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Ewing sarcoma (ES) is the second most common bone malignancy in children and young adults, with a slight male predominance.1 ES has a high propensity to metastasize, and about 25% of patients show disseminated involvement at the beginning of disease. Multimodal treatment approaches, including surgery, radiotherapy (RT), and intensive multidrug chemotherapy, have led to notable improvement in the outcome of patients with localized disease, up to about 70% of event-free survival (EFS).2,3 In contrast, the prognosis of patients with primary disseminated disease remains very poor, with an EFS of <20%.4 Patients with primary pulmonary metastases show a better outcome than patients with primary bone and/or bone marrow involvement.4–8 Conventional treatment regimens for localized ES generally consist of a combination of vincristine, actinomycin-D, cyclophosphamide, doxorubicin, ifosfamide, and etoposide. Adjunctive surgical resection with or without radiation therapy is used for local control. Most episodes of disease recurrence occur after completion of therapy, and most recurrences (approximately 80%) occur within 2 years from initial diagnosis.9 Cisplatin (CDDP) and other platinum compounds are widely used drugs for the treatment of solid tumors in adults and children, especially when failure of first-line therapy occurs, but data concerning its activity and efficacy are very limited in ES. We describe an impressive response to front-line CDDP as a single drug in a child with metastatic ES with a favorable clinical outcome.

Cisplatin (CDDP) and other platinum compounds are widely used drugs for the treatment of solid tumors in adults and children, especially when failure of first-line therapy occurs, but data concerning its activity and efficacy are very limited in ES. We describe an impressive response to front-line CDDP as a single drug in a child with metastatic ES with a favorable clinical outcome. CASE REPORT A 10-year-old African boy was admitted 10 months after the onset of left elbow pain, followed by progressive swelling. He was diagnosed with an osteosarcoma (small cell variant) of the left arm in a Nigerian Hospital. He did not receive any therapy in Nigeria because they concentrate the few economical resources available on patients with more chances of healing. An international committee organized a transfer abroad to verify the histologic diagnosis and to explore the possibility of a therapeutic approach. On admission the boy showed a huge mass (circumference 43 cm, whereas the contralateral was 15 cm) in the left elbow, and packed lymph nodes, sized 6×8 cm, were palpable in the left armpit (Figs. 1A, B). Other enlarged lymph nodes were palpable just over the left collar-bone. A painful subcutaneous nodule, sized 3×2 cm, was palpable in the left shoulder blade region. The left knee compression was painful. The abdomen, heart, and chest were unremarkable; no hepatosplenomegaly or skin lesions were observed.

Figs. 1A, B). Other enlarged lymph nodes were palpable just over the left collar-bone. A painful subcutaneous nodule, sized 3×2 cm, was palpable in the left shoulder blade region. The left knee compression was painful. The abdomen, heart, and chest were unremarkable; no hepatosplenomegaly or skin lesions were observed. FIGURE 1 A, On admission the boy presented an impressive huge mass of the left elbow, having a circumference of about 43 cm. B, Packed lymph nodes, sized 6×8 cm, were palpable in the left armpit. Laboratory findings showed lactic dehydrogenase levels to be 1951 U/L (normal value <480 U/L). A computed tomography (CT) scan showed a large osteolytic lesion of the distal humerus and proximal radius and ulna surrounded by proliferating soft tissue. Total body CT scan and 99Tc-MDP bone scan showed a large mass of metastatic packed lymph nodes in the left armpit and skeletal metastases to the proximal region of the left humerus, to the fourth left rib, on the inferior region of the left shoulder blade and on the left knee. Bone marrow biopsy and aspirate were normal. An open biopsy of the elbow mass was done. Histologically, the tumor was characterized by a monomorphic proliferation of small round cells with regular nuclei, finely dispersed chromatin, and inconspicuous nucleoli (Figs. 2A, B). Cytoplasm was scant and slightly eosinophilic with no matrix production. The tumor grew in a diffuse and sheet-like pattern infiltrating the cortical bone and lamellar bone of periosteal reaction and assumed lobular and filigree pattern in soft tissue invasion with necrosis (Fig. 2C).

onspicuous nucleoli (Figs. 2A, B). Cytoplasm was scant and slightly eosinophilic with no matrix production. The tumor grew in a diffuse and sheet-like pattern infiltrating the cortical bone and lamellar bone of periosteal reaction and assumed lobular and filigree pattern in soft tissue invasion with necrosis (Fig. 2C). FIGURE 2 A and B, The tissue was characterized by a monomorphic proliferation of small round cells with regular nuclei, finely dispersed chromatin, and inconspicuous nucleoli. The cytoplasm was scant and slightly eosinophilic with no matrix production. C, The tumor grew in a diffuse and sheet-like pattern infiltrating the cortical bone and lamellar bone of periosteal reaction and assumed a lobular and filagree pattern in soft tissue invasion with necrosis. D, The cells showed strong and diffuse membranous immunoreactivity toward cell-surface glycoprotein CD99. The cells showed strong and diffuse membranous immunoreactivity toward cell-surface glycoprotein CD99 (Fig. 2D). Reverse transcriptase polymerase chain reaction analysis of ES was performed from paraffin-embedded tissue. Molecular studies showed the characteristic translocation leading to a fusion of the ES gene (EWS) on 22q12 as to a member of the ES family transcription factors, which in our case was FLI1.

The cells showed strong and diffuse membranous immunoreactivity toward cell-surface glycoprotein CD99 (Fig. 2D). Reverse transcriptase polymerase chain reaction analysis of ES was performed from paraffin-embedded tissue. Molecular studies showed the characteristic translocation leading to a fusion of the ES gene (EWS) on 22q12 as to a member of the ES family transcription factors, which in our case was FLI1. In conclusion, a diagnosis of ES with multiple skeletal and lymph node metastases was reached. The patient was enrolled in a joint trial of ISG and AIEOP for patients with multicenter ES at onset, called ISG-AIEOP/VHR-EW 02 protocol, consisting of a front-line with 2 courses of CDDP 100 mg/m2 by 48-hour continuous infusion 3 weeks apart, followed by 8 courses of combined chemotherapy, a consolidation phase with myeloablative busulfan+melphalan, and local treatment on the site of the primary tumor, according to the same strategy adopted in the previous ISG/AIEOP VHR-EW 01 protocol, in which the front-line consisted of melphalan.10 The front-line CDDP courses were well tolerated without any severe side effect. After the second course of therapy with CDDP, the elbow circumference decreased to 28 cm (Fig. 3A); supraclavicular lymph nodes disappeared and axillary reduced to <1 cm. Levels of lactic dehydrogenase fell below 500 IU/L.

In conclusion, a diagnosis of ES with multiple skeletal and lymph node metastases was reached. The patient was enrolled in a joint trial of ISG and AIEOP for patients with multicenter ES at onset, called ISG-AIEOP/VHR-EW 02 protocol, consisting of a front-line with 2 courses of CDDP 100 mg/m2 by 48-hour continuous infusion 3 weeks apart, followed by 8 courses of combined chemotherapy, a consolidation phase with myeloablative busulfan+melphalan, and local treatment on the site of the primary tumor, according to the same strategy adopted in the previous ISG/AIEOP VHR-EW 01 protocol, in which the front-line consisted of melphalan.10 The front-line CDDP courses were well tolerated without any severe side effect. After the second course of therapy with CDDP, the elbow circumference decreased to 28 cm (Fig. 3A); supraclavicular lymph nodes disappeared and axillary reduced to <1 cm. Levels of lactic dehydrogenase fell below 500 IU/L. FIGURE 3 A, The boy showed a dramatic response to 2 courses of cisplatin therapy; elbow circumference reduced to 28 cm and the armpit lymphadenopathy regressed completely. B, Evaluation after the last course of chemotherapy, before the myeloablative treatment, showed an elbow circumference of 17 cm.

The front-line CDDP courses were well tolerated without any severe side effect. After the second course of therapy with CDDP, the elbow circumference decreased to 28 cm (Fig. 3A); supraclavicular lymph nodes disappeared and axillary reduced to <1 cm. Levels of lactic dehydrogenase fell below 500 IU/L. FIGURE 3 A, The boy showed a dramatic response to 2 courses of cisplatin therapy; elbow circumference reduced to 28 cm and the armpit lymphadenopathy regressed completely. B, Evaluation after the last course of chemotherapy, before the myeloablative treatment, showed an elbow circumference of 17 cm. The reevaluation by CT scan of the chest and left arm, total body scan with 99Tc-MDP, and magnetic resonance imaging of the left arm, showed a very good response to treatment, with a very impressive reduction of the primary tumor size and a complete disappearance of the metastatic lymphadenopathy. A residual abnormal uptake in the 99Tc-MDP bone scan on the left knee and left humerus was documented. The patient underwent a subsequent phase with 8 intensive courses of chemotherapy. Collection of hematopoietic stem cells was done by leukapheresis after first course of cyclophosphamide 4 g/m2+etoposide 600 mg/m2 (overall the fourth course), followed by filgrastim at a dose of 10 µg/m2/d, according to the protocol. A total of 4×108 nucleated cells and 8×106 CD34+ cells were collected.

chemotherapy. Collection of hematopoietic stem cells was done by leukapheresis after first course of cyclophosphamide 4 g/m2+etoposide 600 mg/m2 (overall the fourth course), followed by filgrastim at a dose of 10 µg/m2/d, according to the protocol. A total of 4×108 nucleated cells and 8×106 CD34+ cells were collected. Evaluation after the last course of chemotherapy, preceding the myeloablative treatment, showed an elbow circumference of 17 cm (Fig. 3B), whereas CT scan and magnetic resonance imaging of the elbow confirmed marked reduction of the tumor. Total body bone scan with 99Tc-MDP showed a residual uptake on the left elbow without other metastatic locations. To obtain the best control of the disease, we proposed demolitive surgery consisting of amputation of the left arm. The mother of the patient refused surgery, and for this reason we decided to give exclusive RT for local control, which was delayed until after the myeloablative phase. The patient received the consolidation phase with busulfan (1 mg/kg×16 doses) and melphalan (140 mg/m2), followed by autologous peripheral blood stem cell transplantation, with a normal take of polymorphonucleates and thrombocytes and without acute complications. RT on the site of the primary tumor concluded the treatment program and consisted of 2 fractions of 1.2 Gy/d, up to a total dose of 55.2 Gy. Now, 42 months after the beginning of treatment and 30 months after the end of therapy, the child is well and in continuous complete remission.

