effects [10]. The frequency of toxicity by study types was similar in both the dose-escalation and FIH studies (12.4% and 10.4%, respectively) (Table 4), possibly because FIH studies start at much lower dosage levels (e.g., 0.1 LD10 of mice) compared to the doses used in dose-escalation studies. Severe toxicity in dose-finding studies was low (4.7%), likely because the dose level in these studied may not reach the MTD. Consequently, in phase I oncology dose-escalation and dose-finding studies, hospitalization during the first cycle should not be mandatory. In contrast, in FIH studies conducted to determine MTD, careful observation is required because the drug’s toxicity profile is not well characterized, and hospitalization is one option to ensure patient safety. In FIH studies in Japan for drugs studied in earlier Western trials, it can be useful to use the Western clinical data to design phase I trials that reduce the risk of severe toxicity because it has been observed that the both the MTD and DLT frequency tends to be very similar between Western and Japanese patients [11].

n FIH studies in Japan for drugs studied in earlier Western trials, it can be useful to use the Western clinical data to design phase I trials that reduce the risk of severe toxicity because it has been observed that the both the MTD and DLT frequency tends to be very similar between Western and Japanese patients [11]. The present study has some limitations. It is a retrospective study, and there is no analysis by design (e.g., conventional “3 + 3” design, accelerated titration design, etc.) or by dosage levels. Of particular note, there is no standardization regarding the type of toxicity requiring hospitalization in Japan compared to other countries. We employed the definition described in the Methods, although it should be noted that hematological toxicity can be managed in an outpatient setting by blood transfusion with close monitoring in some cases. An effort between clinical investigators, regulatory authority, and other key stakeholders to identify a consensus definition of “toxicity requiring hospitalization” is warranted. Conclusion Our results indicate that there is a low rate of severe toxicity requiring hospitalization in phase I oncology trials in Japan. Consequently, the conventional approach requiring participants to be hospitalized during the first cycle of the phase I trial is not necessary. Rather, the outpatient setting should be considered for phase I clinical trials in Japan. Compliance with ethical standards Conflict of interest All authors have no conflict of interests to declare.

Introduction Adenocarcinoma of the small bowel (SBA) is a rare form of gastrointestinal cancer. SBA accounts for approximately one-third of all small intestinal malignancies, with the other major tumor types being neuroendocrine carcinomas, sarcomas, and lymphomas [1, 2]. The age-standardized incidence rates of SBA were reported to be 8.1 per million men and 5.5 per million women in the USA in 1995–2008 [3]. In Sweden, the age-standardized incidence of all malignant small bowel tumors (including adenocarcinomas, carcinoids, sarcomas, and lymphomas) increased from 14.2−19.7 per million people. In particular, the incidence of duodenal adenocarcinoma increased dramatically from 0.7−4.2 per million people during the period 1960–2009 [4]. As symptoms of SBA are usually nonspecific, diagnosis is difficult. Most affected patients present with advanced-stage disease and either lymph node involvement or distant metastatic disease [5]. Surgical management, with regional lymph node dissection is the only therapeutic modality with curative potential for localized SBA. For unresectable or recurrent tumors, chemotherapy regimens are generally used, as indicated for other gastrointestinal malignancies. Although several retrospective studies suggested that chemotherapy prolonged the survival of patients with unresectable or recurrent SBA [6–10], there are still no standard treatment protocols, and no randomized controlled trials have been carried out for these types of tumor [6–17].

ther gastrointestinal malignancies. Although several retrospective studies suggested that chemotherapy prolonged the survival of patients with unresectable or recurrent SBA [6–10], there are still no standard treatment protocols, and no randomized controlled trials have been carried out for these types of tumor [6–17]. Fluorouracil (5-FU) is the most commonly used agent for the treatment of unresectable or recurrent SBA, and various 5-FU-based regimens have been used [8–20]. In a retrospective analysis, Overman et al. reported that combination therapy with 5-FU and platinum compounds showed better results than other regimens [18]. Among the various types of combinations of 5-FU and platinum, oxaliplatin-containing regimens showed better efficacy in several studies. To date, two multicenter retrospective studies have been conducted. Zaanan et al. and Tsushima et al. reported median progression-free survival (PFS) times of 6.9 and 9.3 months, respectively, and median overall survival (OS) times of 17.8 and 22.2 months, respectively, with leucovorin + 5-FU + oxaliplatin (FOLFOX) therapy [19, 20]. In addition, two prospective studies have been reported. Overman et al. and Xiang et al. reported on CAPOX (capecitabine + oxaliplatin) therapy and FOLFOX4 therapy and found median times to treatment failure of 11.3 and 7.8 months, respectively, and median OS times of 20.4 and 15.2 months, respectively [21, 22]. Although combination therapy with 5-FU and oxaliplatin appears promising, to date the efficacy and safety of the mFOLFOX6 regimen (defined below) for SBA have not been demonstrated in any study. Therefore, we investigated the efficacy and safety of this regimen for Japanese patients with unresectable or recurrent SBA.

Although combination therapy with 5-FU and oxaliplatin appears promising, to date the efficacy and safety of the mFOLFOX6 regimen (defined below) for SBA have not been demonstrated in any study. Therefore, we investigated the efficacy and safety of this regimen for Japanese patients with unresectable or recurrent SBA. Patients and methods Patients All eligible patients were required to have histologically confirmed unresectable or recurrent SBA, excluding any ampullary carcinomas. Inclusion criteria were age 20–80 years; Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–2; and adequate hematologic parameters (white blood cell count ≥3000 and ≤12,000/mm3, neutrophil count ≥1500 cells/mm3, platelet count ≥100,000 cells/mm3, and hemoglobin ≥8 g/dL), normal hepatic function (total bilirubin ≤2.0 g/dL, and transaminases ≤100 IU/L), and normal renal function (creatinine ≤1.5 mg/dL). Prior chemotherapy or radiotherapy was not allowed, but prior use of adjuvant chemotherapy at least 6 months before evidence of recurrence was permitted. Patients with peripheral neuropathy of grade ≥1, brain metastases, concurrent therapeutic warfarin use, uncontrolled concurrent serious medical illnesses, pregnant or breast-feeding women, or patients with gastrointestinal malabsorption were not eligible to participate in the study.

Patients and methods Patients All eligible patients were required to have histologically confirmed unresectable or recurrent SBA, excluding any ampullary carcinomas. Inclusion criteria were age 20–80 years; Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–2; and adequate hematologic parameters (white blood cell count ≥3000 and ≤12,000/mm3, neutrophil count ≥1500 cells/mm3, platelet count ≥100,000 cells/mm3, and hemoglobin ≥8 g/dL), normal hepatic function (total bilirubin ≤2.0 g/dL, and transaminases ≤100 IU/L), and normal renal function (creatinine ≤1.5 mg/dL). Prior chemotherapy or radiotherapy was not allowed, but prior use of adjuvant chemotherapy at least 6 months before evidence of recurrence was permitted. Patients with peripheral neuropathy of grade ≥1, brain metastases, concurrent therapeutic warfarin use, uncontrolled concurrent serious medical illnesses, pregnant or breast-feeding women, or patients with gastrointestinal malabsorption were not eligible to participate in the study. Written informed consent was obtained from all patients, and the institutional review board of each participating hospital approved the study. This study was registered in the University Hospital Medical Network Clinical Trials Registry in Japan (UMIN000002797; http://www.umin.ac.jp/ctr/).

Patients with peripheral neuropathy of grade ≥1, brain metastases, concurrent therapeutic warfarin use, uncontrolled concurrent serious medical illnesses, pregnant or breast-feeding women, or patients with gastrointestinal malabsorption were not eligible to participate in the study. Written informed consent was obtained from all patients, and the institutional review board of each participating hospital approved the study. This study was registered in the University Hospital Medical Network Clinical Trials Registry in Japan (UMIN000002797; http://www.umin.ac.jp/ctr/). Study design This was an open-label, single-arm, multicenter, phase II study conducted at 24 academic centers in Japan. Treatment consisted of intravenous oxaliplatin (85 mg/m2) and l-leucovorin (l-LV; 200 mg/m2) administered intravenously over a 2-h period on day 1, followed by a bolus of 5-FU (400 mg/m2) and a 46-h infusion of 5-FU (2400 mg/m2), defined as the mFOLFOX6 regimen. Treatment cycles were repeated every 14 days. Staging procedures were conducted every 8 weeks. Patients were removed from the study if they withdrew their consent, if they experienced unacceptable toxicity, if they had a treatment delay of >2 weeks because of toxicity from the treatment, or if the investigator deemed that withdrawal was in the patient’s best interest.

ing procedures were conducted every 8 weeks. Patients were removed from the study if they withdrew their consent, if they experienced unacceptable toxicity, if they had a treatment delay of >2 weeks because of toxicity from the treatment, or if the investigator deemed that withdrawal was in the patient’s best interest. Dose reductions All toxicities were graded according to the National Cancer Institute Common Toxicity Criteria, version 3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm), except for neurotoxicity. Initiation of a cycle of mFOLFOX6 required grade ≤1 granulocytopenia, grade ≤1 thrombocytopenia, and recovery from any treatment-related nonhematologic toxicity (excluding alopecia and neurosensory toxicity) to baseline or to grade ≤1.

.gov/protocolDevelopment/electronic_applications/ctc.htm), except for neurotoxicity. Initiation of a cycle of mFOLFOX6 required grade ≤1 granulocytopenia, grade ≤1 thrombocytopenia, and recovery from any treatment-related nonhematologic toxicity (excluding alopecia and neurosensory toxicity) to baseline or to grade ≤1. Treatments with 5-FU and oxaliplatin were interrupted during a cycle if there was grade 3 or 4 hematologic toxicity (excluding anemia), or grade ≥2 nonhematologic toxicity (excluding nausea, vomiting, fatigue, or anorexia). The 5-FU dosage was reduced by 17% for grade 2 hand–foot syndrome, by 50% for grade 3 hand–foot syndrome, by 25% for grade 3 nonhematologic toxicity, by 50% for grade 4 nonhematologic toxicity, or by 25% for a delay in hematologic recovery of >1 week (excluding anemia). The 5-FU and oxaliplatin dosages were reduced, respectively, by 20 and 25% for grade 3 or 4 hematologic toxicity (excluding anemia), grade 3 or 4 nonhematologic toxicity (excluding nausea, vomiting, fatigue, or anorexia), or a delay in hematologic recovery of >1 week (excluding anemia). The oxaliplatin dosage was reduced by 25% for paresthesia with pain or functional impairment >7 days, and was discontinued if paresthesia with pain or functional impairment persisted throughout any treatment cycle.

ausea, vomiting, fatigue, or anorexia), or a delay in hematologic recovery of >1 week (excluding anemia). The oxaliplatin dosage was reduced by 25% for paresthesia with pain or functional impairment >7 days, and was discontinued if paresthesia with pain or functional impairment persisted throughout any treatment cycle. Statistical analysis All analysis followed the intent-to-treat principle. The primary endpoint was 1-year PFS as assessed by the treating investigator. Responses were determined according to Response Evaluation Criteria In Solid Tumors (RECIST) (version 1.1) (http://www.irrecist.com). Secondary endpoints included overall response rate (ORR), OS, PFS, and safety. PFS and OS were defined as the time from the date of registration to the date of disease progression or death, respectively. PFS and OS were analyzed using the Kaplan–Meier method. The log-rank test was used to compare survival rates between groups. Multivariate analyses were performed using the Cox proportional hazards test. Toxicity data were analyzed in all patients who received at least one dose of study medication. Statistical analysis was performed by a statistician (KY) at an independent academic research organization. All statistical analysis was performed using SAS Release 9.13 (SAS Institute Inc., Cary, NC, USA). Initially, a total of 31 patients was determined to reject the 1-year PFS of 25% under the expectation of 45% with a power of 0.80 and a one-sided alpha of 0.10. However, on 23 October 2012, because of the slow accrual of patients, the sample size was amended to 24 patients for final analysis to provide a one-sided 90% confidence interval (CI) for the 1-year PFS rate, which would exclude a threshold value of 25% if the observed 1-year PFS was ~45%. Following the protocol amendment to sample size, the primary analysis was an evaluation of 1-year PFS using the Kaplan−Meier method relative to the pre-specified threshold values of 25%.

% confidence interval (CI) for the 1-year PFS rate, which would exclude a threshold value of 25% if the observed 1-year PFS was ~45%. Following the protocol amendment to sample size, the primary analysis was an evaluation of 1-year PFS using the Kaplan−Meier method relative to the pre-specified threshold values of 25%. Immunohistochemical analysis Immunohistochemical (IHC) staining was performed on 5-μm-thick unstained sections from tissue microarray blocks using antibodies to cytokeratin 7 (CK7) (clone OV-TL12/30; Dako, Carpinteria, CA, USA; 1:300 dilution), to cytokeratin 20 (CK20) (clone Ks20.8; Dako; 1:200 dilution), to homeobox protein CDX2 (clone CDX2-88; Abcam, Cambridge, MA, USA; 1:100 dilution), and to epidermal growth factor receptor(EGFR) (clone 3C6; Roche Diagnostics, Mannheim, Germany; 1:100 dilution). IHC staining of human epidermal growth factor receptor 2 (HER2) was performed using the Ventana Ultra View DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA) and the Ventana PATHWAY HER2/neu rabbit monoclonal antibody (4B5) on a Ventana BenchMark XT immunostainer (Ventana Medical Systems). All slides from each tumor were evaluated by a single pathologist independently based on the following criteria. Expression of CK7, CK20, and CDX2 was considered positive if 10% of the tumor cells showed immunoreactivity. For EGFR, both the percentage of positive tumor cells and the intensity of positive staining were graded according to a previous report [23]. Total grades were generated on a scale of 0–6 and considered positive if the score was 2–6. The staining for HER2 was graded according to the guidelines for such testing in gastric cancers [24].

he percentage of positive tumor cells and the intensity of positive staining were graded according to a previous report [23]. Total grades were generated on a scale of 0–6 and considered positive if the score was 2–6. The staining for HER2 was graded according to the guidelines for such testing in gastric cancers [24]. Results Baseline characteristics Between April 2010 and November 2012, 24 patients with advanced SBA were enrolled from 12 institutions in Japan. The baseline characteristics of the intention-to-treat population are listed in Table 1. Metastatic disease was present in 22 of the patients, and the ECOG performance status was 0 or 1 in all patients. There were 14 patients with SBA in the duodenum (58%) and 10 patients with SBA in the jejunum (42%). The liver was the most common site of metastasis in 10 patients (40%). Three patients showed recurrence after curative resection of the SBA without receiving adjuvant therapy. Seven patients including the above recurrence cases underwent primary resection, and six underwent bypass therapy to resolve stenosis of the primary tumor. Eleven patients (46%) had not undergone any prior surgery. Four patients were excluded from the analysis of response rate (RR) because of lack of target lesion of RECST ver 1.1. The median follow-up time was 14.7 months (range 3.7–40.3).Table 1 Clinical and pathological characteristics of the patients

he primary tumor. Eleven patients (46%) had not undergone any prior surgery. Four patients were excluded from the analysis of response rate (RR) because of lack of target lesion of RECST ver 1.1. The median follow-up time was 14.7 months (range 3.7–40.3).Table 1 Clinical and pathological characteristics of the patients N = 24 (%) Gender: male/female 18/6 75/25 Age: median, years (range) 63 (31–79) ECOG PS: 0/1/2 17/7/0 71/29/0 Disease status: locally advanced/metastatic 2/22 8/92 Metastatic site: liver/lung/peritoneum/distant lymph node/other 10/3/2/9/4 42/13/8/39/17 Primary tumor site: duodenum/jejunum/ileum 14/10/0 58/42/0 Histology: well to moderate/poor/muc/sig 17/4/2/1 71/17/8/4 CEA <5/5 15/9 63/38 CA19-9 <37/37 10/14 42/58 Prior surgery: primary resection/bypass/none 7/6/11 29/25/46 Prior adjuvant chemotherapy: none/yes 3/0 ECOG Eastern Cooperative Oncology Group, CA19-9 carbohydrate antigen 19-9, CEA carcinoembryonic antigen, PS performance status Chemotherapy and response The ORR was 9/20 (45%) and the disease control rate was 16/20 (80%). One patient with liver metastasis had a complete response (CR) to mFOLFOX6. This patient started study treatment after undergoing resection of the SBA (pancreaticoduodenectomy), and we determined a CR after 20 cycles of chemotherapy. This patient was currently alive without evidence of disease at 12 months after the initiation of treatment. Second-line chemotherapy was received by 12 patients, while six patients received additional mFOLFOX6 therapy.