To obtain the best control of the disease, we proposed demolitive surgery consisting of amputation of the left arm. The mother of the patient refused surgery, and for this reason we decided to give exclusive RT for local control, which was delayed until after the myeloablative phase. The patient received the consolidation phase with busulfan (1 mg/kg×16 doses) and melphalan (140 mg/m2), followed by autologous peripheral blood stem cell transplantation, with a normal take of polymorphonucleates and thrombocytes and without acute complications. RT on the site of the primary tumor concluded the treatment program and consisted of 2 fractions of 1.2 Gy/d, up to a total dose of 55.2 Gy. Now, 42 months after the beginning of treatment and 30 months after the end of therapy, the child is well and in continuous complete remission. DISCUSSION ES is the second most common bone malignancy in children and young adults, and prognosis of patients with disseminated disease other than pulmonary remains very poor, with an EFS of <25%.

RT on the site of the primary tumor concluded the treatment program and consisted of 2 fractions of 1.2 Gy/d, up to a total dose of 55.2 Gy. Now, 42 months after the beginning of treatment and 30 months after the end of therapy, the child is well and in continuous complete remission. DISCUSSION ES is the second most common bone malignancy in children and young adults, and prognosis of patients with disseminated disease other than pulmonary remains very poor, with an EFS of <25%. The introduction of combined modality treatment has determined a relevant improvement in the prognosis for many patients with ES. However, the results identified a bad prognosis of patients with large-volume primary tumors and metastatic disease. Our patient had both of these unfavorable prognostic characteristics, because he was admitted to our unit >10 months after the first signs and symptoms. The child, who was referred with a doubtful diagnosis of small cell osteosarcoma, was globally reevaluated, and histologic and molecular diagnosis of ES was correctly reached. The child was enrolled in the ISG-AIEOP/VHR-EW 02 protocol, consisting of a double course of high dose of CDDP window therapy, followed by intensive chemotherapy, myeloablative regimen, and autologous stem cell rescue. Response to CDDP alone, which is not generally used in clinical trials in first-line therapy, was very impressive, with a marked reduction of primary tumor size and metastatic disease.

course of high dose of CDDP window therapy, followed by intensive chemotherapy, myeloablative regimen, and autologous stem cell rescue. Response to CDDP alone, which is not generally used in clinical trials in first-line therapy, was very impressive, with a marked reduction of primary tumor size and metastatic disease. In the literature, few phase II studies with CDDP in refractory solid tumors in children are reported, including ES. Overall, in these studies, of 27 patients with ES, only 2 objective responses were observed.11–14 Platinum compounds have been used as first-line therapy in patients with ES, in a very limited number of studies, mostly in combination with other drugs, generally administered at doses of 70 to 100 mg/m2, and some authors reported its effectiveness in the preoperative treatment of ES.15–17 In this patient the local treatment on the site of the primary consisted of RT alone. We are aware that a factor influencing the outcome in ES is surgery, which gives better results than RT alone in a nonlocalized ES setting, but surgery was not possible because his mother refused a demolitive surgical approach—arm amputation—because of religious reasons.8

the site of the primary consisted of RT alone. We are aware that a factor influencing the outcome in ES is surgery, which gives better results than RT alone in a nonlocalized ES setting, but surgery was not possible because his mother refused a demolitive surgical approach—arm amputation—because of religious reasons.8 In conclusion, the peculiarity of our case was the great sensitivity of the ES to CDDP. The patient had a favorable outcome and is now a long-term survivor. It is not possible to assess the contribution of the front-line CDDP to this favorable outcome, as this phase was part of an intensive and prolonged treatment approach, but our experience suggests that an aggressive treatment option may be justified even if the possibilities of cure seem extremely slim. ACKNOWLEGDMENT The authors thank Prof. Frank Adamo for revising the manuscript. Supported by the parents’ Association “A.S.L.T.I.- Liberi di crescere” Onlus. The authors declare no conflict of interest. Reprints: Paolo D’Angelo, MD, U.O. di Oncoematologia Pediatrica, A.R.N.A.S. Ospedali Civico, Di Cristina e Benfratelli, Piazza Nicola Leotta 4, 90127 Palermo, Italy (e-mail: oncoematoped@ospedalecivicopa.org).

We welcome the letter by Prof Barr.1 Indeed, we consider our study on a sample of adolescent survivors of ALL2 to provide empirical support for the prediction made in a study by Webber et al3: interpreting dual energy x-ray (DXA) bone health data requires caution. In particular, failure to adjust DXA bone mineral density (BMD) data for body size risks erroneous judgments about bone health, which could be clinically important,3 particularly in children and adolescents with chronic disease.1,2 Although DXA is a valuable component of the assessment of bone health,4 and knowledge of the need to adjust BMD values appropriately for body size is increasing, this point is not as widely known as it should be at present.2 Barr1 also highlights the potentially valuable role for DXA in the assessment of body composition. We agree that DXA has great potential as an accurate and precise measure of fat and lean mass in children and adolescents. However, DXA is not a “gold standard” for body composition measurement,5 and we would add that caution is also necessary here. Different combinations of DXA hardware and software are likely to have different accuracy, and the errors inherent in DXA estimates of body composition can be very large.6 In summary, DXA, when used appropriately, can be extremely valuable in the assessment of both bone health and body composition, and the technique can inform both research and clinical management.

ware are likely to have different accuracy, and the errors inherent in DXA estimates of body composition can be very large.6 In summary, DXA, when used appropriately, can be extremely valuable in the assessment of both bone health and body composition, and the technique can inform both research and clinical management. John J. Reilly, PhD*Sheila K. Shepherd, PhD†Fahad K. Aldhafiri, MSc‡Faisal Ahmed, MD, RCPCH‡ *School of Psychological Sciences and Health, University of Strathclyde †Yorkhill Hospitals ‡College of Medical, Veterinary, and Life Sciences University of Glasgow, Yorkhill Hospitals, Glasgow, Scotland

β-Thalassemia is a heterogenous family of inherited disorders affecting hemoglobin synthesis. It is characterized by the complete absence or reduced synthesis of the β chain of hemoglobin, resulting in increased, but ineffective, erythropoiesis. The β-thalassemia phenotypes are variable, ranging from severe conditions requiring blood transfusions (thalassemia major [TM]) to milder forms (intermediate thalassemia [TI]). Patients affected by TM always present a severe microcytic and hypochromic anemia, associated with hepatosplenomegaly, and usually come to medical attention within the first 2 years of life. Treatment with regular blood transfusions and chelation therapy, which is aimed at reducing transfusion-induced iron overload, allows normal growth and development of these children. This therapeutic approach has extended patients’ life expectancy up to the age of 30 or 40 years, with a substantially improved survival and quality of life.1

r blood transfusions and chelation therapy, which is aimed at reducing transfusion-induced iron overload, allows normal growth and development of these children. This therapeutic approach has extended patients’ life expectancy up to the age of 30 or 40 years, with a substantially improved survival and quality of life.1 Subjects presenting TI show a later onset of the disease, characterized by milder anemia rarely requiring blood transfusion. These patients also present liver and spleen enlargement, typical bone modifications, and mild to moderate jaundice.2 Current epidemiological data show that approximately 7% of the global population present genes associated with hemoglobin disorders (healthy carrier of the disease). It is estimated that 300,000 to 500,000 children suffering from TM are delivered annually, 80% of who are living in developing countries.3 As the longevity of patients with TM increases, osteoporosis has emerged as an important cause of morbidity and disability in adult patients.4 The etiology of bone diseases in patients with thalassemia is more complex than in individuals without thalassemia. Factors such as anemia, massive ineffective erythropoiesis, bone marrow expansion with thinning of cortical bone, endocrine dysfunction, iron overload, desferrioxamine toxicity, metabolic factors (ie, deficiencies in calcium, vitamin D, and zinc), inadequate physical activity, and genetic factors may all play a role in the reduction of bone mass in these patients. In the last decade, the presence of osteopenia and osteoporosis in thalassemic patients receiving all the current gold standard treatments for this disease has been described in different studies, showing a high prevalence of up to 50%.5,6 Two main techniques are commonly used for evaluation of bone mineral density and fragility fracture risk: dual energy x-ray absorptiometry (DXA) bone densitometry is the “gold standard” method for diagnosis of osteoporosis, with this condition being defined by a reduction of bone mineral density (BMD)>2.5 SD below the values observed in healthy young subjects (T score <−2.5 SD). DXA has been widely accepted as a reference method for BMD measurement in adults and in pediatric subjects.7 However, it seems that other factors, in addition to BMD reduction, such as elasticity and bone microarchitectural bone characteristics are also important in determining bone fragility.