ection of the SBA (pancreaticoduodenectomy), and we determined a CR after 20 cycles of chemotherapy. This patient was currently alive without evidence of disease at 12 months after the initiation of treatment. Second-line chemotherapy was received by 12 patients, while six patients received additional mFOLFOX6 therapy. Efficacy The primary endpoint for this study was 1-year PFS as assessed by the treating investigator. With a median follow-up of 14.7 months, the PFS rate at 1 year was 23% (95% CI 8.6–44.2%). The median PFS and OS were 5.4 months (95% CI 4.8–6.0 Fig. 1), and 17.3 months (95% CI 11.7–19.0 Fig. 2), respectively. An exploratory analysis was conducted to determine the prognosis factors that might have influenced OS (Table 2). Parameters studied included age, PS, primary site and histological grade of the tumor, resection of the primary tumor or bypass, numbers of metastatic organs, and serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 levels. Upon univariate analysis, good PS (0), location in the jejunum, resection of the primary tumor or bypass, and a low level of serum CEA (<5 ng/mL) were all significantly associated with longer OS. However, upon multivariate analysis, only resection of primary tumor or bypass was an independent predictor of better OS (P = 0.023). The RR was slightly higher in patients with a tumor in the jejunum (4/7; 57%) than in the duodenum (5/13; 38%).Fig. 1 The survival rate in this study: progression-free survival (PFS) curve Fig. 2 The survival rate in this study: overall survival (OS) curve

Efficacy The primary endpoint for this study was 1-year PFS as assessed by the treating investigator. With a median follow-up of 14.7 months, the PFS rate at 1 year was 23% (95% CI 8.6–44.2%). The median PFS and OS were 5.4 months (95% CI 4.8–6.0 Fig. 1), and 17.3 months (95% CI 11.7–19.0 Fig. 2), respectively. An exploratory analysis was conducted to determine the prognosis factors that might have influenced OS (Table 2). Parameters studied included age, PS, primary site and histological grade of the tumor, resection of the primary tumor or bypass, numbers of metastatic organs, and serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 levels. Upon univariate analysis, good PS (0), location in the jejunum, resection of the primary tumor or bypass, and a low level of serum CEA (<5 ng/mL) were all significantly associated with longer OS. However, upon multivariate analysis, only resection of primary tumor or bypass was an independent predictor of better OS (P = 0.023). The RR was slightly higher in patients with a tumor in the jejunum (4/7; 57%) than in the duodenum (5/13; 38%).Fig. 1 The survival rate in this study: progression-free survival (PFS) curve Fig. 2 The survival rate in this study: overall survival (OS) curve Table 2 Univariate and multivariate analysis of factors associated with survival Univariate Multivariate N HR (95% CI) P HR (95% CI) P

Efficacy The primary endpoint for this study was 1-year PFS as assessed by the treating investigator. With a median follow-up of 14.7 months, the PFS rate at 1 year was 23% (95% CI 8.6–44.2%). The median PFS and OS were 5.4 months (95% CI 4.8–6.0 Fig. 1), and 17.3 months (95% CI 11.7–19.0 Fig. 2), respectively. An exploratory analysis was conducted to determine the prognosis factors that might have influenced OS (Table 2). Parameters studied included age, PS, primary site and histological grade of the tumor, resection of the primary tumor or bypass, numbers of metastatic organs, and serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 levels. Upon univariate analysis, good PS (0), location in the jejunum, resection of the primary tumor or bypass, and a low level of serum CEA (<5 ng/mL) were all significantly associated with longer OS. However, upon multivariate analysis, only resection of primary tumor or bypass was an independent predictor of better OS (P = 0.023). The RR was slightly higher in patients with a tumor in the jejunum (4/7; 57%) than in the duodenum (5/13; 38%).Fig. 1 The survival rate in this study: progression-free survival (PFS) curve Fig. 2 The survival rate in this study: overall survival (OS) curve Table 2 Univariate and multivariate analysis of factors associated with survival Univariate Multivariate N HR (95% CI) P HR (95% CI) P ECOG PS 0 17 1 7 3.69 (1.31–10.38) 0.014 2.60 (0.73–9.28) 0.14 Primary site Jejunum 10 Duodenum 14 2.88 (0.89–9.27) 0.077 2.85 (0.85–9.52) 0.090 Prior surgery Yes 13 No 11 2.83 (1.02–7.84) 0.046 3.98 (1.21–13.07) 0.023 Serum CEA (ng/mL) <5 15 ≥5 9 2.51 (0.90–7.01) 0.079 1.61 (0.43–6.00) 0.48

N HR (95% CI) P HR (95% CI) P ECOG PS 0 17 1 7 3.69 (1.31–10.38) 0.014 2.60 (0.73–9.28) 0.14 Primary site Jejunum 10 Duodenum 14 2.88 (0.89–9.27) 0.077 2.85 (0.85–9.52) 0.090 Prior surgery Yes 13 No 11 2.83 (1.02–7.84) 0.046 3.98 (1.21–13.07) 0.023 Serum CEA (ng/mL) <5 15 ≥5 9 2.51 (0.90–7.01) 0.079 1.61 (0.43–6.00) 0.48 CEA carcinoembryonic antigen, CI confidence interval, ECOG Eastern Cooperative Oncology Group, HR hazard ratio, PS performance status An exploratory analysis was conducted to determine the characteristic factors of the patients that might have influenced response. However, no difference between the responder and non-responder patients was identified for age, gender, PS, histological grade, tumor resection or bypass, location of primary tumor and metastasis, serum CEA and CA19-9 levels. Toxicity All 24 patients who received one dose of the study treatment were evaluated for toxicity. The common treatment-related grade 3–4 adverse events are listed in Table 3. The most common events were neutropenia (38%), anemia/peripheral neuropathy (25%), stenosis (17%), fatigue/anorexia/bilirubin increase (8%), and diarrhea (4%). One nonhematological grade 4 toxicity occurred; this patient had symptomatic cerebrovascular ischemia and was under treatment for type 2 diabetes and hypertension, and discontinued chemotherapy. There were no treatment-related deaths.Table 3 The most common treatment-related toxicities based on the Common Terminology Criteria for Adverse Events (CTCAE; Ver. 3.0)

ed; this patient had symptomatic cerebrovascular ischemia and was under treatment for type 2 diabetes and hypertension, and discontinued chemotherapy. There were no treatment-related deaths.Table 3 The most common treatment-related toxicities based on the Common Terminology Criteria for Adverse Events (CTCAE; Ver. 3.0) Any grade N (%) Grade 3/4 N (%) Neutropenia 12 (50) 9 (38) Anemia 13 (54) 6 (25) Thrombocytopenia 10 (42) 0 (0) Fatigue 18 (75) 2 (8) Nausea 18 (75) 0 (0) Vomiting 9 (38) 0 (0) Diarrhea 11 (46) 1 (4) Stomatitis 5 (21) 0 (0) Bilirubin elevation 4 (17) 2 (8) Peripheral neuropathy 19 (79) 6 (25) Hand–foot syndrome 4 (17) 0 (0) Pneumonitis 2 (8) 0 (0) Stenosis 9 (38) 4 (17) Hemorrhage 1 (4) 1 (4) Cerebrovascular ischemia 1 (4) 1 (4) Immunohistochemistry We were able to collect 13 tumor samples from the primary site and performed IHC analysis. The most commonly expressed immunophenotypic marker was CDX2, observed in seven patients (54%). Expression of CK20 occurred in 5 (38%) patients, and expression of CK7 occurred in 4 (31%) patients. The tissues demonstrated great variability for CK7, CK20, and CDX2. There was no significant difference in the expression of CK7, CK20, and CDX2 between the duodenal and nonduodenal SBA. Expression of EGFR score 2–6 was observed in 3 (23%) patients. Expression of HER2 was not observed in this analysis. There was no significant association between immunophenotypes and patient survival.

The most commonly expressed immunophenotypic marker was CDX2, observed in seven patients (54%). Expression of CK20 occurred in 5 (38%) patients, and expression of CK7 occurred in 4 (31%) patients. The tissues demonstrated great variability for CK7, CK20, and CDX2. There was no significant difference in the expression of CK7, CK20, and CDX2 between the duodenal and nonduodenal SBA. Expression of EGFR score 2–6 was observed in 3 (23%) patients. Expression of HER2 was not observed in this analysis. There was no significant association between immunophenotypes and patient survival. Discussion In the treatment of patients with advanced SBA, no prospective studies evaluated the benefit of chemotherapy compared with best supportive care. Single-institution retrospective studies have suggested that palliative chemotherapy confers a survival benefit to such patients [6, 8, 9]. In the largest retrospective analysis that evaluated 113 patients with advanced SBA, palliative chemotherapy predicted OS in a multivariate analysis (hazard ratio [HR] 0.47) [8]. Therefore, although no prospective study has evaluated outcomes, palliative chemotherapy is considered a standard treatment for patients with unresectable or recurrent SBA. In the past decades, these patients had been treated with the same chemotherapy regimen used for colorectal cancer (CRC) or gastric cancer. Therefore, in retrospective studies on patients with SBA, the most common regimen was 5-FU or 5-FU with a platinum agent (Table 4). Among them, combination chemotherapy of 5-FU with platinum compounds including oxaliplatin seemed to be more effective than other regimens.Table 4 Summary of previous studies on patients with SBA

re, in retrospective studies on patients with SBA, the most common regimen was 5-FU or 5-FU with a platinum agent (Table 4). Among them, combination chemotherapy of 5-FU with platinum compounds including oxaliplatin seemed to be more effective than other regimens.Table 4 Summary of previous studies on patients with SBA Authors Study Type Pts No. Regimen RR (%) PFS/TTP (M) MST (M) Xiang et al. [22] P II 33 FOLFOX 48.5 7.8 15.2 Tsushima et al. [20] Retro 22 FOLFOX 42 9.6 22.2 Zaanan et al. [19] Retro 38 FOLFOX 34 6.9 17.8 Overman et al. [21] P II 30 CAPOX 50 11.3 20.4 Suenaga et al. [15] Retro 10 5-FU-based 10 2.9 12 Overman et al. [18] Retro 29 5-FU and Platinum 46 8.7 14.8 Aparicio et al. [32] Retro 21 FOLFOX NR 7 NR Czaykowski et al. [9] Retro 37 5-FU-based 5 NR 15.6 Fishman et al. [8] Retro 44 Various 29 NR 18.6 Gibson et al. [11] P II 39 FAM 18 5.0 8 Locher et al. [13] Retro 20 5-FU and Platinum 21 8.0 14 Dabaja et al. [6] Retro 48 NR NR NR 11 Crawley et al. [14] Retro 8 ECF or 5-FU 38 7.8 13 Jigyasu et al. [16] Retro 14 5-FU-based 7 NR 9 Morgan and Busuttil [33] Retro 7 5-FU-based 0 NR NR Rochlin et al. [34] Retro 11 5-FU 36 3.8 NR 5-FU fluorouracil, ECF epirubicin, cisplatin and 5-FU, FAM 5-FU adriamycin and mitomycin, FOLFOX oxaliplatin, leucovorin and 5-FU, CAPOX capecitabine and oxaliplatin, RR response rate, PFS progression-free survival, TTP time to progression, MST median survival time

Authors Study Type Pts No. Regimen RR (%) PFS/TTP (M) MST (M) Xiang et al. [22] P II 33 FOLFOX 48.5 7.8 15.2 Tsushima et al. [20] Retro 22 FOLFOX 42 9.6 22.2 Zaanan et al. [19] Retro 38 FOLFOX 34 6.9 17.8 Overman et al. [21] P II 30 CAPOX 50 11.3 20.4 Suenaga et al. [15] Retro 10 5-FU-based 10 2.9 12 Overman et al. [18] Retro 29 5-FU and Platinum 46 8.7 14.8 Aparicio et al. [32] Retro 21 FOLFOX NR 7 NR Czaykowski et al. [9] Retro 37 5-FU-based 5 NR 15.6 Fishman et al. [8] Retro 44 Various 29 NR 18.6 Gibson et al. [11] P II 39 FAM 18 5.0 8 Locher et al. [13] Retro 20 5-FU and Platinum 21 8.0 14 Dabaja et al. [6] Retro 48 NR NR NR 11 Crawley et al. [14] Retro 8 ECF or 5-FU 38 7.8 13 Jigyasu et al. [16] Retro 14 5-FU-based 7 NR 9 Morgan and Busuttil [33] Retro 7 5-FU-based 0 NR NR Rochlin et al. [34] Retro 11 5-FU 36 3.8 NR 5-FU fluorouracil, ECF epirubicin, cisplatin and 5-FU, FAM 5-FU adriamycin and mitomycin, FOLFOX oxaliplatin, leucovorin and 5-FU, CAPOX capecitabine and oxaliplatin, RR response rate, PFS progression-free survival, TTP time to progression, MST median survival time Our study demonstrated an RR of 45%, a median PFS of 5.4 months, and a median OS of 17.3 months in patients given the mFOLFOX6 regimen. To our knowledge, only two prospective phase II studies have used the combination of fluoropyrimidine and oxaliplatin. Overman et al. reported on a capecitabine and oxaliplatin (CAPOX) regimen [21] and Xiang et al. reported on a mFOLFOX6 regimen but omitted a bolus 5-FU treatment [22]. The RR and median OS were similar in these studies. However, the PFS of our study was worse than that of the CAPOX regimen. Ono possibility is that patients with duodenal cancer were more frequently seen in our study than in the CAPOX study (58 vs 23%). Although there was no statistical difference in multivariate analysis for OS, the RR for patients with a tumor in the duodenum appeared lower than in the jejunum (38 vs 57%, respectively), and location in the jejunum was significantly associated with a longer OS in univariate analysis (P = 0.077). The primary site was demonstrated as a predictive or as a worse factor for OS in previous analyses and our study, respectively [25, 26]. This difference might reflect the heterogeneous nature of the epithelium of tumor origin between the duodenum and jejunum.

ociated with a longer OS in univariate analysis (P = 0.077). The primary site was demonstrated as a predictive or as a worse factor for OS in previous analyses and our study, respectively [25, 26]. This difference might reflect the heterogeneous nature of the epithelium of tumor origin between the duodenum and jejunum. This study did not meet the primary endpoint because of the small sample size. However, the differences in PFS between the two prospective studies (above) and our result are small. Therefore, the survival benefit of combining fluoropyrimidine and oxaliplatin combination was confirmed as a first-line chemotherapy regimen for patients with unresectable or recurrent SBA.

se of the small sample size. However, the differences in PFS between the two prospective studies (above) and our result are small. Therefore, the survival benefit of combining fluoropyrimidine and oxaliplatin combination was confirmed as a first-line chemotherapy regimen for patients with unresectable or recurrent SBA. In multivariate analysis, only resection of the primary tumor or bypass was an independent predictor of better OS (P = 0.023). Severe stenosis of grade ≥3 which requests resection of the primary tumor or bypass occurred in 4 patients (17%, 4/23) in this study. Resection of the primary tumor was also demonstrated to be a factor predictive of a better OS in other reports [18]. Palliative tumor resection has been traditionally advocated in metastatic CRC to prevent symptoms and complications linked to the primary tumor, such as obstruction, perforation, or bleeding. The risk of obstruction caused by the tumor during initial chemotherapy was 6–29% in patients with CRC [27, 28]. In this study, severe stenosis (grade ≥3) occurred in 3 patients (27%, 3/11) among the patients without resection of the primary tumor or bypass. All cases occurred within 2 months after starting chemotherapy. If the lumen of the small intestine is narrower than the colorectum, then we should consider that stenosis is likely to occur at an early stage of chemotherapy.

in 3 patients (27%, 3/11) among the patients without resection of the primary tumor or bypass. All cases occurred within 2 months after starting chemotherapy. If the lumen of the small intestine is narrower than the colorectum, then we should consider that stenosis is likely to occur at an early stage of chemotherapy. There were some limitations to our study. First, the small sample size hampered comparisons between subgroups. Nevertheless, to separate the heterogeneity in the survival outcomes associated with the primary site (duodenum vs jejunum) and prior surgery (resection of the primary tumor or bypass vs without resection), an exploratory analysis of the subgroups were performed. Second, IHC analysis used only a small subsample of the patients. No information was obtained from this analysis. Consequently, firm conclusions cannot be drawn from the subgroup and IHC analyses because the rarity of this disease hampers large-scale studies. Importantly, this trial demonstrates the feasibility of the completion of phase II studies in such rare tumor types, and should promote more robust research on these orphan tumors. In addition, given the overall tolerability of the regimen, it is logical to investigate the role of targeted therapies in combination with mFOLFOX6.

y, this trial demonstrates the feasibility of the completion of phase II studies in such rare tumor types, and should promote more robust research on these orphan tumors. In addition, given the overall tolerability of the regimen, it is logical to investigate the role of targeted therapies in combination with mFOLFOX6. The main challenge for the future will be to identify a molecular marker involved in small-bowel carcinogenesis that can predict chemosensitivity, and thus improve patient survival. In patients with unresectable CRC, the addition of bevacizumab to mFOLFOX6 was found to prolong PFS compared with mFOLFOX6 alone, and clinical trials that investigate mFOLFOX6 in combination with agents targeting angiogenesis would be reasonable for patients with SBA [29]. In addition, activation of mutations in the KRAS (or RAS) oncogene occur at a similar frequency in both SBA and colorectal adenoma tumors, which suggests a potential role for EGFR inhibition in a subset of patients with SBA [30, 31].

ion with agents targeting angiogenesis would be reasonable for patients with SBA [29]. In addition, activation of mutations in the KRAS (or RAS) oncogene occur at a similar frequency in both SBA and colorectal adenoma tumors, which suggests a potential role for EGFR inhibition in a subset of patients with SBA [30, 31]. The infrequency of SBA made it difficult to conduct a prospective study. It is extremely unlikely that a randomized trial comparing two chemotherapy regimens could be undertaken. However, we have conducted this prospective phase II trial within a new cooperative group in Japan. It provides important insights for the treatment of patients with SBA. The mFOLFOX6 regimen proved effective and probably represents a new standard treatment for patients with an unresectable or recurrent SBA. In the future, the mFOLFOX6 regimen in combination with molecular targeting therapy should be evaluated prospectively to improve the outcomes even for patients with SBA; however, worldwide multi-institutional cooperation will be necessary for investigating such a rare disease. Acknowledgements We especially thank the patients and their family members. We also thank all investigators and clinical research coordinators who participated in this study at the 12 participating centers; and Data and Safety monitoring (K Furukawa, and J Shimizu), and IHC analysis (T Funakoshi, and S Minamiguchi). Compliance with ethical standards Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this paper.

Introduction Prostate cancer is the second most common cancer in males worldwide and accounts for 15% of all cancers diagnosed in men. It represents the fifth leading cause of death from cancer in men and 6.6% of total male mortality [1]. Among patients with localized prostate cancer, treatments are effective, and 5-year survival rates are approximately 100%. Nevertheless, those with distant metastases often become resistant to treatment, and the 5-year survival rate is considerably lower at 31% among this patient population [2]. The standard therapy for patients with advanced prostate cancer is androgen deprivation therapy, which includes medical or surgical castration [2, 3]. The disease is defined to be castrate-resistant prostate cancer (CRPC) if it progresses, either biochemically or radiologically, despite serum testosterone levels of <1.7 nmol/L. Within 5 years of follow-up, 10–20% of patients with prostate cancer develop CRPC [4]. In patients with CRPC, the most frequent site of metastases is bone, and comorbidities or skeletal-related events (SREs) caused by bone metastases are associated with deterioration of the quality of life and an increased risk of death [5]. Therefore, the treatment goal for patients with CRPC and bone metastases should be maintaining quality of life, preventing SREs, and improving survival [6].

orbidities or skeletal-related events (SREs) caused by bone metastases are associated with deterioration of the quality of life and an increased risk of death [5]. Therefore, the treatment goal for patients with CRPC and bone metastases should be maintaining quality of life, preventing SREs, and improving survival [6]. While a number of different treatment approaches are available for the management of metastatic CRPC, including abiraterone, enzalutamide, docetaxel, cabazitaxel, and sipuleucel-T, the effects of these drugs on bone metastases has not been thoroughly investigated [7]. The active form of radium-223 dichloride is an α-emitting radionuclide and a calcium mimetic that forms complexes with the bone mineral hydroxyapatite at areas of high bone turnover, a typical characteristic of bone metastases. Once at the site of bone metastases, radium-223 dichloride emits α particles and induces breaks in double-stranded DNA, killing tumor cells in a targeted fashion [8, 9]. Radium-223 dichloride was approved by the U.S. Food and Drug Administration in 2013 for the treatment of patients with CRPC and symptomatic bone metastases with no known visceral metastases [8].

223 dichloride emits α particles and induces breaks in double-stranded DNA, killing tumor cells in a targeted fashion [8, 9]. Radium-223 dichloride was approved by the U.S. Food and Drug Administration in 2013 for the treatment of patients with CRPC and symptomatic bone metastases with no known visceral metastases [8]. Clinical trials in Caucasian patients with CRPC and bone metastases have shown that radium-223 dichloride is well tolerated, improves overall survival, and reduces symptomatic skeletal events (SSEs) [10–12]. The aim of this phase I study was to investigate the pharmacokinetics, dosimetry, safety, and efficacy of radium-223 dichloride in Japanese patients with CRPC and bone metastases. The pharmacokinetic results of this study have been published [13]; we report here the safety and efficacy (biomarker) outcomes of the study.

he aim of this phase I study was to investigate the pharmacokinetics, dosimetry, safety, and efficacy of radium-223 dichloride in Japanese patients with CRPC and bone metastases. The pharmacokinetic results of this study have been published [13]; we report here the safety and efficacy (biomarker) outcomes of the study. Patients and methods Selection of patients Inclusion criteria The study population included male patients aged ≥20 years with histologically-confirmed adenocarcinoma of the prostate, with ≥2 bone metastases confirmed by scintigraphic imaging within the 4 weeks preceding the start of radium-223 dichloride treatment, and who had failed initial hormonal therapy. Other inclusion criteria were: (1) castrate levels of testosterone of <50 ng/dL (1.7 nmol/L) and continued treatment to maintain castrate levels of testosterone; (2) progressive castration-resistant metastatic disease, defined as at least one of the following: new osseous lesions observed via radionuclide bone scan, a ≥20% increase in the sum of the longest diameter of target lesions, or ≥3 rising prostate specific antigen (PSA) values from baseline; (3) Eastern Cooperative Oncology Group performance status 0–2; (4) alkaline phosphatase (ALP) level greater than the upper institutional limit of normal range.

radionuclide bone scan, a ≥20% increase in the sum of the longest diameter of target lesions, or ≥3 rising prostate specific antigen (PSA) values from baseline; (3) Eastern Cooperative Oncology Group performance status 0–2; (4) alkaline phosphatase (ALP) level greater than the upper institutional limit of normal range. Exclusion criteria Patients were excluded from the study if they had (1) received an investigational drug in the 4 weeks immediately preceding the start of radium-223 dichloride treatment, or were scheduled to receive one during the treatment or 8 weeks after study drug administration; (2) received chemo-, immuno-, or radiotherapy within the last 4 weeks prior to entry in the study, or had not recovered from acute adverse events (AEs) as a result of such therapy; (3) started or stopped systemic steroids within 1 week prior to study drug administration, or were expected to change systemic steroids; (4) had a history of gastrointestinal bleeding or ulcer within 3 months prior to study entry; (5) had small cell carcinoma; predominant visceral metastases (≥3 lung or liver lesions) or symptomatic lymphadenopathy which was characterized by scrotal or pedal edema. Written informed consent was obtained from all the patients or their legally authorized representatives prior to the study.

Exclusion criteria Patients were excluded from the study if they had (1) received an investigational drug in the 4 weeks immediately preceding the start of radium-223 dichloride treatment, or were scheduled to receive one during the treatment or 8 weeks after study drug administration; (2) received chemo-, immuno-, or radiotherapy within the last 4 weeks prior to entry in the study, or had not recovered from acute adverse events (AEs) as a result of such therapy; (3) started or stopped systemic steroids within 1 week prior to study drug administration, or were expected to change systemic steroids; (4) had a history of gastrointestinal bleeding or ulcer within 3 months prior to study entry; (5) had small cell carcinoma; predominant visceral metastases (≥3 lung or liver lesions) or symptomatic lymphadenopathy which was characterized by scrotal or pedal edema. Written informed consent was obtained from all the patients or their legally authorized representatives prior to the study. Study design This study was an open-label, uncontrolled, non-randomized, multicenter phase I trial (Trial registration: ClinicalTrials.gov number NCT01565746) conducted at three study centers in Japan (National Cancer Center Hospital East, Yokohama City University Hospital, and Kinki University Hospital).

Written informed consent was obtained from all the patients or their legally authorized representatives prior to the study. Study design This study was an open-label, uncontrolled, non-randomized, multicenter phase I trial (Trial registration: ClinicalTrials.gov number NCT01565746) conducted at three study centers in Japan (National Cancer Center Hospital East, Yokohama City University Hospital, and Kinki University Hospital). All patients received a single intravenous bolus of radium-223 dichloride. A single 50 kBq/kg body weight (BW) dose (equivalent to 55 kBq/kg BW after implementation of the National Institute of Standards and Technology (NIST) update [14]; hereafter described as 55 kBq/kg) was given to patients in cohort 1, and if the incidence of critical toxicity was lower than 33%, a single dose of 100 kBq/kg BW (equivalent to 110 kBq/kg BW after the NIST update, and hereafter described as 110 kBq/kg) was given to cohort 2 (cycle 1). Cycle 2 and subsequent 4-week cycles (at a dose of 55 kBq/kg) continued for up to five additional doses for cohort 1 and up to four additional doses for cohort 2. Patients were allowed to receive the next dose only if they did not have definitive progressive disease and did not show critical toxicity.

hort 2 (cycle 1). Cycle 2 and subsequent 4-week cycles (at a dose of 55 kBq/kg) continued for up to five additional doses for cohort 1 and up to four additional doses for cohort 2. Patients were allowed to receive the next dose only if they did not have definitive progressive disease and did not show critical toxicity. Additional patients were enrolled in the expansion cohort provided the safety of radium-223 dichloride was confirmed in cohort 1. The patients in cohorts 1 and 2 were hospitalized for the first 28 days, while those in the expansion cohort were hospitalized for the first 8 days for safety observations. All patients were followed up at 4, 8 and 12 weeks after the last treatment, plus every 6 months after the last treatment for up to 36 months after the first treatment. The study was conducted according to four internal manuals outlining a standard protocol for the proper use of radium-223 dichloride, describing (1) the safe and efficient use of medical radiation [15], (2) proper use of radionuclide therapy in clinical trials 1, (3) protection from medical radiation [16], and (4) quantifying shielding and radiation exposure in the atmosphere, exhaust air and exhaust fluid 2.

the proper use of radium-223 dichloride, describing (1) the safe and efficient use of medical radiation [15], (2) proper use of radionuclide therapy in clinical trials 1, (3) protection from medical radiation [16], and (4) quantifying shielding and radiation exposure in the atmosphere, exhaust air and exhaust fluid 2. All study protocols were approved by the Institutional Review Boards of the National Cancer Center Hospital East, Yokohama City University Hospital and Kinki University Hospital before commencing the study. In addition to all local legal and regulatory requirements, the study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization guideline E6: Good Clinical Practice. Study outcomes The primary study endpoint/outcome was safety (AEs), while the secondary endpoints included treatment efficacy (determined via biochemical bone markers).

All study protocols were approved by the Institutional Review Boards of the National Cancer Center Hospital East, Yokohama City University Hospital and Kinki University Hospital before commencing the study. In addition to all local legal and regulatory requirements, the study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization guideline E6: Good Clinical Practice. Study outcomes The primary study endpoint/outcome was safety (AEs), while the secondary endpoints included treatment efficacy (determined via biochemical bone markers). Safety assessments All AEs that occurred in the patients during the study treatment and within 12 weeks after the last dose were recorded. Any causal relationship between the given treatment and observed AEs was assessed. All AEs were coded by MedDRA Version 16.1 (https://www.meddra.org/sites/default/files/.../intguide_16_1_english.pdf) and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0) (https://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf). The critical toxicities were defined as (1) grade 3 or higher non-hematologic toxicity or (2) hematologic toxicity, such as grade 3 neutropenia with fever or grade 4 neutropenia that failed to recover to grade 2 or less after treatment with granulocyte-colony stimulating factor within 2 weeks or (3) grade 4 thrombocytopenia.

itical toxicities were defined as (1) grade 3 or higher non-hematologic toxicity or (2) hematologic toxicity, such as grade 3 neutropenia with fever or grade 4 neutropenia that failed to recover to grade 2 or less after treatment with granulocyte-colony stimulating factor within 2 weeks or (3) grade 4 thrombocytopenia. A serious AE (SAE) was one that was life-threatening, required inpatient hospitalization or prolongation of existing hospitalization, or resulted in persistent or significant disability/incapacity, a congenital anomaly, serious event, or death. Treatment-emergent AEs (TEAEs) were defined as all events occurring or worsening after the first injection and within 30 days after the last injection of radium-223 dichloride. Post-treatment follow-up AEs were recorded for 30 days after the last dose up to 12 weeks after the last dose. AEs which occurred and were considered to be related to treatment with radium-223 dichloride were reported every 6 months after the last dose for up to 36 months after the first dose. Efficacy assessments Levels of PSA and bone markers, including serum total ALP, serum bone ALP, procollagen 1 N-terminal propeptide (P1NP), C-terminal crosslinked telopeptide of type I collagen (CTX-1), and carboxyterminal telopeptide of type I collagen (ICTP) were used for the efficacy assessment. All markers were measured at screening, at baseline before injection, on day 15 in cycle 1, on day 1 in cycle 2 and subsequent cycles, at the end of treatment (EOT), and at 4, 8 and 12 weeks after the last treatment or the end of follow-up.

al telopeptide of type I collagen (ICTP) were used for the efficacy assessment. All markers were measured at screening, at baseline before injection, on day 15 in cycle 1, on day 1 in cycle 2 and subsequent cycles, at the end of treatment (EOT), and at 4, 8 and 12 weeks after the last treatment or the end of follow-up. Statistical analyses Statistical analyses for the study were performed using the Statistical Analysis System (SAS; SAS Institute Inc., Raleigh, NC). The safety analysis included all patients who received at least one dose of study medication, while the efficacy analysis included all patients who received at least one dose and who had post-baseline efficacy data available. Demographic and other baseline characteristics were summarized using descriptive statistics. The PSA values, changes from baseline, and percentage changes from baseline were summarized by visit. Response rates (≥30% reduction and ≥50% reduction) were estimated at 12 weeks and at the EOT for PSA and bone markers. Results Patient disposition and baseline characteristics A total of 19 patients were enrolled in the study. All received at least one dose of radium-223 dichloride and were included in the safety and efficacy analysis set (three in cohort 1, three in cohort 2, and 13 in the expansion cohort). Demographic and baseline characteristics and any prior treatment received by the study patients are shown in Table 1.Table 1 Demographics, baseline characteristics, and prior treatments

1 The Japanese Society of Nuclear Medicine (NA) manual for proper use in clinical trials relating to radionuclide therapy with radium-223 dichloride (Ra-223) injection. 2 The Japanese Society of Nuclear Medicine (NA) Addendum to the manual for proper use in clinical trials relating to radionuclide therapy with Ra-223 dichloride (Ra-223) injection: methods of calculating radiation shielding and levels of radioactivity in the atmosphere, exhaust air and exhaust fluid. Acknowledgements Medical writing assistance in drafting this manuscript under direction of the authors was provided by Sheridan Henness, Ph.D., and Matt Weitz of Springer Healthcare Communications. This assistance was funded by Bayer, Japan. Author contributions All authors were involved in the implementation of the study and preparation of the manuscript. All authors declare that they have full control of all primary data and agree to allow the International Journal of Clinical Oncology to review this data if requested. Compliance with ethical standards Conflict of interest Seigo Kinuya received lecture fees from Bayer, Japan. Yoko Yajima is an employee of Bayer Yakuhin, Ltd. Hiroji Uemura, Hirotsugu Uemura, Nobuaki Matsubara, Makoto Hosono and Toshihiko Doi have no conflicts of interest to declare.