jects (T score <−2.5 SD). DXA has been widely accepted as a reference method for BMD measurement in adults and in pediatric subjects.7 However, it seems that other factors, in addition to BMD reduction, such as elasticity and bone microarchitectural bone characteristics are also important in determining bone fragility. Therefore, different quantitative ultrasound (QUS) methods have been developed for the assessment of fracture risk.8,9 QUS measured at the phalanx is a novel kind of noninvasive, radiation-free, and portable method that can be used extensively to get both qualitative and quantitative information.10–12 As comorbidities (ie, hypogonadism, hypothyroidism, and other illnesses) might play a relevant role in inducing bone demineralization, the aim of this study was to assess the ability of DXA and QUS to evaluate fracture risk in thalassemic patients, with particular focus on the effect of age and concurrent diseases on T score values. MATERIALS AND METHODS Within the Prevention Osteoporosis and Fractures Project, which is an innovative disease management program aimed at preventing osteoporotic fractures in the Salento region, we have enrolled 88 young thalassemic patients being followed up at SS Annunziata Hospital in Taranto.

Therefore, different quantitative ultrasound (QUS) methods have been developed for the assessment of fracture risk.8,9 QUS measured at the phalanx is a novel kind of noninvasive, radiation-free, and portable method that can be used extensively to get both qualitative and quantitative information.10–12 As comorbidities (ie, hypogonadism, hypothyroidism, and other illnesses) might play a relevant role in inducing bone demineralization, the aim of this study was to assess the ability of DXA and QUS to evaluate fracture risk in thalassemic patients, with particular focus on the effect of age and concurrent diseases on T score values. MATERIALS AND METHODS Within the Prevention Osteoporosis and Fractures Project, which is an innovative disease management program aimed at preventing osteoporotic fractures in the Salento region, we have enrolled 88 young thalassemic patients being followed up at SS Annunziata Hospital in Taranto. Of the 88 enrolled patients, 42 (48%) were male patients (aged 22 to 69 y; mean age: 34.1 y; SD±7.9), and 46 (52%) were female patients (aged 13 to 48 y; mean age: 34.5 y; SD±6.7). The majority of patients was affected by TM (n=58; 66% of the sample), whereas the remaining 30 patients (34% of the sample) were affected by TI. These 2 groups were following different kinds of treatments based on the severity of the disease: patients with TM assumed iron-chelating therapy (desferrioxamine, deferasirox, or deferiprone) every day and underwent blood transfusion 2 times per month. TI patients followed a variable therapeutic regimen based on daily iron-chelating therapy, with blood transfusion being infrequently necessary.

rity of the disease: patients with TM assumed iron-chelating therapy (desferrioxamine, deferasirox, or deferiprone) every day and underwent blood transfusion 2 times per month. TI patients followed a variable therapeutic regimen based on daily iron-chelating therapy, with blood transfusion being infrequently necessary. For all patients, QUS measurements at the phalanx were performed using ultrasound densitometer DBM Sonic 1200 Bone Profiler (IGEA S.r.l.; Carpi, MO, Italy). DXA measurements at the lumbar spine (L1-L4) were also obtained for all patients by using Hologic QDR 4000 machine (Bedford, MA). All measurements were performed between 2008 and 2010, and informed consent was obtained from all the patients. T score and Z score values were obtained for all patients. For the DXA method, osteoporosis and osteopenia were defined according to the World Health Organization definitions (osteoporosis: T score <−2.5; osteopenia: T score between >−2.5 and <−1; normal: T score >−1).13 For QUS of the phalanx, different cutoff values have been directly provided by the manufacturer (osteoporosis: T score <−3.2; osteopenia: T score between >−3.2 and <−1; normal: T score >−1).14 Calibration of QUS densitometer was carried out daily using manufacturer’s verification phantom for quality control and assurance. T score values obtained with both methods (DXA and QUS) were correlated to age, sex, body mass index, type of thalassemia (TM or TI), previous fragility fractures, and presence of the following concurrent diseases: diabetes, hypothyroidism, hypogonadotrophic hypogonadism, and hepatitis (HBV). Fragility fractures were defined as resulting from low-energy trauma at different skeletal sites (ie, hip, humerus, vertebrae, wrist, leg, and ankle). Fractures that occurred at workplace or as a consequence of car accidents, as well as fractures of fingers and big toe, were excluded from statistical analyses because they were likely to be related to high-energy trauma. Statistical analyses were performed using STATA 11.0 (StataCorp LP, College Station, TX). Paired and unpaired Student t test was used to compare the comparisons. Ratios were compared by the χ2 or Fischer exact test. Linear regressions were performed to evaluate the effect of age, height, and weight on T scores obtained with both methods.

s were performed using STATA 11.0 (StataCorp LP, College Station, TX). Paired and unpaired Student t test was used to compare the comparisons. Ratios were compared by the χ2 or Fischer exact test. Linear regressions were performed to evaluate the effect of age, height, and weight on T scores obtained with both methods. RESULTS Nine of 88 patients (10% of the sample) presented a previous diagnosis of diabetes mellitus, whereas 10 patients had hypothyroidism (11%), 43 were affected by HBV (49%), and 52 (59%) presented hypogonadotrophic hypogonadism. There were 23 (26%) patients with a previous fragility fracture. Table 1 shows the characteristics of the study population. Patients’ height and weight were higher in the male than in the female patients, with this difference reaching statistical significance (P<0.05). T score values did not show any statistically difference when adjusted by height and weight (P=0.487 and 0.283, respectively). All the other examined parameters showed no correlation with sex. In Table 2, we report T score and Z score values obtained by using both DXA and QUS according to the type of thalassemia, presence of hypothyroidism, hypogonadotrophic hypogonadism, diabetes, HBV, and previous fragility fractures. The comparison of T score and Z score values between TM and TI patients did not show statistically significant differences for both techniques. TABLE 1 Characteristics of Study Population (n=88) TABLE 2 T Scores Obtained With Both Methods According to Different Clinical Variables

Table 1 shows the characteristics of the study population. Patients’ height and weight were higher in the male than in the female patients, with this difference reaching statistical significance (P<0.05). T score values did not show any statistically difference when adjusted by height and weight (P=0.487 and 0.283, respectively). All the other examined parameters showed no correlation with sex. In Table 2, we report T score and Z score values obtained by using both DXA and QUS according to the type of thalassemia, presence of hypothyroidism, hypogonadotrophic hypogonadism, diabetes, HBV, and previous fragility fractures. The comparison of T score and Z score values between TM and TI patients did not show statistically significant differences for both techniques. TABLE 1 Characteristics of Study Population (n=88) TABLE 2 T Scores Obtained With Both Methods According to Different Clinical Variables The 52 subjects suffering from hypogonadotrophic hypogonadism showed lower T score values compared with the 36 patients not affected by hypogonadism. Lower values were observed also in case of hypothyroidism, although the number of patients (n=10) is too little to extend this observation. Patients with HBV (almost 50% of the sample) showed lower T score values compared with patients not affected by HBV.

ues compared with the 36 patients not affected by hypogonadism. Lower values were observed also in case of hypothyroidism, although the number of patients (n=10) is too little to extend this observation. Patients with HBV (almost 50% of the sample) showed lower T score values compared with patients not affected by HBV. Similarly, also diabetes was associated with low T score values, although no statistical significance was reached due to the small number of subjects affected (n=9). As reported in Table 3, both DXA and QUS were equally able to detect the demineralization status in almost all patients with previous fragility fractures (91% for DXA and 83% for QUS). TABLE 3 Ability of DXA and QUS to Detect Patients With Previous Fragility Fracture

Similarly, also diabetes was associated with low T score values, although no statistical significance was reached due to the small number of subjects affected (n=9). As reported in Table 3, both DXA and QUS were equally able to detect the demineralization status in almost all patients with previous fragility fractures (91% for DXA and 83% for QUS). TABLE 3 Ability of DXA and QUS to Detect Patients With Previous Fragility Fracture The overall prevalence of osteopenia and osteoporosis defined by DXA and QUS are presented in Tables 4 and 5, respectively. Using DXA, 60% of the male patients (25/42) and 45% of the female patients (21/46) were found to be osteoporotic, whereas osteopenia was reported in 28% of male patients (12/42) and in 35% of female patients (16/46). Overall, osteoporosis showed a global prevalence of 52% (46/88) by DXA and only 10% by QUS (9/88), whereas osteopenia was found in 32% (28/88) and in 60% (53/88) of cases using DXA or QUS, respectively. Up to 12% of male and 20% of female patients presented DXA T score values higher than −1 SD (normality). Normal findings were more frequent when QUS was used (28% of male patients: 12/42; 31% of female patients: 14/46). When performing the Fisher exact test, there were no differences in the ratios between the male and female patients both for DXA (P=0.420) and QUS (P=0.184). TABLE 4 Prevalence of Osteopenia and Osteoporosis Derived by the DXA Method TABLE 5 Prevalence of Osteopenia and Osteoporosis Derived by the QUS Method

The overall prevalence of osteopenia and osteoporosis defined by DXA and QUS are presented in Tables 4 and 5, respectively. Using DXA, 60% of the male patients (25/42) and 45% of the female patients (21/46) were found to be osteoporotic, whereas osteopenia was reported in 28% of male patients (12/42) and in 35% of female patients (16/46). Overall, osteoporosis showed a global prevalence of 52% (46/88) by DXA and only 10% by QUS (9/88), whereas osteopenia was found in 32% (28/88) and in 60% (53/88) of cases using DXA or QUS, respectively. Up to 12% of male and 20% of female patients presented DXA T score values higher than −1 SD (normality). Normal findings were more frequent when QUS was used (28% of male patients: 12/42; 31% of female patients: 14/46). When performing the Fisher exact test, there were no differences in the ratios between the male and female patients both for DXA (P=0.420) and QUS (P=0.184). TABLE 4 Prevalence of Osteopenia and Osteoporosis Derived by the DXA Method TABLE 5 Prevalence of Osteopenia and Osteoporosis Derived by the QUS Method DISCUSSION This is the first study addressing the issue of DXA and QUS ability to detect fragility fractures and concurrent pathologies in thalassemic patients. In a previous study aimed at comparing DXA and quantitative computed tomography (QCT), Mylona and colleagues observed a strong difference in the prevalence of osteopenia and osteoporosis in patients with β-thalassemia according to the technique used. In this study we observed a different prevalence of osteoporosis for DXA and QUS.15 The classification of our patient into normal, osteopenic, and osteoporotic categories is exclusively based on T score values. Osteoporosis and osteopenia are nowadays highly prevalent in patients affected by TM or TI, with increased patient survival and multifactorial pathogenesis being responsible for that (iron inclusions in the bones, desferrioxamine-induced bone dysplasia, ineffective erythropoiesis, and multiple endocrine dysfunctions).

Osteoporosis and osteopenia are nowadays highly prevalent in patients affected by TM or TI, with increased patient survival and multifactorial pathogenesis being responsible for that (iron inclusions in the bones, desferrioxamine-induced bone dysplasia, ineffective erythropoiesis, and multiple endocrine dysfunctions). Hormonal deficiency is an important cause of demineralization in TM and TI, as lower testosterone levels in men are associated with low BMD and an increased incidence of spine and hip fractures.16 It is known that adolescents with hypogonadism do not achieve optimal bone mass.17 In addition, delayed puberty, hypoparathyroidism, and hypothyroidism have been proposed as causes of reduction in BMD.18,19 Another metabolic/endocrine disorder such as diabetes mellitus has been reported to negatively influence bone density in thalassemic subjects,20 although a different study has not confirmed this observation.15 The crucial role of hormonal regulation is confirmed by the high number of thalassemic patients with hypogonadotrophic hypogonadism (52/88) showing low T scores by both DXA and QUS. The prevalence of osteoporosis and osteopenia with DXA was 52% and 32%, respectively. These results are consistent with those of previous studies.20,21 In contrast with other reports,15,22,23 we found a relevant difference between sexes. However, this finding was reported by several authors.24 Although DXA sensitivity for the detection of osteoporosis was higher, both DXA and QUS were able to detect the demineralization status (osteopenia or osteoporosis) in the vast majority of subjects with previous fragility fractures.

vant difference between sexes. However, this finding was reported by several authors.24 Although DXA sensitivity for the detection of osteoporosis was higher, both DXA and QUS were able to detect the demineralization status (osteopenia or osteoporosis) in the vast majority of subjects with previous fragility fractures. With both techniques, osteoporosis was found to be more frequent in the male than in the female patients (60% vs. 45% by DXA, and 17% vs. 4% by QUS), whereas osteopenia was more prevalent among the female compared with the male patients (35% vs. 28% by DXA and 65% vs. 55% by QUS). Normal T score values (>−1 SD) were more frequently obtained by QUS (26 patients of 88; 30% of the sample) than with the DXA method (14 patients of 88; 10% of the sample). Data obtained with the QUS technique were particularly interesting, showing only a 10% prevalence rate for osteoporosis, and a higher number of osteopenic patients (60%) compared with DXA. This result places the QUS method in an intermediate position between DXA and QCT, which identify the majority of patients as osteoporotic and normal,15,25,26 respectively. In their direct comparison study, Mylona et al15 have reported 44% and 6% of patients as osteoporotic with DXA (normal findings only in 4% of the subjects) and QCT (54% of normal findings), respectively. DXA and QCT methods provide a measure of BMD, which is currently considered the best predictor of osteoporotic fractures.27 However, these techniques are able to explain only 60% to 80% of the variability in bone strength, and it has been demonstrated that other mechanical aspects of the bone are important in determining fracture risk. These factors include microarchitectural parameters and geometric and elastic properties of bone tissue, which cannot be assessed using DXA.28 QUS techniques have been developed over the past 10 years to determine bone quality and the state of the skeleton on the basis of various studies suggesting how sonographic parameters are able to provide information on bone density and structure, including elastic properties.29

ue, which cannot be assessed using DXA.28 QUS techniques have been developed over the past 10 years to determine bone quality and the state of the skeleton on the basis of various studies suggesting how sonographic parameters are able to provide information on bone density and structure, including elastic properties.29 Many authors report that low DXA values do not reflect the behavior of vertebrae under stress conditions,30 thus suggesting that BMD represents only 1 factor involved in determining bone strength, and that bone quality is independent from mineral density. Despite the lack of specific correlation studies between DXA and QUS findings, large prospective studies, carried out both in the elderly and early postmenopausal women, indicate that these 2 techniques are equally able to predict patients at risk for future fragility fractures.31–33 The discriminatory power in classifying individuals with or without vertebral fractures was tested in cross-sectional studies using different QUS methods, including QUS of the phalanx, and DXA in a sample of older postmenopausal women.34 In addition, a multicentric study assessed the performance of 5 different ultrasound devices and their association with prevalent vertebral fractures compared with DXA in a group of European women.35 In addition, this study showed that QUS findings are similar to DXA femoral BMD in identifying subjects with prevalent vertebral fractures.

icentric study assessed the performance of 5 different ultrasound devices and their association with prevalent vertebral fractures compared with DXA in a group of European women.35 In addition, this study showed that QUS findings are similar to DXA femoral BMD in identifying subjects with prevalent vertebral fractures. According to our study, the classification of β-thalassemic subjects as osteoporotic or osteopenic yields variable results, and this depends on the technique used. If we consider DXA to be more specific, most of the patients should be classified as osteopenic and/or osteoporotic, a finding not in accordance with the low incidence of previous fragility fractures observed in our sample. Our study demonstrates a “profitable” correlation between QUS and DXA measurements in thalassemic patients. In summary, our study showed that the ability of the QUS method to detect patients with previous fracture outside the range of normal T scores is high, as much as that of DXA. High prevalence of osteoporosis and osteopenia among patients with thalassemia requires more attention to therapeutic approaches for the prevention and treatment of these skeletal disorders. Osteoporosis is a progressive disease that can start in early childhood in this kind of patients. Therefore, prevention and early diagnosis become particularly important, as well as treatment of the established disease. Annual screening of adolescent thalassemic patients and correction of endocrine factors by replacement therapy might be a promising approach to prevent osteoporosis in subjects with thalassemia. Further studies should be performed to clarify the optimal approach to these patients, who are already undergoing a large number of invasive clinical procedures. Our study showed that the ability of QUS to detect thalassemic patients with prevalent fragility fractures is comparable to that of DXA. In fact hypogonadism, hypothyroidism, HBV, and the presence of previous fragility fractures (but not diabetes) were significantly associated with the demineralization status (lower T scores values) both with DXA and QUS.

QUS to detect thalassemic patients with prevalent fragility fractures is comparable to that of DXA. In fact hypogonadism, hypothyroidism, HBV, and the presence of previous fragility fractures (but not diabetes) were significantly associated with the demineralization status (lower T scores values) both with DXA and QUS. ACKNOWLEDGMENTS The authors thank all the physicians and the ISBEM researchers who have contributed to the Prevention Osteoporosis and Fractures project. Authors are particularly grateful to Dr Antonio Marsico, Dr Giulio Franco, and Dr Pierguido Conte (Local Health Authority, ASL Taranto). P.P. has received consulting fees from Amgen-Dompè, Sanofi-Aventis, Eli-Lilly, and Servier. For the remaining authors none were declared. Reprints: Alberto Argentiero, PhD, c/o ISBEM Research Centre, Via Reali di Bulgaria, Mesagne, Brindisi 72023, Italy (e-mail: alberto_argentiero@libero.it).