ride and were included in the safety and efficacy analysis set (three in cohort 1, three in cohort 2, and 13 in the expansion cohort). Demographic and baseline characteristics and any prior treatment received by the study patients are shown in Table 1.Table 1 Demographics, baseline characteristics, and prior treatments Patient characteristics Cohort 1 (n = 3) Cohort 2 (n = 3) Expansion cohort (n = 13) Cohort 1 + expansion cohort (n = 16) Total (n = 19) Demographic characteristics, mean ± SD Age (years) 73.3 ± 6.7 71.7 ± 5.9 71.3 ± 4.7 71.7 ± 4.9 71.7 ± 4.9 Weight (kg) 67.7 ± 4.2 60.1 ± 3.1 62.3 ± 7.8 63.3 ± 7.5 62.8 ± 7.0 Height (cm) 162.5 ± 4.8 165.8 ± 4.1 163.2 ± 4.2 163.1 ± 4.2 163.5 ± 4.2 Body mass index (kg/m2) 25.6 ± 0.5 21.9 ± 1.1 23.4 ± 3.8 23.8 ± 3.2 23.5 ± 3.0 ECOG performance status at baseline, n (%) 0 3 (100.0) 3 (100.0) 11 (84.6) 14 (87.5) 17 (89.5) 1 0 0 2 (15.4) 2 (12.5) 2 (10.5) Prior anticancer therapy/therapeutic procedures, n (%) Prior therapeutic procedurea 1 (33.3) 1 (33.3) 2 (15.4) 3 (18.8) 4 (21.1) Prior diagnostic procedureb 3 (100.0) 3 (100.0) 13 (100.0) 16 (100.0) 19 (100.0) Prior systemic anti-cancer therapy 3 (100.0) 3 (100.0) 13 (100.0) 16 (100.0) 19 (100.0) Prior radiotherapy 0 1 (33.3) 3 (23.1) 3 (18.8) 4 (21.1) Prior local anti-cancer therapyc 0 0 0 0 0 Baseline of tumor markers, mean ± SD PSA (ng/mL) 42.8 ± 25.1 669.6 ± 737.5 379.7 ± 505.5 316.5 ± 472.2 372.3 ± 496.2 ALP (U/L) 198.0 ± 52.8 1354.0 ± 1697.8 1024.1 ± 1015.6 869.2 ± 967.6 945.7 ± 1049.0

(100.0) 19 (100.0) Prior radiotherapy 0 1 (33.3) 3 (23.1) 3 (18.8) 4 (21.1) Prior local anti-cancer therapyc 0 0 0 0 0 Baseline of tumor markers, mean ± SD PSA (ng/mL) 42.8 ± 25.1 669.6 ± 737.5 379.7 ± 505.5 316.5 ± 472.2 372.3 ± 496.2 ALP (U/L) 198.0 ± 52.8 1354.0 ± 1697.8 1024.1 ± 1015.6 869.2 ± 967.6 945.7 ± 1049.0 ALP Alkaline phosphatase, ECOG Eastern Cooperative Oncology Group, PSA prostate specific antigen, SD standard deviation aPrior therapeutic procedure includes orchiectomy and/or prostatectomy bPrior diagnostic procedure includes biopsy and/or prostatectomy cLocal anticancer therapy includes radiotherapy and surgery Treatment exposure The median duration of radium-223 dichloride treatment ranged from 114 to 142 days in all three cohorts, with patients receiving a median of five or six injections. The median total dose of radium-223 dichloride ranged from 15,736 kBq in the expansion cohort to 22,214 kBq in cohort 1. For the 55 kBq/kg treatment (cohort 1 + expansion cohort, n = 16) the median duration of treatment and number of injections was 129 days and 5.5 injections, respectively; the median total dose of radium-223 dichloride that patients received was 18,983 kBq. For the 110 kBq/kg treatment (cohort 2, n = 3) the median duration of treatment and number of injections was 114 days and 5.0 injections, respectively; the median total dose of radium-223 dichloride that patients received was 18,778 kBq.

median total dose of radium-223 dichloride that patients received was 18,983 kBq. For the 110 kBq/kg treatment (cohort 2, n = 3) the median duration of treatment and number of injections was 114 days and 5.0 injections, respectively; the median total dose of radium-223 dichloride that patients received was 18,778 kBq. Safety Almost all patients (n = 18, 94.7%) experienced one or more TEAEs; those TEAEs considered to be drug-related are summarized in Table 2. No grade 4 or grade 5 TEAEs were observed (Table 3). The grade 3 TEAE occurring in the highest proportion of patients was anemia (21.1%, 4/19), while other TEAEs were observed in one patient (5.3%). Three patients died in the post-treatment period in the expansion cohort (23.1%, 3/13), and no deaths were observed in cohort 1 or cohort 2. All deaths were considered to be unrelated to study treatment. SAEs were experienced by three patients in the expansion cohort (23.1%, 3/13) during the treatment period (Table 4). The worst grade of these SAEs was grade 3 (infection, lung infection, bone pain, and prostate cancer), and grade 2 (rectal hemorrhage). No SAEs were related to study treatment.Table 2 List of drug-related treatment-emergent adverse events TEAEs, n (%) Cohort 1 (n = 3) Cohort 2 (n = 3) Expansion cohort (n = 13) Cohort 1 + expansion cohort (n = 16) Total (n = 19) Drug-related TEAEsa Any 1 (33.3) 3 (100.0) 7 (53.8) 8 (50.0) 11 (57.9) Worst grade, grade 5 (death) 0 0 0 0 0 Worst grade, grade 3 or 4b 0 0 2 (15.4) 2 (12.5) 2 (10.5) Drug-related post treatment follow-up AEsc

TEAEs, n (%) Cohort 1 (n = 3) Cohort 2 (n = 3) Expansion cohort (n = 13) Cohort 1 + expansion cohort (n = 16) Total (n = 19) Drug-related TEAEsa Any 1 (33.3) 3 (100.0) 7 (53.8) 8 (50.0) 11 (57.9) Worst grade, grade 5 (death) 0 0 0 0 0 Worst grade, grade 3 or 4b 0 0 2 (15.4) 2 (12.5) 2 (10.5) Drug-related post treatment follow-up AEsc Any 0 0 2 (15.4) 2 (12.5) 2 (10.5) Grade 5 (death) 0 0 0 0 0 Grade 3 or 4b 0 0 1 (7.7) 1 (6.3) 1 (5.3) Long-term toxicityd 0 0 0 0 0 All drug-related TEAEs in treatment period, by MedDRA term (and by CTCAE where different) Any 1 (33.3) 3 (100.0) 7 (53.8) 8 (50.0) 11 (57.9) Anemia 1 (33.3) 0 3 (23.1) 4 (25.0) 4 (21.1) Constipation 0 0 1 (7.7) 1 (6.3) 1 (5.3) Diarrhea 0 3 (100.0) 0 0 3 (15.8) Lymphocytopenia (lymphocyte count decreased) 0 0 2 (15.4) 2 (12.5) 2 (10.5) Thrombocytopenia (platelet count decreased) 1 (33.3) 0 2 (15.4) 3 (18.8) 3 (15.8) Leukopenia (white blood cells decreased) 0 0 1 (7.7) 1 (6.3) 1 (5.3) Bone pain 0 0 1 (7.7) 1 (6.3) 1 (5.3) Dysgeusia 0 0 2 (15.4) 2 (12.5) 2 (10.5) Rash (rash acneiform) 0 1 (33.3) 0 0 1 (5.3) AEs adverse events, CTCAE Common Terminology Criteria for Adverse Events, MedDRA medical dictionary for regulatory activities, TEAEs treatment-emergent adverse events aTEAEs were defined as all events occurring or worsening after the first injection of study treatment and within 12 weeks after the last injection of study treatment bThe worst grade was grade 3; no grade 4 TEAEs were reported

AEs adverse events, CTCAE Common Terminology Criteria for Adverse Events, MedDRA medical dictionary for regulatory activities, TEAEs treatment-emergent adverse events aTEAEs were defined as all events occurring or worsening after the first injection of study treatment and within 12 weeks after the last injection of study treatment bThe worst grade was grade 3; no grade 4 TEAEs were reported cPost-treatment follow-up AEs were defined as AEs considered to be related to the study treatment which occurred between 30 days and 12 weeks after the last treatment or up to the end of the follow-up dLong-term toxicity was defined as AEs considered to be related to the study treatment which occurred between 12 weeks after the last treatment and 36 months after the first treatment Table 3 Grade 3 treatment-emergent adverse events

cPost-treatment follow-up AEs were defined as AEs considered to be related to the study treatment which occurred between 30 days and 12 weeks after the last treatment or up to the end of the follow-up dLong-term toxicity was defined as AEs considered to be related to the study treatment which occurred between 12 weeks after the last treatment and 36 months after the first treatment Table 3 Grade 3 treatment-emergent adverse events Grade 3 or grade 4 TEAEs by MedDRA (and by CTCAE where different), n (%) Worst CTCAE grade Cohort 1 (n = 3) Cohort 2 (n = 3) Expansion cohort (n = 13) Cohort 1 + expansion cohort (n = 16) Total (n = 19) Anemia Grade 3 0 0 4 (30.8) 4 (25.0) 4 (21.1) Nausea Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Rectal stenosis Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Infection (infections and infestations—other) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Lung infection (lung infection) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Lymphocytopenia (lymphocyte count decreased) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Leukopenia (white blood cells decreased) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Decreased appetite (anorexia) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Inadequate control of diabetes mellitus (glucose intolerance) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Hypocalcemia Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Hypophosphatemia Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Bone pain Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Cancer pain (tumor pain) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Prostate cancer (neoplasms benign, malignant and unspecified, including cysts and polyps—other) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Renal impairment (renal and urinary disorders—other) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3)

in Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Cancer pain (tumor pain) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Prostate cancer (neoplasms benign, malignant and unspecified, including cysts and polyps—other) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Renal impairment (renal and urinary disorders—other) Grade 3 0 0 1 (7.7) 1 (6.3) 1 (5.3) Table 4 All treatment-emergent serious adverse events reported during the study Treatment-emergent SAEs, by MedDRA (and by CTCAE where different), n (%) Cohort 1 (n = 3) Cohort 2 (n = 3) Expansion cohort (n = 13) Cohort 1 + expansion cohort (n = 16) Total (n = 19) Rectal hemorrhage 0 0 1 (7.7) 1 (6.3) 1 (5.3) Infection (infections and infestations—other) 0 0 1 (7.7) 1 (6.3) 1 (5.3) Lung infection (lung infection) 0 0 1 (7.7) 1 (6.3) 1 (5.3) Bone pain 0 0 1 (7.7) 1 (6.3) 1 (5.3) Prostate cancer (neoplasms benign, malignant and unspecified incl. cysts and polyps—other) 0 0 1 (7.7) 1 (6.3) 1 (5.3) SAEs Serious adverse events Two patients (10.5%, 2/19) experienced AEs (grade 2 thrombocytopenia and grade 3 gastric hemorrhage, respectively) that led to discontinuation of study treatment in the expansion cohort, but not in cohort 1 or cohort 2. The incidence of serious TEAEs leading to dose interruption and permanent discontinuation of study drug was 15.4% (2/13) and 7.7% (1/13), respectively. One patient from cohort 2 and eight patients from the expansion cohort were withdrawn from the study due to disease progression.

ohort, but not in cohort 1 or cohort 2. The incidence of serious TEAEs leading to dose interruption and permanent discontinuation of study drug was 15.4% (2/13) and 7.7% (1/13), respectively. One patient from cohort 2 and eight patients from the expansion cohort were withdrawn from the study due to disease progression. In the expansion cohort, two patients (10.5%, 2/19) reported drug-related post-treatment AEs, including anemia in two patients (grade 2 and 3, respectively), and platelet count decreased in one patient (grade 4). No long-term toxicity was reported in this study. Efficacy In cohort 1 + the expansion cohort, serum PSA levels remained stable or slightly increased after the injection of radium-223 dichloride at week 12 and at EOT (Table 5; Fig. 1).Table 5 Percentage change from baseline in efficacy markers following injections of radium-223 dichloride in cohort 1 + expansion cohort (n = 16) Markers 12 weeks after treatment End of treatment n Mean ± SD Range n Mean ± SD Range PSA 11 83.5 ± 124.5 −32.4 to 423.8 16 182.0 ± 254.2 −37.8 to 934.5 Bone markers Total ALP 11 −30.4 ± 23.6 −69.1 to 12.1 16 −27.7 ± 25.2 −66.5 to 26.0 Bone ALP 11 −46.2 ± −18.7 −78.0 to −10.5 16 −48.2 ± 17.2 −75.1 to −13.7 P1NP 11 −42.1 ± 25.0 −71.2 to 9.7 16 −29.5 ± 40.0 −81.4 to 45.9 CTX-1 11 −20.8 ± 24.1 −66.7 to 0.0 16 35.9 ± 127.2 −66.7 to 500.0 ICTP 11 14.3 ± 38.3 −12.8 to 116.0 16 69.2 ± 190.4 −24.4 to 763.0 CTX-1 C-terminal crosslinked telopeptide of type I collagen, ICTP carboxyterminal telopeptide of type I collagen, P1NP procollagen 1 N-terminal propeptide,

n Mean ± SD Range n Mean ± SD Range PSA 11 83.5 ± 124.5 −32.4 to 423.8 16 182.0 ± 254.2 −37.8 to 934.5 Bone markers Total ALP 11 −30.4 ± 23.6 −69.1 to 12.1 16 −27.7 ± 25.2 −66.5 to 26.0 Bone ALP 11 −46.2 ± −18.7 −78.0 to −10.5 16 −48.2 ± 17.2 −75.1 to −13.7 P1NP 11 −42.1 ± 25.0 −71.2 to 9.7 16 −29.5 ± 40.0 −81.4 to 45.9 CTX-1 11 −20.8 ± 24.1 −66.7 to 0.0 16 35.9 ± 127.2 −66.7 to 500.0 ICTP 11 14.3 ± 38.3 −12.8 to 116.0 16 69.2 ± 190.4 −24.4 to 763.0 CTX-1 C-terminal crosslinked telopeptide of type I collagen, ICTP carboxyterminal telopeptide of type I collagen, P1NP procollagen 1 N-terminal propeptide, Fig. 1 Percentage changes from baseline in prostate-specific antigen (PSA) levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles Mean ± standard deviation (SD). EOT End of treatment. PSA response rate was defined as the percentage of patients whose PSA blood level was reduced by ≥30 or ≥50% vs. baseline

tate-specific antigen (PSA) levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles Mean ± standard deviation (SD). EOT End of treatment. PSA response rate was defined as the percentage of patients whose PSA blood level was reduced by ≥30 or ≥50% vs. baseline Total ALP levels in blood decreased from baseline to week 12 and EOT in all the cohorts (Table 5; Fig. 2). The total ALP response rates (≥30 and ≥50% reductions) were 54.5% (6/11) and 9.1% (1/11), respectively, at week 12, and 56.3% (9/16) and 25.0% (4/16), respectively, at EOT. Bone ALP levels also decreased from baseline to week 12 and EOT in all cohorts (Table 5; Fig. 3). Bone ALP response rates (≥30 and ≥50% reductions) were 81.8% (9/11) and 36.4% (4/11), respectively, at week 12, and 81.3% (13/16) and 50.0% (8/16), respectively, at EOT.Fig. 2 Percentage changes from baseline in total alkaline phosphatase (ALP) levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles Mean ± SD. ALP response rate was defined as the percentage of subjects whose ALP blood level was reduced by ≥30 or ≥50% vs. baseline Fig. 3 Percentage changes from baseline in bone ALP levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles mean ± SD. ALP response rate was defined as the percentage of subjects whose ALP blood level was reduced by ≥30 or ≥50% vs. baseline

Total ALP levels in blood decreased from baseline to week 12 and EOT in all the cohorts (Table 5; Fig. 2). The total ALP response rates (≥30 and ≥50% reductions) were 54.5% (6/11) and 9.1% (1/11), respectively, at week 12, and 56.3% (9/16) and 25.0% (4/16), respectively, at EOT. Bone ALP levels also decreased from baseline to week 12 and EOT in all cohorts (Table 5; Fig. 3). Bone ALP response rates (≥30 and ≥50% reductions) were 81.8% (9/11) and 36.4% (4/11), respectively, at week 12, and 81.3% (13/16) and 50.0% (8/16), respectively, at EOT.Fig. 2 Percentage changes from baseline in total alkaline phosphatase (ALP) levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles Mean ± SD. ALP response rate was defined as the percentage of subjects whose ALP blood level was reduced by ≥30 or ≥50% vs. baseline Fig. 3 Percentage changes from baseline in bone ALP levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles mean ± SD. ALP response rate was defined as the percentage of subjects whose ALP blood level was reduced by ≥30 or ≥50% vs. baseline The mean percentage change of P1NP from baseline at week 12 and at EOT was −42.1 and −29.5%, respectively. As for the bone resorption markers, the mean percentage change of CTX-I from baseline at week 12 and at EOT was −20.8 and 35.9%, respectively, and that of ICTP was 14.3 and 69.2%, respectively (Table 5).