Osteosarcoma is the most common malignant bone tumor in children, adolescents, and young adults, with about 400 new cases diagnosed each year in the United States. It accounts for approximately 60% of all malignant bone tumors diagnosed in patients before the age of 20 with the peak incidence associated with puberty.1 Although osteosarcoma can occur in the axial skeleton, it typically presents in the metaphysis of long bones, a site of rapid bone growth during adolescence. The standard treatment for high-grade osteosarcoma requires both surgery and chemotherapy given preoperatively (neoadjuvant) and/or postoperatively. Despite improved surgical outcomes and efforts to intensify therapy, the 5-year event-free survival (EFS) remains 65% with no significant improvement in the past 20 years.2 In studies conducted pertaining to osteosarcoma outcome in pediatrics, only 10% to 15% of the cohorts are Hispanics. A study by Mirabello et al3 based on data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results Program, indicates a higher incidence and slightly poorer outcome of osteosarcoma in Hispanics when compared with non-Hispanics. Hispanic patients that are reported in this studies come from a variety of locations including Mexico, South America, Cuba, and Latin America.

te’s Surveillance, Epidemiology, and End Results Program, indicates a higher incidence and slightly poorer outcome of osteosarcoma in Hispanics when compared with non-Hispanics. Hispanic patients that are reported in this studies come from a variety of locations including Mexico, South America, Cuba, and Latin America. In this study we performed a retrospective analysis of the characteristics and outcomes in patients with localized high-grade osteosarcoma of the extremity diagnosed under the age of 30 treated at a single institution, University of Texas Health Science Center at San Antonio (UTHSCSA), over an 11-year period. The patients in this study were 70% Hispanic, homogenously of Mexican American ancestry, therefore giving us a unique cohort to study. To the best of our knowledge, this is the largest series composed of such patients. Interestingly, we found a decreased survival of preadolescent patients compared with the patients who were between 12 and 30 years old at diagnosis. Furthermore, our data suggests that tumor necrosis after neoadjuvant chemotherapy might not be directly predictive of outcome.

he largest series composed of such patients. Interestingly, we found a decreased survival of preadolescent patients compared with the patients who were between 12 and 30 years old at diagnosis. Furthermore, our data suggests that tumor necrosis after neoadjuvant chemotherapy might not be directly predictive of outcome. MATERIALS AND METHODS Patient Selection and Data Elements Our cohort consisted of 50 patients below 30 years of age diagnosed with localized high-grade osteosarcoma of the extremity between January 2000 and December 2010 that were treated by members of the UTHSCSA sarcoma team. Patients with axial primaries or metastatic disease at diagnosis were excluded from this analysis. During this time frame nearly uniform treatment was used for patients with osteosarcoma. Hospital and clinic records from University Hospital, CHRISTUS Santa Rosa Children’s Hospital, and the Cancer Therapy and Research Center were reviewed. A retrospective analysis of patient demographics (age at diagnosis, sex, date of diagnosis, race, and ethnicity), presence of predisposing factors, socioeconomic status (based on family income obtained from institutional survey), and tumor characteristics (location, histology, tumor volume, response to neoadjuvant chemotherapy, and type of primary surgery) was performed.

at diagnosis, sex, date of diagnosis, race, and ethnicity), presence of predisposing factors, socioeconomic status (based on family income obtained from institutional survey), and tumor characteristics (location, histology, tumor volume, response to neoadjuvant chemotherapy, and type of primary surgery) was performed. Ethnicity was assigned based on parental report, and the National Cancer Institute/Children’s Oncology Group (COG) definitions. According to this convention, the term Hispanic can include Mexican Americans, South Americans, or Cubans but our population of Hispanics was exclusively Mexican American. Tumor volume was defined as the absolute tumor volume (ATV) in cm3. ATV was defined as absolute tumor length (ATL)×absolute tumor width (ATW)×absolute tumor depth (ATD) (ATV in cm3=ATL×ATW×ATD×0.52). The diagnosis of osteosarcoma was confirmed by J.H.-H., a pediatric pathologist with expertise in bone tumors. The project was conducted after approval was received from the institutional review board at the respective institution.

Ethnicity was assigned based on parental report, and the National Cancer Institute/Children’s Oncology Group (COG) definitions. According to this convention, the term Hispanic can include Mexican Americans, South Americans, or Cubans but our population of Hispanics was exclusively Mexican American. Tumor volume was defined as the absolute tumor volume (ATV) in cm3. ATV was defined as absolute tumor length (ATL)×absolute tumor width (ATW)×absolute tumor depth (ATD) (ATV in cm3=ATL×ATW×ATD×0.52). The diagnosis of osteosarcoma was confirmed by J.H.-H., a pediatric pathologist with expertise in bone tumors. The project was conducted after approval was received from the institutional review board at the respective institution. Treatment Chemotherapy was utilized in the neoadjuvant and adjuvant settings. The chemotherapy regimens, specifically dosing, were based on body surface area and the 2 groups received equivalent treatment either on study or according to study protocol based on either AOST0331 or POG9754 depending on when the patient was diagnosed. The chemotherapy included combinations of methotrexate, cisplatin, adriamycin, ifosfamide, and etoposide; although not all agents were used in each patient. All 50 patients underwent surgery for local control and had negative surgical margins as confirmed by pathology. The type of surgery (limb salvage, amputation, rotationplasty) was determined by the extent of disease, involvement of neurovascular bundle, presence of pathologic fracture, and appraisal for best limb functionality after surgical resection. The resected specimens were examined for percentage of tumor necrosis in response to neoadjuvant chemotherapy and were assigned a grade of 1 to 6 as defined by Salzer-Kuntschik et al.4 In this grading system, grade 1 signifies no viable tumor; grade 2, solitary live cells or 1 islet of live cells <0.5 cm; grade 3, <10% viable cells; grade 4, 10% to 50% viable cells; grade 5, >50% alive tumor; and grade 6, 100% viable tumor. This system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual POG9754 and AOST0331 studies. Longitudinal tumor assessment (primary site and lungs) was performed according to POG9754 or AOST0331 recommendations.

ve tumor; and grade 6, 100% viable tumor. This system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual POG9754 and AOST0331 studies. Longitudinal tumor assessment (primary site and lungs) was performed according to POG9754 or AOST0331 recommendations. Statistical Analysis The primary focus of this analysis was EFS and overall survival (OS). EFS was calculated from the date of diagnosis until recurrence, secondary malignancy, death, or most recent follow-up examination showing absence of an event. OS was calculated from the date of diagnosis to death or most recent follow-up examination. The survival curves were calculated using the Kaplan-Meier method and the differences of survival curves were assessed using the log-rank test. Adjusted estimates were obtained from proportional hazards models with age, sex, ethnicity, family income, tumor volume, and tumor necrosis included as covariates. Power analysis was completed and all statistical testing was 2-sided with a significance level of 5%. SAS Version 9.2 (SAS Institute, Cary, NC) was used.

t. Adjusted estimates were obtained from proportional hazards models with age, sex, ethnicity, family income, tumor volume, and tumor necrosis included as covariates. Power analysis was completed and all statistical testing was 2-sided with a significance level of 5%. SAS Version 9.2 (SAS Institute, Cary, NC) was used. RESULTS Demographic Data Fifty patients with localized osteosarcoma of the extremity below 30 years of age were diagnosed between January 2000 and December 2010 at UTHSCSA. The complete demographic and clinicopathologic characteristics are shown in Table 1. The cohort was composed of 35 Hispanics (70%), 10 whites (20%), 4 blacks (8%), and 1 other (2%). Ethnicity was assigned based on parental report, and the National Cancer Institute/COG definitions. Therefore, although our cohort is comprised of mainly Hispanics, it is not exclusively Hispanics. However, as described in the Methods section our Hispanic population is uniquely composed of only Mexican Americans, whereas most of the studies that include Hispanics are more heterogenous. TABLE 1 Patient Demographics The mean age at diagnosis was 15 years (range, 2 to 28 y). Male to female ratio was approximately 3:1. Socioeconomic status based on family income was available for 68% of the cohort. Family income was <$25,000 for 13 patients and ≥$25,000 for 21 patients. There was no clinical or statistical correlation between socioeconomic status data and tumor size or extent of disease at presentation.

e ratio was approximately 3:1. Socioeconomic status based on family income was available for 68% of the cohort. Family income was <$25,000 for 13 patients and ≥$25,000 for 21 patients. There was no clinical or statistical correlation between socioeconomic status data and tumor size or extent of disease at presentation. Clinicopathologic Characteristics All patients presented with localized disease of an extremity. Tumor location was as follows: 41 (82%) in the lower extremity and 9 (18%) in the upper extremity. High-grade, conventional osteosarcoma was diagnosed in all patients. Tumor size was available for 34 (85%) patients. Eighteen (53%) patients had a tumor volume >150 cm3 and 16 (47%) patients had a tumor volume ≤150 cm3. No significant association could be found between Hispanic ethnicity and large tumor size (P=0.73). All patients had surgery for local control; 60% had ablative surgery (amputation, disarticulation, or rotationplasty), whereas 40% had limb salvage procedures. We acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement. All patients had a complete resection.

disarticulation, or rotationplasty), whereas 40% had limb salvage procedures. We acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement. All patients had a complete resection. Histologic response to neoadjuvant chemotherapy was available for 40 (80%) patients. Seventeen (42%) patients were good responders with grades 1 to 3 necrosis (>90%). Twelve (30%) patients had grade 4 necrosis (between 50% and 90%) and 11 (28%) patients had a very poor response (<50% necrosis) including 2 patients with 100% viable tumor after neoadjuvant chemotherapy. Although the cohort of patients for which histologic response was available was 70% Hispanic, 10/11 (91%) of the patients with a very poor response were Hispanics. Univariate Survival Analysis The OS at 3 and 5 years was 73% and 65%, respectively, for the entire cohort. The 3- and 5-year EFS were 50% and 48%, respectively. Eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year EFS and OS relative to those diagnosed between the ages of 12 and 29 (11% vs. 57%, respectively, P=<0.001 for EFS and 25% vs. 76%, respectively, P=<0.001 for OS) (Table 2). There was no statistically significant difference in outcomes based on ethnicity, income, or tumor volume. TABLE 2 Patient Characteristics and Univariate Analysis

Univariate Survival Analysis The OS at 3 and 5 years was 73% and 65%, respectively, for the entire cohort. The 3- and 5-year EFS were 50% and 48%, respectively. Eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year EFS and OS relative to those diagnosed between the ages of 12 and 29 (11% vs. 57%, respectively, P=<0.001 for EFS and 25% vs. 76%, respectively, P=<0.001 for OS) (Table 2). There was no statistically significant difference in outcomes based on ethnicity, income, or tumor volume. TABLE 2 Patient Characteristics and Univariate Analysis Although response to neoadjuvant chemotherapy has traditionally been used as a prognostic marker for both EFS and OS, it was of prognostic significance only for EFS (Table 2 and Figs. 1, 2) in our population. We observed an increased percentage of patients with <50% necrosis after neoadjuvant chemotherapy and chose to grade necrosis based on 6 categories as defined by Salzer-Kuntschik et al4 to further categorize the “poor responders.” The 5-year EFS when compared between groups categorized by grades 1 to 3, grade 4, and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis (61% vs. 42% vs. 21%, respectively, P=0.03). The 5-year OS for these patients was similar across the groups suggesting that our patient population was salvageable after initial relapse.

to 3, grade 4, and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis (61% vs. 42% vs. 21%, respectively, P=0.03). The 5-year OS for these patients was similar across the groups suggesting that our patient population was salvageable after initial relapse. FIGURE 1 Event-free survival (EFS) of localized/extremity tumor patients based on tumor necrosis. The 5-year EFS for patients with grades 5 to 6 was significantly different than the 5-year EFS of patients with grades 1 to 3 and grade 4 tumor necrosis (P=0.03). FIGURE 2 Overall survival (OS) of localized/extremity tumor patients based on tumor necrosis. There was no significant difference in OS based on tumor necrosis in this population.

FIGURE 1 Event-free survival (EFS) of localized/extremity tumor patients based on tumor necrosis. The 5-year EFS for patients with grades 5 to 6 was significantly different than the 5-year EFS of patients with grades 1 to 3 and grade 4 tumor necrosis (P=0.03). FIGURE 2 Overall survival (OS) of localized/extremity tumor patients based on tumor necrosis. There was no significant difference in OS based on tumor necrosis in this population. Twenty-three of the 50 patients relapsed. There was no difference in the location of relapse when comparing the preadolescent patients to the older patients (young patients [n=9]: 22%, local; 55%, lung; 22%, lung/local; older patients [n=14]: 21%, local; 57%, lung; 21%, lung/local). For the complete cohort, isolated lung recurrence was observed in 13 patients. Overall, 4 patients experienced a local recurrence, 5 had combined local/lung recurrence, and 1 patient had progressive disease, meaning nonresponsive to neoadjuvant chemotherapy. The mean time to relapse was 19 months (range, 2 to 37 mo). Salvage treatment included surgical resection for the majority of patients, with many receiving postoperative chemotherapy. Eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence/progression were successfully salvaged for an overall salvage rate of 52% for first relapses.

Salvage treatment included surgical resection for the majority of patients, with many receiving postoperative chemotherapy. Eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence/progression were successfully salvaged for an overall salvage rate of 52% for first relapses. Multivariate Survival Analysis Multivariate Cox regression analysis was performed to assess the association between EFS or OS and predictors, such as age, ethnicity, income, tumor volume, and tumor necrosis. The results are summarized in Table 3. Patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis (hazard ratio [HR], 4.77; 95% CI, 2.03-11.17; P=<0.001 for EFS; HR, 5.23; 95% CI, 1.86-14.59; P=0.002 for OS). Grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased EFS (HR, 3.76; 95% CI, 1.29-10.94; P=0.02) and there was a trend toward lower OS (HR, 3.45; 95% CI, 0.91-12.99; P=0.07). None of ethnicity, income, or tumor volume was significantly correlated with either EFS or OS. TABLE 3 Multivariate Analysis (Cox Models) of EFS and OS

Multivariate Survival Analysis Multivariate Cox regression analysis was performed to assess the association between EFS or OS and predictors, such as age, ethnicity, income, tumor volume, and tumor necrosis. The results are summarized in Table 3. Patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis (hazard ratio [HR], 4.77; 95% CI, 2.03-11.17; P=<0.001 for EFS; HR, 5.23; 95% CI, 1.86-14.59; P=0.002 for OS). Grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased EFS (HR, 3.76; 95% CI, 1.29-10.94; P=0.02) and there was a trend toward lower OS (HR, 3.45; 95% CI, 0.91-12.99; P=0.07). None of ethnicity, income, or tumor volume was significantly correlated with either EFS or OS. TABLE 3 Multivariate Analysis (Cox Models) of EFS and OS DISCUSSION The population of localized osteosarcoma of the extremity patients treated at UTHSCSA was 70% Hispanic (35/50), homogenously of Mexican American ancestry, giving us a unique cohort to study. Of the 50 patients in our cohort, 11 were categorized as preadolescent (below 12 y of age at diagnosis). Nine of the 35 Mexican American patients were preadolescents at diagnosis and to the best of our knowledge this is the largest series composed of such patients. In this study, the 5-year EFS of 48% was similar to results from the Brazilian Osteosarcoma Treatment Group who reported a 5-year EFS of only 53%.5 In this cohort, the 5-year EFS was inferior to a smaller study completed on patients in low-income countries in Latin America.6 These data suggest that there might be some similarities and differences in outcome among other Hispanic groups. Although the 5-year OS of 65% was comparable with that reported by large European groups,2,7 the EFS in this cohort was lower.2,8–12 In this regard, the data indicated that our patients had a higher risk of relapse after primary treatment, but were salvageable as denoted by OS rates.

utcome among other Hispanic groups. Although the 5-year OS of 65% was comparable with that reported by large European groups,2,7 the EFS in this cohort was lower.2,8–12 In this regard, the data indicated that our patients had a higher risk of relapse after primary treatment, but were salvageable as denoted by OS rates. Notably, our predominantly Mexican American preadolescent patients (below 12 y of age at diagnosis of which 9/11 are Hispanic) had an overall poor outcome with 5-year EFS of 11% and a 5-year OS of 25%. In stark contrast, many published studies report the outcome of preadolescent patients to be equivalent to those diagnosed after puberty.13–17 Bacci et al14 reported results on a large cohort of patients treated on protocol between 1972 and 1999, comparing the outcome of patients aged 12 years and below at diagnosis to that of patients between 13 and 40 years of age. The 2 groups have equivalent clinicopathologic features and similar histologic response to chemotherapy. The 5-year EFS is 60% versus 58% in favor of the preadolescents.14

ol between 1972 and 1999, comparing the outcome of patients aged 12 years and below at diagnosis to that of patients between 13 and 40 years of age. The 2 groups have equivalent clinicopathologic features and similar histologic response to chemotherapy. The 5-year EFS is 60% versus 58% in favor of the preadolescents.14 In our analysis, the older patients (12 to 30 y of age at diagnosis) had a better 5-year EFS compared with the preadolescent patients. However, there was no difference in the response to neoadjuvant chemotherapy between the 2 age groups and thus would not explain the inferior outcome of the preadolescent patients. Moreover, our patients were very compliant and therefore received the recommended chemotherapy with no difference between preadolescents and older patients with regard to chemoreduction for toxicities. There is a recent report by Sharib et al18 that mentioned increased toxicity in young patients and in their Latino population with Ewing Sarcoma. We did not find an increase in toxicity in either of these groups within our cohort and thus could not use it as an explanation for the trend toward inferior outcome.