Fig. 3 Percentage changes from baseline in bone ALP levels after treatment with radium-223 dichloride at 50 kBq/kg (cohort 1 + expansion cohort, n = 16). Filled circles mean ± SD. ALP response rate was defined as the percentage of subjects whose ALP blood level was reduced by ≥30 or ≥50% vs. baseline The mean percentage change of P1NP from baseline at week 12 and at EOT was −42.1 and −29.5%, respectively. As for the bone resorption markers, the mean percentage change of CTX-I from baseline at week 12 and at EOT was −20.8 and 35.9%, respectively, and that of ICTP was 14.3 and 69.2%, respectively (Table 5). Discussion The safety results of this study show that radium-223 dichloride was well tolerated by the Japanese patients with CRPC and bone metastases who were enrolled in the trial. As such, these results are comparable with those from previous studies in Caucasian patients and confirm the safety results obtained in the early development studies (BC1-05, BC1-08) [17, 18] and the ALSYMPCA study [10]. Of the 19 subjects participating in the study, 18 experienced an AE during the study period, with anemia, diarrhea, and thrombocytopenia being the most frequently observed AEs. The severity of the AEs were grade 1 or 2 in most cases, and no grade 4 or 5 TEAEs were observed. None of the observed treatment-emergent SAEs or AEs that led to discontinuation of study treatment were considered to be drug related. In the ALYSYMPCA study, the overall incidence of AEs in the radium-223 dichloride arm was comparable to or lower than that in the placebo arm [10]. Myelosuppression was rare in patients enrolled in the ALSYMPCA study, with a similar incidence of anemia between patients receiving radium-223 dichloride and those receiving placebo (31% for all grades), and the incidence of thrombocytopenia and neutropenia was 12 and 5%, respectively, in the radium-223 dichloride arm, and 6 and 1%, respectively, in the placebo arm [10]. In a phase II study, radium-223 dichloride improved overall survival, while there were no drug-related AEs or long-term hematological toxicity reported during the 12- to 24-month follow-up period after treatment [12]. In addition, there were no significant differences between the radium-233 dichloride and placebo groups in hematological parameters [12]. Both total and bone ALP are known bone markers that are associated with the diagnosis of bone metastases, SRE outcomes, disease progression, and prognosis in cancer patients [19–22]. High levels of bone markers predict bone-related complications or SRE among cancer patients with bone metastases [23, 24]. In addition to increased risk of SRE occurrence, high bone ALP level before treatment is indicative of the progression of bone lesions and mortality [19].

and prognosis in cancer patients [19–22]. High levels of bone markers predict bone-related complications or SRE among cancer patients with bone metastases [23, 24]. In addition to increased risk of SRE occurrence, high bone ALP level before treatment is indicative of the progression of bone lesions and mortality [19]. Levels of total and bone ALP are significant predictors of prostate cancer-related death [21], and high bone ALP level is associated with shorter overall survival [25, 26]. In a retrospective analysis of the TAX327 study that included data from men with CRPC, bone metastases, and high baseline total ALP level who were receiving docetaxel or mitoxantrone, normalization of ALP level by day 90 predicted better survival while an increase in ALP level by day 90 predicted poor survival, both factors being independent of PSA decline [27]. Although the clinical significance of these bone markers is not well established, they do respond promptly and profoundly to bone-modulating agents (BMAs) and antineoplastic therapy, and this response appears to be associated with a favorable clinical outcome in patients with bone metastases [28].

of PSA decline [27]. Although the clinical significance of these bone markers is not well established, they do respond promptly and profoundly to bone-modulating agents (BMAs) and antineoplastic therapy, and this response appears to be associated with a favorable clinical outcome in patients with bone metastases [28]. From the biomarker analysis results in this study, total ALP levels in blood decreased by approximately 30% after the administration of radium-223 dichloride in all the cohorts at week 12, with approximately 55% of patients having a ≥30% reduction in total ALP. These results are comparable to those of the ALSYMPCA study: the mean percentage change in total ALP level from baseline at week 12 and EOT was −32.2 and −30.0%, respectively, and the ≥30% reductions of total ALP at week 12 and EOT were 46.9 and 60.1%, respectively [10]. No data on bone ALP are available in the ALSYMPCA study, but in this study the mean percentage change from baseline at week 12 and EOT was >45%, and a ≥30% reduction in bone ALP was seen in >81% of patients at week 12 and EOT. Since bone ALP is a specific marker for osteogenesis and is considered to be a reliable and established bone formation marker for prostate cancer with bone metastases [29], the decrease in the level of bone ALP of up to 50% during the treatment and high response rates are indicative of the anti-cancer activity of radium-223 dichloride against bone metastatic lesions, as well as the clinical benefit in this population.

ed bone formation marker for prostate cancer with bone metastases [29], the decrease in the level of bone ALP of up to 50% during the treatment and high response rates are indicative of the anti-cancer activity of radium-223 dichloride against bone metastatic lesions, as well as the clinical benefit in this population. Compared with markers of bone formation (total ALP, bone-specific ALP, P1NP), which were clearly decreased at 12 weeks after radium-223 dichloride administration (by ≥30%), the bone resorption markers CTX-1 and ICTP decreased to a lesser degree (by –20.8) or increased (by 14.3%), respectively. The lesser responsiveness of bone resorption markers is likely due to the use of BMAs, including denosumab and/or zoledronic acid, both prior to and during the study. BMAs inhibit bone resorption [30–33], and in cohort 1 and the expansion cohort, 12 of the 16 (75%) patients were pre-treated with BMAs before starting radium-223 dichloride therapy (data not shown). Pre-clinical studies have shown that bone-seeking α-emitters accumulate in the osteoblastic bone matrix [34]; therefore, the radium-223 dichloride-induced anti-tumor effects are expected to be concentrated in these lesions. The strengths of this study include the rigorous methodology (inclusion and exclusion criteria, as well as the consistency achieved via the standard protocols defined in the internal manuals). This study, despite its small sample size, does confirm the results observed in ALSYMPCA, which was a large and controlled study [10].

engths of this study include the rigorous methodology (inclusion and exclusion criteria, as well as the consistency achieved via the standard protocols defined in the internal manuals). This study, despite its small sample size, does confirm the results observed in ALSYMPCA, which was a large and controlled study [10]. Based on the results of the safety and efficacy analyses presented here, together with the report that there were no differences in the pharmacokinetics or the absorbed radiation dose in organs and tissues between Japanese and non-Japanese patients with CRPC and bone metastases receiving a single dose of radium-223 dichloride [35], the rational next step is to proceed to a Japanese phase II study for further efficacy evaluation. While the present study illustrates that treatment with radium-223 dichloride in Japanese patients decreased ALP, previous clinical trials in Caucasian patients have demonstrated that treatment of patients with CRPC and bone metastases with radium-223 dichloride confers a significant survival advantage, prolongs the time to SSEs and reduces the risk pf SSEs [10–12, 36]. Thus, further studies in Japanese patients should examine measures of survival and quality of life. 1 The Japanese Society of Nuclear Medicine (NA) manual for proper use in clinical trials relating to radionuclide therapy with radium-223 dichloride (Ra-223) injection.

Based on the results of the safety and efficacy analyses presented here, together with the report that there were no differences in the pharmacokinetics or the absorbed radiation dose in organs and tissues between Japanese and non-Japanese patients with CRPC and bone metastases receiving a single dose of radium-223 dichloride [35], the rational next step is to proceed to a Japanese phase II study for further efficacy evaluation. While the present study illustrates that treatment with radium-223 dichloride in Japanese patients decreased ALP, previous clinical trials in Caucasian patients have demonstrated that treatment of patients with CRPC and bone metastases with radium-223 dichloride confers a significant survival advantage, prolongs the time to SSEs and reduces the risk pf SSEs [10–12, 36]. Thus, further studies in Japanese patients should examine measures of survival and quality of life. 1 The Japanese Society of Nuclear Medicine (NA) manual for proper use in clinical trials relating to radionuclide therapy with radium-223 dichloride (Ra-223) injection. 2 The Japanese Society of Nuclear Medicine (NA) Addendum to the manual for proper use in clinical trials relating to radionuclide therapy with Ra-223 dichloride (Ra-223) injection: methods of calculating radiation shielding and levels of radioactivity in the atmosphere, exhaust air and exhaust fluid.

Introduction Chronic myeloid leukemia (CML), a neoplastic disorder of hematopoietic stem cells, is caused by a BCR-ABL1 fusion protein that results from t(9;22)(q43;q11). With the introduction of imatinib, a first-generation tyrosine kinase inhibitor (TKI), to inhibit BCR-ABL1 kinase, the outcome of chronic-phase CML (CP-CML) has improved dramatically [1, 2]. However, only up to 10–15% of CP-CML patients could achieve a deep molecular response (DMR) after 2 years of imatinib treatment [3, 4]. Although definitions of DMR are varied among clinical trials, it is considered to be BCR-ABL1 transcript levels <0.01% (MR4) on the International Scale (IS), as measured by the standardized quantitative real-time polymerase chain reaction (RQ-PCR) method [5]. Treatment with second-generation TKIs, dasatinib and nilotinib, has shown faster and deeper responses in newly diagnosed CP-CML patients than imatinib [6, 7]. Dasatinib is 325 times more potent than imatinib in the inhibition of BCR-ABL1 tyrosine kinase in vitro [8]. In addition, dasatinib shows unique immunological activity which is not observed in other TKIs. An increased number of large granular lymphocytes (LGLs) is observed in a substantial proportion of CML patients treated with dasatinib [9–12] and is associated with a superior clinical response [9–11, 13–15]. These facts indicate that dasatinib has immunological anti-leukemic effects in addition to the direct action on leukemic cells thorough BCR-ABL1 tyrosine kinase inhibition.

served in a substantial proportion of CML patients treated with dasatinib [9–12] and is associated with a superior clinical response [9–11, 13–15]. These facts indicate that dasatinib has immunological anti-leukemic effects in addition to the direct action on leukemic cells thorough BCR-ABL1 tyrosine kinase inhibition. Dasatinib is effective and induces a deeper response in CP-CML patients who do not achieve an optimal response with imatinib treatment. We hypothesized that dasatinib can induce DMR in CP-CML patients who achieve a major molecular response (MMR) corresponding to 0.1% IS (MR3), but not DMR with imatinib treatment, through more potent TKI activity and unique immunological properties related to anti-leukemic effects. Therefore, we conducted a clinical study in which dasatinib replaced imatinib in CP-CML patients who achieved MMR but not DMR after at least two years of imatinib treatment.

(MR3), but not DMR with imatinib treatment, through more potent TKI activity and unique immunological properties related to anti-leukemic effects. Therefore, we conducted a clinical study in which dasatinib replaced imatinib in CP-CML patients who achieved MMR but not DMR after at least two years of imatinib treatment. Patients and methods Patients Inclusion criteria for the study were patients with CP-CML who were aged ≥20 years, treated with imatinib for at least 24 months and achieved MMR with detectable levels of BCR-ABL1 transcripts by RQ-PCR, and a sensitivity of at least MR4 below the standardized line. Exclusion criteria were patients with a performance status (PS) of grade ≥3 by ECOG definition, with active second primary cancer, with clinically defined pleural effusion, with a history of critical cardiovascular events, including acute myocardial infarction within 6 months, angina pectoris within 3 months, and congestive heart failure within 3 months, with prolonged QTc of ≥450 ms or the possibility of congenital prolonged QT syndrome, with a history of treatment by dasatinib, or with ABL1 mutations (T315I, F317L and V299L) for which dasatinib is ineffective, at the time of study inclusion. Pregnant or breastfeeding women were also ineligible.

ilure within 3 months, with prolonged QTc of ≥450 ms or the possibility of congenital prolonged QT syndrome, with a history of treatment by dasatinib, or with ABL1 mutations (T315I, F317L and V299L) for which dasatinib is ineffective, at the time of study inclusion. Pregnant or breastfeeding women were also ineligible. RQ-PCR analysis for BCR-ABL1 transcripts The BCR-ABL1 transcript level in peripheral blood was monitored before and at 1, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib. To measure the BCR-ABL1 transcript level, the RQ-PCR method with at least MR4 sensitivity (i.e., 0.0069% on IS) was carried out by Bio Medical Laboratories (BML, Inc., Tokyo, Japan) as described previously [14, 16, 17]. In this analysis, we defined DMR as a peripheral major BCR-ABL1/ABL1 transcript ratio below the detection limit of RQ-PCR analysis. Screening for BCR-ABL1 mutations Before switching to dasatinib, we screened for 25 clinically important BCR-ABL1 mutations at 18 nucleotide positions by RT-PCR using PCR-Invador assay (BML, Inc., Tokyo, Japan) in all enrolled patients [18, 19].

RQ-PCR analysis for BCR-ABL1 transcripts The BCR-ABL1 transcript level in peripheral blood was monitored before and at 1, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib. To measure the BCR-ABL1 transcript level, the RQ-PCR method with at least MR4 sensitivity (i.e., 0.0069% on IS) was carried out by Bio Medical Laboratories (BML, Inc., Tokyo, Japan) as described previously [14, 16, 17]. In this analysis, we defined DMR as a peripheral major BCR-ABL1/ABL1 transcript ratio below the detection limit of RQ-PCR analysis. Screening for BCR-ABL1 mutations Before switching to dasatinib, we screened for 25 clinically important BCR-ABL1 mutations at 18 nucleotide positions by RT-PCR using PCR-Invador assay (BML, Inc., Tokyo, Japan) in all enrolled patients [18, 19]. Study design and treatment After confirmation of MMR with detectable levels of BCR-ABL1 transcripts by RQ-PCR, dasatinib therapy was initiated for eligible patients at a dose of 100 mg once a day. The study treatment was continued until disease progression or development of unacceptable adverse events. During the study, any treatment for CML other than dasatinib was not allowed. Therapy for comorbidities and/or adverse events was permitted. Interruption, dose reduction, or dose re-escalation of dasatinib due to adverse events were allowed.

inued until disease progression or development of unacceptable adverse events. During the study, any treatment for CML other than dasatinib was not allowed. Therapy for comorbidities and/or adverse events was permitted. Interruption, dose reduction, or dose re-escalation of dasatinib due to adverse events were allowed. Evaluation of efficacy The primary endpoint in this study was the achievement of DMR at 12 months after switching to dasatinib. Secondary endpoints included the dose intensity of dasatinib at 12 months, progression-free survival, safety profiles, and immunophenotypic alterations of peripheral lymphocytes after switching to dasatinib. Progressive disease was defined as loss of complete hematological response, loss of MMR, progression to accelerated/blastic phases, or death from any cause during the treatment period. Evaluation of safety Adverse events were monitored and assessed according to the Common Terminology Criteria for Adverse Events version 4.0 in all participating patients throughout the study. Chest X-ray was carried out to check pleural effusion at baseline, at 2 weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary. An ECG was carried out to screen for arrhythmia and to monitor the QTc interval at baseline, at 2 weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary.

weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary. An ECG was carried out to screen for arrhythmia and to monitor the QTc interval at baseline, at 2 weeks and at 1, 2, 3, 6, 9, 12, 15 and 18 months after switching to dasatinib or more frequently if necessary. Monitoring immunophenotypes in peripheral blood lymphocytes during dasatinib treatment Immunophenotypes of peripheral blood lymphocytes were monitored before, at 2 weeks and at 1, 3, 6, 9 and 12 months after switching to dasatinib by two- or three-color flow cytometry performed by BML Inc. using monoclonal antibodies against the following antigens—CD3, CD4, CD8, CD16, CD56, CD57, CD25 and CD127. Statistical analyses Differences in immunophenotypes in peripheral lymphocytes between paired samples before and at 6 months after switching to dasatinib were evaluated statistically by paired t test using JMP Pro 11.2 (SAS Institute Inc.). A statistically significant result was considered to have a P value of <0.05.

Monitoring immunophenotypes in peripheral blood lymphocytes during dasatinib treatment Immunophenotypes of peripheral blood lymphocytes were monitored before, at 2 weeks and at 1, 3, 6, 9 and 12 months after switching to dasatinib by two- or three-color flow cytometry performed by BML Inc. using monoclonal antibodies against the following antigens—CD3, CD4, CD8, CD16, CD56, CD57, CD25 and CD127. Statistical analyses Differences in immunophenotypes in peripheral lymphocytes between paired samples before and at 6 months after switching to dasatinib were evaluated statistically by paired t test using JMP Pro 11.2 (SAS Institute Inc.). A statistically significant result was considered to have a P value of <0.05. Ethics and study management The study was conducted by the Kanto CML study group, which consists of 13 institutions in Japan, according to the Declaration of Helsinki, and the protocol was reviewed by institutional review boards or ethics committees for each participating center. Before entry into the study, all candidates were informed about the aim of the present study, and also merits and demerits of switching to dasatinib. Merits include induction of deeper response and leading to future TKI discontinuation. Demerits include newly developed adverse events caused by dasatinib, although certain adverse events due to imatinib treatment may be solved. Written informed consent was obtained from all participating patients before registration. This trial was registered at www.clinicaltrials.gov as # NCT01342679.

discontinuation. Demerits include newly developed adverse events caused by dasatinib, although certain adverse events due to imatinib treatment may be solved. Written informed consent was obtained from all participating patients before registration. This trial was registered at www.clinicaltrials.gov as # NCT01342679. Results Patients and treatment A total of 19 patients were registered for the study from April 2011 to March 2013. Three patients were excluded from the study before switching to dasatinib treatment owing to withdrawal of consent. Subsequently, treatment was switched from imatinib to dasatinib in the remaining 16 patients (11 males and 5 females; median age 50 years, range 25−72 years). The characteristics of the 16 patients are summarized in Table 1. No BCR-ABL1 mutations were detected at 18 nucleotide positions in all 16 participating patients before switching to dasatinib. The median time from CML diagnosis to dasatinib treatment was 56 months (range 26−173 months). All patients were PS 0 when the dasatinib treatment started. For the current analysis, all patients had follow-up periods of at least 12 months after switching to dasatinib, and the median follow-up period was 27.6 months (range 12.9–38.5 months).Table 1 Characteristics of the 16 patients in the study Age (years) Median 50 Range 25–72 Sex Male/female 11/5 Time since diagnosis of CML (months) Median 56 Range 26–173 ECOG performance status, n (%) 0 16 (100) 1 0 (0) 2 0 (0) Base-line BCR-ABL1 transcript level (IS%) Median 0.014 Range 0.0054–0.078

Results Patients and treatment A total of 19 patients were registered for the study from April 2011 to March 2013. Three patients were excluded from the study before switching to dasatinib treatment owing to withdrawal of consent. Subsequently, treatment was switched from imatinib to dasatinib in the remaining 16 patients (11 males and 5 females; median age 50 years, range 25−72 years). The characteristics of the 16 patients are summarized in Table 1. No BCR-ABL1 mutations were detected at 18 nucleotide positions in all 16 participating patients before switching to dasatinib. The median time from CML diagnosis to dasatinib treatment was 56 months (range 26−173 months). All patients were PS 0 when the dasatinib treatment started. For the current analysis, all patients had follow-up periods of at least 12 months after switching to dasatinib, and the median follow-up period was 27.6 months (range 12.9–38.5 months).Table 1 Characteristics of the 16 patients in the study Age (years) Median 50 Range 25–72 Sex Male/female 11/5 Time since diagnosis of CML (months) Median 56 Range 26–173 ECOG performance status, n (%) 0 16 (100) 1 0 (0) 2 0 (0) Base-line BCR-ABL1 transcript level (IS%) Median 0.014 Range 0.0054–0.078 The median and average daily doses of dasatinib at 12 months after switching to dasatinib were 99.5 mg per day and 88.0 mg per day, respectively (range 25.6–100 mg per day), and eight patients received 100% doses of dasatinib. The median treatment duration was 550 days (range 120− 691 days). Two patients (13%) discontinued the treatment within 12 months because of adverse events and patient request.

satinib were 99.5 mg per day and 88.0 mg per day, respectively (range 25.6–100 mg per day), and eight patients received 100% doses of dasatinib. The median treatment duration was 550 days (range 120− 691 days). Two patients (13%) discontinued the treatment within 12 months because of adverse events and patient request. Efficacy The rates of achievement of DMR at 1, 3, 6 and 12 months after switching to dasatinib treatment in 16 patients were 44% (7/16), 56% (9/16), 63% (10/16) and 75% (12/16), respectively (Fig. 1). The cumulative rate of achieving DMR at 12 months was 93.8% (15/16) (Fig. 2). In six patients who achieved DMR, detectable levels of BCR-ABL1 transcripts were observed to have re-emerged during dasatinib treatment (Fig. 1). Among these six patients, three patients subsequently obtained DMR again by continuing dasatinib treatment. At 18 months after switching to dasatinib, 10 patients (55.6%) maintained DMR. The two patients (no. 10 and no. 13) who discontinued dasatinib within 12 months achieved DMR at 6 months, although RQ-PCR data at 12 months were not available. One of these two patients (patient no. 10) was switched to nilotinib treatment and maintained DMR at 35.5 months after the initiation of dasatinib treatment. No death or disease progression was observed during the study period, and progression-free survival at 12 months was 100%.Fig. 1 Undetectable level of BCR-ABL1 transcripts during dasatinib therapy. Results of monitoring levels of BCR-ABL1 fusion transcripts measured by quantitative RT-PCR with sensitivity of at least MR4.0 in 16 patients. Closed circles indicate detectable BCR-ABL1 transcripts. Open circles indicate undetectable BCR-ABL1 transcripts. Patients no. 10 and no. 13 discontinued dasatinib therapy within 12 months after switching to dasatinib

fusion transcripts measured by quantitative RT-PCR with sensitivity of at least MR4.0 in 16 patients. Closed circles indicate detectable BCR-ABL1 transcripts. Open circles indicate undetectable BCR-ABL1 transcripts. Patients no. 10 and no. 13 discontinued dasatinib therapy within 12 months after switching to dasatinib Fig. 2 Cumulative rate of achievement of DMR by dasatinib treatment Safety profile Therapy-related adverse events are summarized in Table 2. Hematologic toxicities were common adverse events, although grade 3 or 4 toxicities were less frequent. Anemia, neutropenia and thrombocytopenia were observed in 13 (81.3%), 8 (50%) and 8 (50%) patients, respectively. The most common non-hematologic adverse event was grade 1 or 2 pleural effusion, which occurred in 6 patients. The median time of appearance of pleural effusion after switching to dasatinib treatment was 9 months (range 0.5–12 months). The second most common non-hematologic adverse events were fever and skin rash, each of which occurred in 3 patients. Although grade 3 or 4 non-hematologic toxicities were rare, one male patient (patient no. 10) suffered from septicemia. He fully recovered, but subsequently discontinued dasatinib therapy. Dose reduction and/or treatment interruption due to adverse events was required in eight patients.Table 2 Therapy-related toxicities

lthough grade 3 or 4 non-hematologic toxicities were rare, one male patient (patient no. 10) suffered from septicemia. He fully recovered, but subsequently discontinued dasatinib therapy. Dose reduction and/or treatment interruption due to adverse events was required in eight patients.Table 2 Therapy-related toxicities Event All grades Grade 3 or 4 No. of patients % No. of patients % Hematologic toxicities Neutropenia 8 50 1 6.3 Thrombocytopenia 8 50 1 6.3 Anemia 13 81.3 3 18.8 Non-hematologic toxicities Pleural effusion 6 37.5 0 0 Fever 3 18.8 0 0 Rash 3 18.8 0 0 Diarrhea 2 12.5 0 0 Gastro-intestinal bleeding 2 12.5 0 0 Edema 2 12.5 0 0 Septicemia 1 6.3 1 6.3 Biochemical Elevated AST 7 43.8 0 0 Elevated ALT 5 31.3 0 0 Elevated BUN 3 18.8 0 0 Decreased albumin 3 18.8 0 0 Hyperkalemia 2 12.5 0 0 Hypokalemia 2 12.5 0 0 Any grade 3 or 4 toxicities and/or toxicities of all grades that occurred in at least 10% of the treated patients are listed in this table

epticemia 1 6.3 1 6.3 Biochemical Elevated AST 7 43.8 0 0 Elevated ALT 5 31.3 0 0 Elevated BUN 3 18.8 0 0 Decreased albumin 3 18.8 0 0 Hyperkalemia 2 12.5 0 0 Hypokalemia 2 12.5 0 0 Any grade 3 or 4 toxicities and/or toxicities of all grades that occurred in at least 10% of the treated patients are listed in this table Immunophenotypic changes in peripheral blood lymphocytes during dasatinib therapy Immunophenotypes of peripheral blood lymphocytes were analyzed by flow cytometry in 16 patients. The median CD4/CD8 ratios before and at 6 months after switching to dasatinib treatment were 1.74 and 1.11, respectively. The CD4/CD8 ratio decreased from baseline to 6 months after the switch with a statistically significant difference (P = 0.008) (Fig. 3a). The median percentages of natural killer cells, defined as CD3-CD56+ cells, in peripheral blood lymphocytes before and at 6 months after switching to dasatinib were 19.0 and 27.5%, respectively (P = 0.0009) (Fig. 3b). The proportion of NK T cells, defined as CD3+CD56+ lymphocytes, was also elevated during dasatinib treatment. A significant difference between the median percentages of NKT cells before and at 6 months after switching to dasatinib was also observed (median percentages 4.0 vs 5.2%, P = 0.0002) (Fig. 3c). The proportion of regulatory T cells (Tregs), defined as CD25+CD127+ cells, in CD4+ lymphocytes was analyzed by three-color flow cytometry before and at 6 months after treatment initiation. The median percentages of Tregs in CD4+ lymphocytes before and at 6 months after switching to dasatinib were 7.7 and 6.7%, respectively, with a statistically significant difference (P = 0.0123) (Fig. 3d).Fig. 3 Immunophenotype alterations in peripheral blood lymphocytes before and after 6 months of dasatinib treatment. (a) CD4/8 ratio, (b) percentages of NK (CD3−/CD56+) cells and (c) NK (CD3+/CD56+) T cells, (d) percentage of Treg in CD4 lymphocytes

ly, with a statistically significant difference (P = 0.0123) (Fig. 3d).Fig. 3 Immunophenotype alterations in peripheral blood lymphocytes before and after 6 months of dasatinib treatment. (a) CD4/8 ratio, (b) percentages of NK (CD3−/CD56+) cells and (c) NK (CD3+/CD56+) T cells, (d) percentage of Treg in CD4 lymphocytes Discussion Dasatinib induces a notable response in imatinib-intolerant and -resistant CP-CML patients and also a faster and deeper response in newly diagnosed CP-CML patients [4, 7, 20]. The present study further demonstrated that dasatinib rapidly induced DMR in most CP-CML patients who had received at least two years of imatinib treatment and obtained MMR but not DMR. Switching to nilotinib, a second-generation TKI, after long-term imatinib treatment has been previously reported, showing that nilotinib enabled more patients with CML-CP to gain a DMR compared to remaining on imatinib [21]. To our knowledge, this is the first report of switching to dasatinib from imatinib with the aim of achieving DMR.

inib, a second-generation TKI, after long-term imatinib treatment has been previously reported, showing that nilotinib enabled more patients with CML-CP to gain a DMR compared to remaining on imatinib [21]. To our knowledge, this is the first report of switching to dasatinib from imatinib with the aim of achieving DMR. There are several possible explanations for rapid DMR induction by dasatinib in CP-CML patients who achieved MMR, but not DMR by imatinib treatment. First, the strong TKI activity of dasatinib, which is 325 times more potent against BCR-ABL1 tyrosine kinase than imatinib in vitro, may contribute to decreasing the number of residual leukemic cells in patients treated with imatinib. In addition, dasatinib has different pharmacokinetics from imatinib. Decreased expression of human organic cationic transporter 1 (hOCT1) causes reduced influx of imatinib into leukemia cells, leading to decreased intracellular concentrations of imatinib and inferior clinical outcome in CP-CML patients treated with imatinib [22, 23]. In contrast, dasatinib is not affected by hOCT1 [24]. Although we did not analyze serum imatinib concentrations and hOCT1 expression in the present study, the different pharmacokinetics and pharmacodynamics between dasatinib and imatinib may be one of the reasons for the induction of DMR in the patients.

inib [22, 23]. In contrast, dasatinib is not affected by hOCT1 [24]. Although we did not analyze serum imatinib concentrations and hOCT1 expression in the present study, the different pharmacokinetics and pharmacodynamics between dasatinib and imatinib may be one of the reasons for the induction of DMR in the patients. In the molecular pathogenesis of CML, molecules other than BCR-ABL1 tyrosine kinase play certain roles. For example, dysregulation of SRC family kinases, mTOR, and p53 in CML cells has been reported, and these molecular pathways also mediate imatinib resistance [25–27]. Dasatinib inhibits multiple tyrosine kinases, including SRC family kinases, while imatinib does not [28]. Through inhibitory effects on CML cells as a multi-kinase inhibitor, dasatinib may induce a deeper response.