cities. There is a recent report by Sharib et al18 that mentioned increased toxicity in young patients and in their Latino population with Ewing Sarcoma. We did not find an increase in toxicity in either of these groups within our cohort and thus could not use it as an explanation for the trend toward inferior outcome. Traditionally, the histologic response to neoadjuvant chemotherapy has been used as a prognostic factor. In previous studies, good responders (>90% necrosis) had a 5-year EFS of 67% to 78%, whereas the poor responders had a 5-year EFS of 49% to 51%.8–10 In our cohort, tumor necrosis assessment was available for 40 patients. Of these patients 43% were “good responders.” Surprisingly, the 5-year EFS in the good responders was only 61%. The 5-year OS was 70% and this is comparable with that reported in larger studies8–10 as described above. The patients who were deemed “good responders” to neoadjuvant chemotherapy relapsed at a higher rate than expected but were salvageable. Eleven patients (91% Hispanic) in our cohort had a very poor response with <50% necrosis. Nine of the 11 patients relapsed, were lost to follow-up, or were censored within 3 years due to the time frame of the study. The patients that relapsed were salvageable and had a 5-year OS of 51%. Although our sample size is small (n=50), our analysis suggested that tumor necrosis in response to neoadjuvant chemotherapy regimens might not be a valid prognostic marker for EFS in our study.

ed within 3 years due to the time frame of the study. The patients that relapsed were salvageable and had a 5-year OS of 51%. Although our sample size is small (n=50), our analysis suggested that tumor necrosis in response to neoadjuvant chemotherapy regimens might not be a valid prognostic marker for EFS in our study. In this study, the same orthopedic surgeon provided surgical management over the 11-year period. As well, the same pathologist confirmed diagnosis over the 11-year period. Chemotherapy was delivered according to standard protocols from the COG by pediatric or medical oncologists from different COG institutions. All patients were treated at institutions by medical teams trained in delivering treatment on osteosarcoma protocols, whether enrolled on study or not. Therefore, the inferior outcome in this population might not be the result of poor adherence to treatment or a decrease in treatment because of toxicity. In our cohort, the male to female ratio was much higher than expected and a disproportionate amount of patients received ablative surgical procedures. The observation of an increase in male to female ratio may in part be due to our small sample size. The higher proportion of patients receiving ablative surgical procedures was in part because our patients presented with more invasive disease at diagnosis.

onate amount of patients received ablative surgical procedures. The observation of an increase in male to female ratio may in part be due to our small sample size. The higher proportion of patients receiving ablative surgical procedures was in part because our patients presented with more invasive disease at diagnosis. In conclusion, we reviewed the clinicopathologic characteristics and disease outcome of 50 patients with localized high-grade osteosarcoma of the extremity treated by ≥1 members of the UTHSCSA sarcoma team over an 11-year period. Our cohort was comprised of 70% Hispanics of Mexican American descent and we found a decreased EFS but similar OS to what is reported. More importantly, we found a strikingly increased rate of relapse in young patients diagnosed before the age of 12. We also found that the percentage of tumor necrosis after neoadjuvant chemotherapy was not directly predictive of outcome in our population. The possibility exists that this difference in outcome is secondary to a difference in pharmacodynamics or pharmacogenomics leading to a difference in the metabolism of the various drugs used to treat osteosarcoma. It is also possible that a difference in tumor biology does exist and could be explored further in a larger study. A larger, multi-institutional study including patients with similar demographics to our study is warranted. More data related to outcomes in patients of Mexican American ancestry will potentially aid in future treatment decision making and management concerning this fast-growing population.

d further in a larger study. A larger, multi-institutional study including patients with similar demographics to our study is warranted. More data related to outcomes in patients of Mexican American ancestry will potentially aid in future treatment decision making and management concerning this fast-growing population. Supported in part by a National Institutes of Health NIH-NCATS UL1TR000149 CTSA grant through the Institute of Medicine and Science (IIMS) at the University of Texas Health Science Center at San Antonio to J.Y.H. and A.-M.L., and a Fellowship Training Grant through the Cancer Prevention & Research Institute of Texas (CPRIT) to A.J.S. The authors declare no conflict of interest.

Recent advances in treatment strategies for childhood acute lymphoblastic leukemia (ALL) have improved the overall survival rate by 80% to 90%.1–3 Enhanced chemotherapeutic agents, refined risk classification criteria, and improved supportive care have contributed to these high cure rates, but significant toxicity remains a major risk factor that causes long-term morbidity and decreased quality of life. Osteonecrosis (ON) has been increasingly documented in pediatric ALL and presents a challenging complication during modern chemotherapy.4–7 ON can result in joint dysfunction and subsequent impairments in activities of daily living among long-term survivors.8,9 Well-known risk factors for ON include age above 10 years, female sex, and use of dexamethasone (DEX).5,7 Although the precise pathophysiology of ON remains unknown, corticosteroid administration has been shown to induce ischemia, upregulate apoptosis of osteoblasts and osteocytes, and prolong osteoclast lifespans.10

9 Well-known risk factors for ON include age above 10 years, female sex, and use of dexamethasone (DEX).5,7 Although the precise pathophysiology of ON remains unknown, corticosteroid administration has been shown to induce ischemia, upregulate apoptosis of osteoblasts and osteocytes, and prolong osteoclast lifespans.10 Most previous studies regarding ON in children with ALL have been limited to European and North American study groups, as there is little data concerning Japanese or Asian patients. Therefore, the aim of the present study was to assess the incidence, risk factors, and morbidity of corticosteroid-induced ON in ALL studies conducted by the Japanese Childhood Cancer and Leukemia Study Group (JCCLSG). We retrospectively analyzed the data of 1095 patients enrolled in 3 consecutive ALL studies (ALL941, ALL2000, and ALL2004) conducted by the JCCLSG. Prednisolone (PSL) was used as the primary corticosteroid in all studies, with DEX acting as a partial substitute for PSL in ALL2004. ON patients were practically identified by symptoms and ON was confirmed with imaging studies in all patients.

ecutive ALL studies (ALL941, ALL2000, and ALL2004) conducted by the JCCLSG. Prednisolone (PSL) was used as the primary corticosteroid in all studies, with DEX acting as a partial substitute for PSL in ALL2004. ON patients were practically identified by symptoms and ON was confirmed with imaging studies in all patients. MATERIALS AND METHODS Patients and Treatment ALL941, ALL2000, and ALL2004 were conducted between 1994 and 2000, 2000 and 2004, and 2004 and 2010, respectively. The therapies on these studies were risk adjusted but not randomized. Patients enrolled in ALL941 and ALL2000 were aged 1 to 15 years, whereas those enrolled in ALL2004 were aged 1 to 18 years. All participants were newly diagnosed with B-precursor ALL or T-cell ALL, and those with a mature B-cell phenotype and Philadelphia chromosome-positive ALL were excluded. All studies were conducted across 18 hospitals that were members of the JCCLSG. The study protocol was approved by the institutional review board of each study, and written informed consent was provided by the patients or their legal guardians before treatment.

pe and Philadelphia chromosome-positive ALL were excluded. All studies were conducted across 18 hospitals that were members of the JCCLSG. The study protocol was approved by the institutional review board of each study, and written informed consent was provided by the patients or their legal guardians before treatment. The treatment protocols adopted in ALL941 and ALL2000 were reported previously.11,12 The patients were stratified according to leukocyte count and age at the time of diagnosis into standard-risk (SR), high-risk (HR), and high-high-risk (HHR) groups. The ALL941 and ALL2000 study protocols were almost identical except for the addition of doxorubicin administration to patients with a leukocyte count <10,000/µL and age below 5 years in ALL2000. Treatment schedules and adopted drugs are briefly described in the supplement (see Supplemental Digital Content 1, Table 1,http://links.lww.com/JPHO/A55).

protocols were almost identical except for the addition of doxorubicin administration to patients with a leukocyte count <10,000/µL and age below 5 years in ALL2000. Treatment schedules and adopted drugs are briefly described in the supplement (see Supplemental Digital Content 1, Table 1,http://links.lww.com/JPHO/A55). The ALL2004 treatment protocols are described in Figure 1 and Table 1. Previous risk classification criteria were modified according to the National Cancer Institute criteria,13 resulting in a shift from HR to SR in 6- to 9-year-old patients with leukocyte counts of 5000 to 10,000 cells/µL. After a 7-day PSL regimen, induction therapy in the SR and HR groups was almost identical to that in previous studies.11,12 In the HHR group, cyclophosphamide was added on day 8. After achieving complete remission, all risk groups received the same intensification therapy (Int-1). At week 15, SR patients with MRD levels <10−3 received further intensification therapy (Int-2) that was followed by maintenance therapy (M-1 and M-2) until week 110. In the HR and HHR groups, patients with MRD levels <10−3 at week 15 received 2 cycles of reinduction/intensification therapy (Rc1/Int-2 and Rc1/Int-3 in HR, Rc2/Int-4 and Rc2/Int-5 in HHR group) that was followed by the same maintenance therapy (M-3 and M-4) until week 165. Patients with MRD levels ≥10−3 at week 12 in the SR and HR/HHR groups were assigned to salvage arms 1 and 2, respectively. In the salvage regimen, patients received intensification therapy comprising 2 cycles of Rc-2/etopside+cytarabine+l-asparaginase. For CNS prophylaxis, SR and HR patients received extended TIT injections beginning from day 1. When IT was combined with a high dose of methotrexate, only cytarabine and hydrocortisone were injected (double intrathecal [DIT] injection). TIT injections were repeated every 6 weeks in the first year, every 8 weeks in the second year, and every 12 weeks in the third year. The HHR group patients included in salvage arm 2 received 18 Gy of CRT in addition to 6 and 7 doses of TIT injections until week 22 and 32 of therapy, respectively.