53 in CML cells has been reported, and these molecular pathways also mediate imatinib resistance [25–27]. Dasatinib inhibits multiple tyrosine kinases, including SRC family kinases, while imatinib does not [28]. Through inhibitory effects on CML cells as a multi-kinase inhibitor, dasatinib may induce a deeper response. Dasatinib has unique properties related to immunological anti-leukemic effects. Lymphocytosis, especially an increased proportion of LGLs, is often observed in peripheral blood during dasatinib therapy and is associated with a favorable response in CP-CML patients treated with dasatinib [9–11]. This phenomenon is rarely observed during treatment with other TKIs. The immunophenotypes of the increased LGLs are either CD3−CD56+ natural killer cells or CD3+CD8+ cytotoxic T cells. A possible hypothesis for the association between a favorable response and the increased proportion of LGLs is an immunological effect on leukemic cells mediated by natural killer and/or cytotoxic T cells induced by dasatinib. Indeed, a significant difference in the immunophenotypes of peripheral lymphocytes was observed before and after switching to dasatinib in this study, which may contribute to the achievement of DMR in patients who could not obtained it by imatinib treatment. Previously, it was shown that the proportion of Tregs, which are negative regulators of the immune response in peripheral blood, was decreased in dasatinib-treated CP-CML patients. In particular, this phenomenon was observed in patients with large granular lymphocytosis [29]. In the present study, the proportion of Tregs was decreased after switching to dasatinib. Although the mechanism has not been elucidated, the decreased proportion of Tregs may result in enhancing the anti-leukemic immune response.

ticular, this phenomenon was observed in patients with large granular lymphocytosis [29]. In the present study, the proportion of Tregs was decreased after switching to dasatinib. Although the mechanism has not been elucidated, the decreased proportion of Tregs may result in enhancing the anti-leukemic immune response. Toxicities from dasatinib therapy were generally not serious and were manageable. Hematologic toxicities in the present study were mostly comparable with previous studies, in which newly diagnosed CP-CML patients received dasatinib as a first-line therapy with median follow-up periods of 12 months [7, 30]. Interruption and/or dose reduction of dasatinib due to adverse events was required in half of the patients, which was comparable with previous reports [31, 32]. At 12 months, 14 of the 16 patients continued dasatinib treatment, indicating that switching from imatinib to dasatinib was tolerable in most patients. Grade 1or 2 pleural effusion occurred in six patients during the follow-up period and was manageable in all cases. Five of the six patients with pleural effusion achieved DMR at 12 months and subsequently maintained the DMR at 18 months, demonstrating that the appearance of mild pleural effusion could be associated with better clinical outcomes in dasatinib-treated patients, as reported previously [13].

was manageable in all cases. Five of the six patients with pleural effusion achieved DMR at 12 months and subsequently maintained the DMR at 18 months, demonstrating that the appearance of mild pleural effusion could be associated with better clinical outcomes in dasatinib-treated patients, as reported previously [13]. In clinical practice, MMR is defined as a treatment target of CP-CML according to the guidelines [33, 34]. It has not been fully elucidated whether induction of DMR leads to the improvement of longer clinical outcomes in CP-CML patients. Several studies demonstrated that CP-CML patients who obtained DMR by TKIs were less likely to lose MMR and showed better clinical outcomes, although the definition of DMR was different among the studies [35–37]. In the German CML-Study IV, MR4.5 at 4 years was associated with better overall survival than MR3 to MR2 (IS 1.0%). However, there were no statistical differences in survival probabilities between patients achieving MR4.5 and those achieving MR4 to MR3, which corresponds to MMR. In a study conducted by a French group, significant differences were reported in long-term clinical outcomes between the patients who obtained DMR and those with MMR but not DMR during the study [36]. More importantly, achieving DMR is a necessary requirement for discontinuation of TKI therapy, which is currently of great interest in CP-CML treatment. In clinical practice, lifelong TKI treatment is recommended for CP-CML patients who obtained an optimal response, unless severe adverse events occur [33, 34]. However, long-term continuous TKI treatment implies several issues in the patients. Low-grade non-serious toxicities, including edema, GI symptoms, and muscular clumps, which affect the quality of life of the patients, are not negligible [38]. Young female patients treated with TKI should give up being pregnant. It has been shown that TKI treatment is associated with an increased risk of cardiovascular events [39]. In addition, lifelong TKI treatment substantially results in a growing medical financial burden. A French group conducted a clinical trial for imatinib discontinuation in CP-CML patients who achieved DMR by imatinib and maintained it for at least two years [40]. The results indicated that approximately 40% of patients were in treatment-free remission without molecular relapse. Several other research groups conducted imatinib discontinuation studies, and similar results have been obtained [41–45].

ients who achieved DMR by imatinib and maintained it for at least two years [40]. The results indicated that approximately 40% of patients were in treatment-free remission without molecular relapse. Several other research groups conducted imatinib discontinuation studies, and similar results have been obtained [41–45]. Cessation of dasatinib treatment in CP-CML patients who obtained DMR has been reported by a Japanese group [17]. The achievement of DMR is essential for treatment discontinuation and treatment-free remission. However, only a small proportion of CP-CML patients substantially achieve DMR by imatinib treatment. The cumulative rate of DMR by 24 months and 36 months in CP-CML patients treated with standard doses of imatinib was up to 10 and 15%, respectively [3, 4]. The present results suggest that by switching to dasatinib, patients who achieved MMR but not DMR with imatinib treatment would become candidates for TKI discontinuation. In conclusion, switching to dasatinib would be a therapeutic option for CP-CML patients who achieved MMR but not DMR by imatinib therapy, especially for patients who wish to discontinue TKI therapy. A comment to this article is available at https://doi.org/10.1007/s10147-017-1214-y. Acknowledgements This study was supported by the Epidemiological and Clinical Research Information Network (ECRIN). We also thank Yumi Miyashita at ECRIN for collecting the data and Yoshinori Yamamoto at BML for analyzing the data.

In conclusion, switching to dasatinib would be a therapeutic option for CP-CML patients who achieved MMR but not DMR by imatinib therapy, especially for patients who wish to discontinue TKI therapy. A comment to this article is available at https://doi.org/10.1007/s10147-017-1214-y. Acknowledgements This study was supported by the Epidemiological and Clinical Research Information Network (ECRIN). We also thank Yumi Miyashita at ECRIN for collecting the data and Yoshinori Yamamoto at BML for analyzing the data. Compliance with ethical standards Conflict of interest TK received honoraria from Bristol-Myers Squibb, Novartis and Pfizer; SM received honorarium from Bristol-Myers Squibb; IK received a grant from Bristol-Myers Squibb. No other author has any conflict of interest related to this study.

Introduction Lung cancer is a malignant tumor with high morbidity and mortality and is the leading cause of cancer death worldwide [1]. In China, there is a similar situation regarding lung cancer, with official statistics showing that of 10 common cancers, lung cancer has the highest morbidity and mortality rates [2]. Depending on the different locations of the lesion, lung cancer can be divided into central type and peripheral type. According to the different biological characteristics, lung cancer can also be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) using the World Health Ognization (WHO) pathological staging system [3].

tions of the lesion, lung cancer can be divided into central type and peripheral type. According to the different biological characteristics, lung cancer can also be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) using the World Health Ognization (WHO) pathological staging system [3]. The central type of lung cancer refers to tumors that occur above the lung segment bronchus, and the peripheral type of lung cancer refers to tumors that occur below the lung segment bronchus. Peripheral lung cancer accounts for >70% of all lung cancers [4]. As a specific clinical and histological lung cancer, SCLC accounts for approximately 15–20% of all lung cancers [5]; it is characterized by short doubling time, early lymph node metastasis and distant metastasis. The incident rate of distant metastases is approximately 60–70% in those patients who were initially diagnosed as SCLC [6]. Although the annual incidence of SCLC in industrialized countries has decreased over the past 30 years, the incidence of SCLC has increased in countries where smoking prevalence remains high, such as Eastern Europe and Asia [7]. A multicenter epidemiological study conducted in China shows that the incidence of SCLC in all lung cancers increased from 13.7 to 18.3% between 2005 and 2010 [8].

creased over the past 30 years, the incidence of SCLC has increased in countries where smoking prevalence remains high, such as Eastern Europe and Asia [7]. A multicenter epidemiological study conducted in China shows that the incidence of SCLC in all lung cancers increased from 13.7 to 18.3% between 2005 and 2010 [8]. In SCLC, the rate of central type is approximately 70–85%. Generally, central type is not considered for surgical treatment. In contrast, the rate of peripheral type is approximately 15–30%. For these patients, surgical treatment may be useful to improve overall survival. Only 4–12% of tumors present as solitary nodules in all SCLCs [9], and for patients whose tumor is diagnosed as stage I (T1-2N0M0), there is a possibility to undergo surgery with curative intent [10]. For NSCLC, if the patient is strongly clinically suspected of having stage I or II cancer, a histological test is not required before surgery [11]. However, for peripheral lung cancer patients, the treatment for NSCLC and SCLC may be completely different. The operation is likely to be the best choice for early stage NSCLC, but unlike NSCLC, most SCLC patients cannot benefit from surgery [12]. It is a problem for clinicians to identify between these two diseases, and there is a necessity for a convenient and accurate diagnosis method.

nd SCLC may be completely different. The operation is likely to be the best choice for early stage NSCLC, but unlike NSCLC, most SCLC patients cannot benefit from surgery [12]. It is a problem for clinicians to identify between these two diseases, and there is a necessity for a convenient and accurate diagnosis method. The most inexpensive and effective noninvasive diagnostic facility for lung cancer, i.e., the CT scanner, has been installed in most hospitals in China. Although there are many studies on the CT features of peripheral SCLC (PSCLC) [4, 6, 9, 13–15], there is few case−control study investigating the differences between PSCLC and peripheral NSCLC (PNSCLC) images. To date, there is still no easy way to distinguish between PSCLC from PNSCLC on CT images. In this study, we will introduce a simple and effective diagnostic prediction model based on CT features for distinguishing between PSCLC and PNSCLC.

vestigating the differences between PSCLC and peripheral NSCLC (PNSCLC) images. To date, there is still no easy way to distinguish between PSCLC from PNSCLC on CT images. In this study, we will introduce a simple and effective diagnostic prediction model based on CT features for distinguishing between PSCLC and PNSCLC. Materials and methods Study population The retrospective database was from the Zhongnan Hospital of Wuhan University. All patients provided signed informed consent before undergoing the CT examination. We selected PSCLC and PNSCLC cases that were confirmed by pathological diagnosis at Zhongnan Hospital of Wuhan University from January 2004 to September 2014. Inclusion criteria were (a) preoperative and continuous CT scans of the chest had been performed, and (b) CT scans of the abdomen, bone scan, and magnetic resonance imaging (MRI) or CT scans of the brain had been performed to diagnose tumor metastasis. Exclusion criteria were (a) central type tumors detected by CT, and (b) patients who were previously diagnosed with other cancers.

s of the chest had been performed, and (b) CT scans of the abdomen, bone scan, and magnetic resonance imaging (MRI) or CT scans of the brain had been performed to diagnose tumor metastasis. Exclusion criteria were (a) central type tumors detected by CT, and (b) patients who were previously diagnosed with other cancers. Acquisition of CT images All CT features were performed with CT scanners (Somatom Sensation16 and Somatom Definition 64; Siemens Healthcare, Forchheim, Germany) in helical mode from the apex to the lung base. The patient was positioned in the supine position. Technical parameters were X-ray tube current 100 mA; tube voltage 120 kV; collimation 5 mm; rotation speed 0.5 s; matrix 512 × 512. All image data were interfaced directly to our picture archiving and communication system. Monitors were used to view both mediastinal and lung window images.

ioned in the supine position. Technical parameters were X-ray tube current 100 mA; tube voltage 120 kV; collimation 5 mm; rotation speed 0.5 s; matrix 512 × 512. All image data were interfaced directly to our picture archiving and communication system. Monitors were used to view both mediastinal and lung window images. CT image analysis Descriptions of imaging features conform to the Nomenclature Committee of the Fleischner Society (NCFS) chest image definition [16]. All CT images were reviewed by the same two senior radiologists, who assessed and recorded lesion size and shape, single/multifocal lesion, halo sign, lobulation sign, spiculated sign, calcification, cavity, air bronchogram, the bronchovascular convergence sign, pleural indentation, pleural effusion, lymphadenectasis, distant metastasis and other signs (see Fig. 1 for definitions).Fig. 1 CT features. a The halo sign is a CT finding of ground-glass opacity surrounding a nodule or mass. b The spiculated sign is when the edge of a nodule or mass extends to the surrounding lung parenchyma, which contains linear strands that extend into the tissue of the lung but not into the pleural margin. c The lobulation sign is when the edge of the nodule shows uneven lobulated contour. d The cavity sign is a gas-filled space, seen as a lucency or low-attenuation area, within a pulmonary consolidation, mass, or nodule. e The calcification showed high attenuation, the average CT value of non-enhanced CT is >100 Hu. f The air bronchogram is a pattern of air-filled (low-attenuation) bronchi on a background of an opaque (high-attenuation) airless lung. g Pleural indentation is showed as a tapered or linear extension of the lesion to pleura, reflect the pulmonary fibrosis with adjacent pleural retraction. h The bronchovascular convergence sign is when one or more vessels reach the edge of the tumor or cross the tumor. i Lymphadenectasis means the size of a mediastinal or hilar lymph node is >1 cm in short axis diameter

ar extension of the lesion to pleura, reflect the pulmonary fibrosis with adjacent pleural retraction. h The bronchovascular convergence sign is when one or more vessels reach the edge of the tumor or cross the tumor. i Lymphadenectasis means the size of a mediastinal or hilar lymph node is >1 cm in short axis diameter Reference standard Peripheral lung cancer in this study was determined using the pathological results from various pathological biopsies. All the specimens were performed by immunohistochemistry and diagnosed as SCLC or NSCLC.

ar extension of the lesion to pleura, reflect the pulmonary fibrosis with adjacent pleural retraction. h The bronchovascular convergence sign is when one or more vessels reach the edge of the tumor or cross the tumor. i Lymphadenectasis means the size of a mediastinal or hilar lymph node is >1 cm in short axis diameter Reference standard Peripheral lung cancer in this study was determined using the pathological results from various pathological biopsies. All the specimens were performed by immunohistochemistry and diagnosed as SCLC or NSCLC. Statistical analysis To evaluate the predictive factors for the pathological types of peripheral lung cancers, statistical differences were analyzed using the Mann–Whitney Wilcoxon test for continuous data and chi-squared test for categorical data, using a significance level of P = 0.05. To identify significant predictors of CT features in PSCLC, multivariate logistic regression analysis was conducted (forward LR method). The removal of variables was based on likelihood ratio statistics with a probability of 0.10. The regression coefficients of significant CT features which were detected by multivariate logistic regression analysis were regarded as independent variable factors, multiplied by ten and rounding to the nearest integer. We calculated all significant features together in every peripheral lung cancer patient using these numbers as scores. We then took the sum of the scores to draw receiver operating characteristic (ROC) curves and calculated the available area under the curve (AUC). We used the Youden index to calculate the best cut-off.

e calculated all significant features together in every peripheral lung cancer patient using these numbers as scores. We then took the sum of the scores to draw receiver operating characteristic (ROC) curves and calculated the available area under the curve (AUC). We used the Youden index to calculate the best cut-off. Results Between January 2004 and September 2015 (the date of patients diagnosed with non-small cell lung cancer ranged from September 2014 to September 2015), 655 patients were diagnosed with lung cancer. The screening criterion was untreated primary lung cancer patients with completed CT and pathological data. There were 220 cases of SCLC (169 cases of central type, 51 cases of peripheral type) and 435 cases of NSCLC (226 cases of central type, 207 cases of peripheral type). In 207 cases of PNSCLC, there were 48 squamous cell carcinomas, 139 adenocarcinomas, seven low differentiated carcinomas, five sarcomatoid carcinomas, one neuroendocrine carcinoma (non-small cell), three adenosquamous carcinomas and four undifferentiated non-small cell lung cancer. According to the TNM for the 7th edition of the AJCC staging system, there were 11 cases of stage I, 10 cases of stage II, 10 cases of stage III and 20 cases of stage IV in 51 cases of PSCLC; there were 59 cases of stage I, 29 cases of stage II, 31 cases of stage III and 88 cases of stage IV in 207 cases of PNSCLC.

Results Between January 2004 and September 2015 (the date of patients diagnosed with non-small cell lung cancer ranged from September 2014 to September 2015), 655 patients were diagnosed with lung cancer. The screening criterion was untreated primary lung cancer patients with completed CT and pathological data. There were 220 cases of SCLC (169 cases of central type, 51 cases of peripheral type) and 435 cases of NSCLC (226 cases of central type, 207 cases of peripheral type). In 207 cases of PNSCLC, there were 48 squamous cell carcinomas, 139 adenocarcinomas, seven low differentiated carcinomas, five sarcomatoid carcinomas, one neuroendocrine carcinoma (non-small cell), three adenosquamous carcinomas and four undifferentiated non-small cell lung cancer. According to the TNM for the 7th edition of the AJCC staging system, there were 11 cases of stage I, 10 cases of stage II, 10 cases of stage III and 20 cases of stage IV in 51 cases of PSCLC; there were 59 cases of stage I, 29 cases of stage II, 31 cases of stage III and 88 cases of stage IV in 207 cases of PNSCLC. CT features and scoring Table 1 shows the CT features of all patients who were diagnosed with peripheral lung cancer. We analyzed the size of peripheral lung nodules and characteristic CT features. By measuring and comparing the size of the lesions, it was found that the size of peripheral lung cancer lesions did not conform to normal distribution—the median was 40 mm in PSCLC, and 37 mm in PNSCLC (P > 0.05), meaning that there was no difference between PSCLC and PNSCLC in lesion size. Statistical analysis showed that six CT features were significant in the diagnosis (P < 0.01) (Table 1).Table 1 Comparison of the CT features between PSCLC and PNSCLC

distribution—the median was 40 mm in PSCLC, and 37 mm in PNSCLC (P > 0.05), meaning that there was no difference between PSCLC and PNSCLC in lesion size. Statistical analysis showed that six CT features were significant in the diagnosis (P < 0.01) (Table 1).Table 1 Comparison of the CT features between PSCLC and PNSCLC CT feature PSCLC (%) PNSCLC (%) Chi-squared P Shape* 18.46 <0.001 Round/ovoid shape 30 (58.8) 113 (54.6) Irregular shape 7 (13.7) 76 (36.7) Multinodular shape 14 (27.5) 18 (8.7) Single lesion 44 (86.3) 188 (90.8) 0.933 0.334 Halo sign 5 (9.80) 13 (6.3) 0.334 0.563 Spiculated sign* 28 (54.9) 178 (86.0) 24.575 0.000 Lobulation sign 42 (82.4) 172 (83.1) 0.016 0.900 Cavity* 21 (41.2) 149 (72.0) 17.276 0.000 Calcification 10 (19.6) 28 (13.5) 1.205 0.272 A|ir bronchogram 5 (9.8) 36 (17.4) 1.762 0.184 Pleural indentation* 19 (37.3) 139 (67.1) 15.406 0.000 Bronchovascular convergence sign* 26 (51.0) 128 (81.5) 18.689 0.000 Pleural effusion 5 (9.8) 19 (9.2) 0.000 1 Lymphadenectasis* 31 (60.8) 82 (39.6) 7.451 0.006 Distant metastasis 20 (39.2) 88 (42.5) 0.183 0.669 Total 51 207 * Differences were statistically significant

ural indentation* 19 (37.3) 139 (67.1) 15.406 0.000 Bronchovascular convergence sign* 26 (51.0) 128 (81.5) 18.689 0.000 Pleural effusion 5 (9.8) 19 (9.2) 0.000 1 Lymphadenectasis* 31 (60.8) 82 (39.6) 7.451 0.006 Distant metastasis 20 (39.2) 88 (42.5) 0.183 0.669 Total 51 207 * Differences were statistically significant Multivariate logistic regression analysis was performed to identify the diagnosis effect of the 6 CT features (shape, spiculated sign, cavity, pleural indentation, bronchovascular convergence sign and lymphadenectasis) with statistical significance. It had a tendency toward PSCLC diagnosis with the CT features of lymphadenectasis and multinodular shape (P < 0.05); however, irregular shape, spiculated sign, cavity and pleural indentation sign had a higher tendency toward PNSCLC diagnosis (P < 0.05). In order to facilitate the clinical application, we set up a mathematical model based on the results of two logistic regression analyses, multiplied the β-coefficients of the significant CT feature by 10 and rounded to the nearest integer for analysis (Table 2). The scores of each CT feature were added together to get the final score of each patient, and we then obtained a group of continuous variables with a score of −51 to 23. The final score was chosen as variable, and the pathological diagnosis as the dependent variable (1 = PSCLC, 0 = PNSCLC), using the statistical software MedCalc to draw the ROC curve (Fig. 2); the AUC was 0.834 (95%CI 0.783–0.877). The maximum value of the Youden index was 0.5584, which corresponded to the most reasonable cut-off of −24. When the final score was more than −24, the diagnosis was PSCLC, and the sensitivity and specificity were 86.3% (95%CI 0.737−0.943) and 69.6% (95%CI 0.628−0.758), respectively.Table 2 The image scoring prediction model of CT features analysis based on logistic regression for diagnosis of peripheral lung cancer

of −24. When the final score was more than −24, the diagnosis was PSCLC, and the sensitivity and specificity were 86.3% (95%CI 0.737−0.943) and 69.6% (95%CI 0.628−0.758), respectively.Table 2 The image scoring prediction model of CT features analysis based on logistic regression for diagnosis of peripheral lung cancer Variable β-Coefficients P value OR (95% CI) Score Multinodular shape 1.208 0.012 3.345 (1.310–8.546) 12 Irregular shape −1.256 0.012 0.285 (0.107–0.758) −13 Spiculated sign −1.386 0.001 0.250 (0.114–0.551) −14 Cavity −1.289 0.001 0.275 (0.132–0.576) −13 Pleural indentation −1.107 0.003 0.331 (0.159–0.690) −11 Lymphadenectasis 1.081 0.005 2.947 (1.382–6.287) 11 Fig. 2 ROC of radiographic scores of peripheral lung cancer on pathological types Discussion Since many surgeons perform surgery without invasive examinations, CT inspection plays an important role in the diagnosis and treatment of SCLC. Although it has been stated that peripherally located SCLC often shows different CT features from other lung cancers [15], there is no unified standard model to evaluate if a CT feature is related to PSCLC. In such an environment, the image scoring prediction model that we have invented can distinguish PSCLC from PNSCLC easier than other noninvasive methods.

rally located SCLC often shows different CT features from other lung cancers [15], there is no unified standard model to evaluate if a CT feature is related to PSCLC. In such an environment, the image scoring prediction model that we have invented can distinguish PSCLC from PNSCLC easier than other noninvasive methods. PSCLC tumors are always described as having homogeneous density, with rounded lobulated nodules and smooth edges in CT images, with little spiculated sign, pleural indentation and internal necrosis [13]. Many articles have studied the CT features of PSCLC and drawn some conclusions [4, 6, 9, 13–15, 17–19], however, their conclusions were varied because of the lack of uniform CT features and the small number of cases and case−control studies for PNSCLC. Most important, however, the conclusions they made were subjective and lacked generality and accuracy. Nevertheless, they also provided many clues from which we were able to draw conclusions to use as a foundation. We then improved the evaluation method and invented a radiological score system to make the diagnosis accurate, simple and practical.

conclusions they made were subjective and lacked generality and accuracy. Nevertheless, they also provided many clues from which we were able to draw conclusions to use as a foundation. We then improved the evaluation method and invented a radiological score system to make the diagnosis accurate, simple and practical. We established a simple and practical mathematical model for CT imaging to distinguish between PSCLC and PNSCLC. This model is based on the pathological type of peripheral lung cancer. The scoring options of the system comprise five CT features—shape, spiculated sign, cavity, pleural indentation and lymphadenectasis, which were selected from thirteen common CT features of peripheral lung cancer. It had a tendency toward PSCLC diagnosis with the CT features of multinodular shape and lymphadenectasis; however, irregular shape, spiculated sign, cavity and pleural indentation sign had a higher tendency toward PNSCLC diagnosis.

ectasis, which were selected from thirteen common CT features of peripheral lung cancer. It had a tendency toward PSCLC diagnosis with the CT features of multinodular shape and lymphadenectasis; however, irregular shape, spiculated sign, cavity and pleural indentation sign had a higher tendency toward PNSCLC diagnosis. In our study, the shape of the peripheral pulmonary nodules had important implications for diagnosing the pathology of tumors. According to the CT, there are three shape characteristics for PSCLC tumors—round/ovoid shape, irregular shape and multinodular shape. Although the round/ovoid shape might be the most common type of PSCLC [15, 20], it is also a common type of PNSCLC and therefore cannot be used as a feature to distinguish between these two diseases. The multinodular shape, defined as spindle-like and whose major axis points to the hilum, performs as a polymer that is aggregated by two or more nodules (Fig. 3), and is characteristic of PSCLC. In previous literature, the multinodular shape was often described as fusiform, vermiform, beaded, etc. [5, 15]. The multinodular shape might be related to the growth pattern of tumors that grow along the bronchial/blood vessel wall with short doubling time. Under these circumstances, the tumor is oppressed by the surrounding tissues, and it grows unbalanced. When the tumor grows surrounding bronchioles, it manifests as adjacent multiple nodules in the axial, which may be the reason why PSCLC is multinodular.Fig. 3 The shape of multinodular tumors

with short doubling time. Under these circumstances, the tumor is oppressed by the surrounding tissues, and it grows unbalanced. When the tumor grows surrounding bronchioles, it manifests as adjacent multiple nodules in the axial, which may be the reason why PSCLC is multinodular.Fig. 3 The shape of multinodular tumors Lymphadenectasis is also reported as a significant CT feature of PSCLC. For histological consideration, the boundary of SCLC is not clear, as it grows as a cluster or nest aggregation pattern, with lack of fibrous tissue inside [3]. The tumor grows along the small bronchial wall or the bronchial artery and can easily invade the lung parenchyma; this expression may be related to local progression of the tumor, including hilar and mediastinal lymph node metastasis [15]. In our study, pleural indentation is a feature that can be diagnosed as being characteristic of PNSCLC. The reason can be revealed by histologic examination which described that the tumor cells of SCLC tend to grow and form clusters or nests in the peripheral alveoli. This conclusion was similar to the results of Nobukata et al. [20], and this may be the reason why cavity formation had a tendency toward PNSCLC diagnosis. An important finding in our study is that TNM staging is not a feature for distinguishing between PSCLC and PNSCLC; this is another one of the reasons why PSCLC is hard to detect from peripheral lung cancer. This finding also gives indirect evidence to the importance of our image scoring system model.

In our study, pleural indentation is a feature that can be diagnosed as being characteristic of PNSCLC. The reason can be revealed by histologic examination which described that the tumor cells of SCLC tend to grow and form clusters or nests in the peripheral alveoli. This conclusion was similar to the results of Nobukata et al. [20], and this may be the reason why cavity formation had a tendency toward PNSCLC diagnosis. An important finding in our study is that TNM staging is not a feature for distinguishing between PSCLC and PNSCLC; this is another one of the reasons why PSCLC is hard to detect from peripheral lung cancer. This finding also gives indirect evidence to the importance of our image scoring system model. The image scoring system model converted the CT features of peripheral lung cancer to qualitative data through logistic regression and ROC curve analysis (Table 2). When the final score was more than −24, the diagnosis was PSCLC, and the sensitivity and specificity were 86.3 and 69.6%, respectively. The AUC was 0.834, indicating that the accuracy of the diagnosis was moderate. There are several limitations in our model. First, as a retrospective design, some bias may exist in our study. Second, the decision on whether patients had distant metastasis was based on the results of clinical and imaging studies instead of pathological investigation. Third, some CT features, such as nodules <1 cm in both PSCLC and PNSCLC, were not typical CT findings. All of these factors might lead to bias in diagnosis.

dy. Second, the decision on whether patients had distant metastasis was based on the results of clinical and imaging studies instead of pathological investigation. Third, some CT features, such as nodules <1 cm in both PSCLC and PNSCLC, were not typical CT findings. All of these factors might lead to bias in diagnosis. In conclusion, we established a scoring system using the CT features of peripheral lung cancer, which provided a basis for clinical treatment selection, and was also a simple and economical method for the noninvasive diagnosis of lung cancer patients. Abbreviations SCLCSmall cell lung cancer NSCLCNon-small cell lung cancer PSCLCPeripheral small cell lung cancer PNSCLCPeripheral non-small cell lung cancer 95% CI95% Confidence interval CTComputed tomography MRIMagnetic resonance imaging PETPositron emission tomography NCFSNomenclature Committee of the Fleischner Society ROCReceiver operating characteristics curves AUCAvailable area under the curve NCCNNational Comprehensive Cancer Network Yanchen Ren and Yiyuan Cao contributed equally to this article. Acknowledgements The authors gratefully acknowledge the excellent technical support of the oncology staff and the radiology staff at Zhongnan Hospital of Wuhan University. Compliance with ethical standards Availability of data and materials All data generated or analyzed during this study are included in this published article. Conflict of interest The authors declare that they have no competing interests. Funding Key projects of Hubei provincial health and Family Planning Commission WJ2017Z006.

Introduction Neoadjuvant chemotherapy is widely employed for patients with locally advanced breast cancer and, sometimes, even in those with operable breast cancer for the purpose of downstaging to facilitate breast-conserving surgery. Survival outcomes of such a strategy were reported to be comparable to those of adjuvant chemotherapy [1]. Patients who achieve pathological complete response (pCR) are known to have better long-term survival than those who achieve a lesser-grade response. Especially in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who received chemotherapy in combination with trastuzumab, an increase in pCR rate was directly related to improved survival outcomes [2, 3]. The significant improvement in outcomes of HER2-positive breast cancer patients achieved with the synergistic use of anti-HER2 drugs with cytotoxic agents in the neoadjuvant setting has prompted the evaluation of various regimens [3–5].

Introduction Neoadjuvant chemotherapy is widely employed for patients with locally advanced breast cancer and, sometimes, even in those with operable breast cancer for the purpose of downstaging to facilitate breast-conserving surgery. Survival outcomes of such a strategy were reported to be comparable to those of adjuvant chemotherapy [1]. Patients who achieve pathological complete response (pCR) are known to have better long-term survival than those who achieve a lesser-grade response. Especially in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer who received chemotherapy in combination with trastuzumab, an increase in pCR rate was directly related to improved survival outcomes [2, 3]. The significant improvement in outcomes of HER2-positive breast cancer patients achieved with the synergistic use of anti-HER2 drugs with cytotoxic agents in the neoadjuvant setting has prompted the evaluation of various regimens [3–5]. A regimen including docetaxel, carboplatin, and trastuzumab (TCbH) has shown promising results in some clinical trials [6–8]. In a large randomized trial (the Breast Cancer International Research Group 006, BCIRG-6), patients with HER2-positive early breast cancer were assigned doxorubicin and cyclophosphamide followed by docetaxel every 3 weeks (AC-T), the same regimen plus trastuzumab (AC-TH), or TCbH; rates of disease-free survival at 5 years were 75, 84, and 81%, respectively, and rates of overall survival were 87, 92, and 91%, respectively [9]. The latter two regimens, which both contained trastuzumab, showed equivalent clinical results. Further, both were found to be superior to AC-T. Owing to the significantly lower rates of congestive heart failure and cardiac dysfunction observed with TCbH, it is expected to become an alternative to the AC-TH regimen.

9]. The latter two regimens, which both contained trastuzumab, showed equivalent clinical results. Further, both were found to be superior to AC-T. Owing to the significantly lower rates of congestive heart failure and cardiac dysfunction observed with TCbH, it is expected to become an alternative to the AC-TH regimen. TCbH has shown promising results in the neoadjuvant setting [10]. In a multicenter neoadjuvant study, GETN(A)-1, 70 patients with HER2-positive stage II/III operable breast cancer received docetaxel (75 mg/m2) and carboplatin [area under curve (AUC) of 6] every 3 weeks plus weekly trastuzumab (initial dose 4 mg/kg, followed by 2 mg/kg). The pCR rate (ypT0/isypN0) was 39%, which translated into an objective response rate (ORR) of 95%, and the breast conservation rate was 64% [11]. To the best of our knowledge, the efficacy and safety of the TCbH regimen in the neoadjuvant setting has not been evaluated among Japanese women with HER2-positive breast cancer. We conducted a neoadjuvant phase II clinical trial of docetaxel (75 mg/m2) and carboplatin (AUC of 6) in combination with trastuzumab (loading dose 8 mg/kg, followed by 6 mg/kg) administered every 3 weeks to Japanese women with stage 1–3 non-inflammatory breast cancer. In this report, we present the efficacy and safety profiles from this study, based on intention-to-treat analysis of data from all 50 patients.

UC of 6) in combination with trastuzumab (loading dose 8 mg/kg, followed by 6 mg/kg) administered every 3 weeks to Japanese women with stage 1–3 non-inflammatory breast cancer. In this report, we present the efficacy and safety profiles from this study, based on intention-to-treat analysis of data from all 50 patients. Patients and methods This prospective clinical study was conducted among treatment-naïve Japanese women (age group 18–75 years) with unilateral breast cancer (T1-3, N0-2, M0), which was confirmed to be HER2-positive invasive carcinoma on histopathological examination. Eligibility criteria included: Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; adequate bone marrow reserve (absolute neutrophil count >1500/µL; platelet count >100,000/µL), renal function (serum creatinine <1.5 × upper limit of normal), and hepatic function (total bilirubin <1.5 × upper limit of normal); left ventricular ejection fraction (LVEF) within normal limits by echocardiography. Exclusion criteria were: patients who had bilateral or metastatic breast disease, inflammatory breast cancer; pregnant or lactating women; another histological subtype or neoplasm; history of atrial or ventricular arrhythmia, congestive heart failure, myocardial infarction; psychiatric disorders; active infection.