cal [DIT] injection). TIT injections were repeated every 6 weeks in the first year, every 8 weeks in the second year, and every 12 weeks in the third year. The HHR group patients included in salvage arm 2 received 18 Gy of CRT in addition to 6 and 7 doses of TIT injections until week 22 and 32 of therapy, respectively. FIGURE 1 Treatment framework and minimal residual disease (MRD) stratification in the ALL2004 study. Patients with MRD levels ≥10−3 at week 12 received salvage therapy (dotted arrows), whereas the remainder continued to receive the initial risk-adapted therapy (solid arrows). Treatment schedules are shown in Table 1. CRT indicates cranial radiotherapy; HR, high-risk group; HHR, high-high-risk group; SR, standard-risk group. TABLE 1 Drug Dosage and Schedule for ALL2004 Cumulative doses of the corticosteroids administered in ALL941/2000 and ALL2004 are listed in Table 2. TABLE 2 Cumulative Dose of Corticosteroid in Trials ALL941/2000 and ALL2004 Identification of ON Patients Because the significance of this therapy-related toxicity had not been fully appreciated until the early 2000s, case report form in the 3 studies did not request data regarding ON. Thus, cases with ON were collected by the questionnaire specified for ON to the investigators of JCCLSG. Most of the ON patients were identified based on clinical symptoms such as bone pain and further confirmed with diagnostic imaging studies (x-ray/magnetic resonance imaging [MRI]) by the each institutional radiologists, except one who was asymptomatic and diagnosed by imaging studies at the discretion of primary physician.

of the ON patients were identified based on clinical symptoms such as bone pain and further confirmed with diagnostic imaging studies (x-ray/magnetic resonance imaging [MRI]) by the each institutional radiologists, except one who was asymptomatic and diagnosed by imaging studies at the discretion of primary physician. Statistical Analysis Differences in the categorical variables of patient characteristics were analyzed using the χ2 test. The cumulative incidence of ON during the study period was estimated using Kaplan-Meier analysis. The median follow-up periods were 147.4, 100.3, and 43.6 months for patients enrolled in ALL941, ALL2000, and ALL2004, respectively (median follow-up period, 78.7 mo for all patients). Differences in cumulative incidence between patient subsets were tested using the log-rank test.

estimated using Kaplan-Meier analysis. The median follow-up periods were 147.4, 100.3, and 43.6 months for patients enrolled in ALL941, ALL2000, and ALL2004, respectively (median follow-up period, 78.7 mo for all patients). Differences in cumulative incidence between patient subsets were tested using the log-rank test. RESULTS A total of 1095 patients were enrolled in the present study (ALL941, n=464; ALL2000, n=305; and ALL2004, n=326). Sixteen of 1095 patients developed ON during or after treatment, 4 (0.86%), 2 (0.65%), and 10 (3.1%) were in ALL941, ALL2000, and ALL2004, respectively. In patients with ON, the median age at diagnosis of ALL was 11.5 years (range, 5 to 16 y) and the male to female ratio was 1:3. When patients were evaluated by risk classification, only 1 patient in the SR group and 15 patients in the HR/HHR groups developed ON. Comparisons of the characteristics of patients with and without ON are presented in Table 3, which shows a predominance of females aged 10 years and above, treatment with ALL2004, and high risk (P<0.01) in patients with ON. Notably, 9 of the 12 female and 3 of the 4 male patients with ON were aged 10 years and above, the latter was marginally significant (P=0.044). ON was diagnosed at median treatment weeks 56.5 (range, 32 to 264) and 66 (range, 37 to 120) in ALL941/2000 and ALL2004, respectively. The median cumulative corticosteroid doses at the time of ON onset were as follows: PSL, 5700 mg/m2 (range, 3480 to 13,880 mg/m2) in ALL941/2000 and PSL, 6030 mg/m2 (range, 3480 to 13,800 mg/m2) and DEX, 330 mg/m2 (range, 240 to 330 mg/m2) in ALL2004. As described in Table 2, SR patients in ALL2004 originally did not receive DEX, and despite the cumulative dose of PSL far exceeded the median doses for patients with ON at onset, none of them eventually developed ON. To obtain total PSL equivalents, DEX was multiplied by a conversion factor of 6.514; therefore, a relatively higher steroid dose was used in ALL2004 compared with that used in ALL941/2000.

spite the cumulative dose of PSL far exceeded the median doses for patients with ON at onset, none of them eventually developed ON. To obtain total PSL equivalents, DEX was multiplied by a conversion factor of 6.514; therefore, a relatively higher steroid dose was used in ALL2004 compared with that used in ALL941/2000. TABLE 3 Comparison of Patient Characteristics Between With and Without ON The cumulative 5-year incidence of ON was 0.76% (SE, 0.43%), 0.35% (SE, 0.35%), and 3.6% (SE, 1.1%) in ALL941, ALL2000, and ALL2004, respectively (Fig. 2), with a significant difference between ALL2004 and ALL941/2000 (P<0.01). To assess the contribution of sex and age to ON incidence in patients receiving DEX-containing protocols, the cumulative incidence of ON was estimated in ALL2004 (Figs. 3A, B). Both sex and age were significantly associated with the 5-year ON incidence rate (P<0.01), whereas female sex and age 10 years and above were HR factors for ON. The cumulative 5-year incidence of ON for girls over 10 years of age was 25.6% (SE, 8.4%), which was extremely higher than the rest of patients in ALL2004 (P<0.0001) (Fig. 3C). FIGURE 2 The cumulative incidence of osteonecrosis (ON) in the 3 Japanese Childhood Cancer and Leukemia Study Group studies on acute lymphoblastic leukemia (ALL). ALL941: 0.76%, SE, 0.43%; ALL2000: 0.35%, SE, 0.35%; ALL2004: 3.6%, SE, 1.1%.

The cumulative 5-year incidence of ON was 0.76% (SE, 0.43%), 0.35% (SE, 0.35%), and 3.6% (SE, 1.1%) in ALL941, ALL2000, and ALL2004, respectively (Fig. 2), with a significant difference between ALL2004 and ALL941/2000 (P<0.01). To assess the contribution of sex and age to ON incidence in patients receiving DEX-containing protocols, the cumulative incidence of ON was estimated in ALL2004 (Figs. 3A, B). Both sex and age were significantly associated with the 5-year ON incidence rate (P<0.01), whereas female sex and age 10 years and above were HR factors for ON. The cumulative 5-year incidence of ON for girls over 10 years of age was 25.6% (SE, 8.4%), which was extremely higher than the rest of patients in ALL2004 (P<0.0001) (Fig. 3C). FIGURE 2 The cumulative incidence of osteonecrosis (ON) in the 3 Japanese Childhood Cancer and Leukemia Study Group studies on acute lymphoblastic leukemia (ALL). ALL941: 0.76%, SE, 0.43%; ALL2000: 0.35%, SE, 0.35%; ALL2004: 3.6%, SE, 1.1%. FIGURE 3 The cumulative incidence of osteonecrosis (ON) in ALL2004 according to sex (a), age (b), and combined (c). A, Boys (n=2/190): 1.3%, SE, 0.9%; girls (n=8/136): 6.7%, SE, 2.3%. B, Age below 10 years (n=1/249): 0.42%, SE, 0.42%; age 10 years and above (n=9/77): 15.6%, SE, 4.8%. C, Girls 10 years and above (n=7/33): 25.6%, SE, 8.42%; others (n=3/293): 1.19%, SE, 0.68%.

in ALL2004 according to sex (a), age (b), and combined (c). A, Boys (n=2/190): 1.3%, SE, 0.9%; girls (n=8/136): 6.7%, SE, 2.3%. B, Age below 10 years (n=1/249): 0.42%, SE, 0.42%; age 10 years and above (n=9/77): 15.6%, SE, 4.8%. C, Girls 10 years and above (n=7/33): 25.6%, SE, 8.42%; others (n=3/293): 1.19%, SE, 0.68%. The characteristics of the 16 patients who eventually developed ON are listed in Table 4. All patients showed typical imaging findings on MRI except 1 (case 941-3) who underwent only x-ray that showed bilateral flattened femoral head. The most commonly affected joints and bones were the hip joint (44%), the knee joint (25%), and the femur (13%). Three patients (19%) exhibited multiple lesions. Nine (56%) continued to receive the planned steroid therapy despite the diagnosis of ON, whereas the doses were decreased or withdrawn in 7 (44%). ON management varied for each patient depending on the physician discretion. Most patients (75%) received supportive care only and were advised to avoid lifting heavy weights (grade 2 according to Common Terminology Criteria for Adverse Event version 4.0). Three patients (19%) underwent surgical intervention (grade 3) and 1 was treated with oral bisphosphonates (grade 2). With the median follow-up times of 33 months (range, 4 to 194), the clinical outcomes of ON were as follows: 12 with amelioration of ON and 3 with stable disease, except 1 who suffered a relapse of leukemia. TABLE 4 Affected Joints and Clinical Course of Patients With ON

The characteristics of the 16 patients who eventually developed ON are listed in Table 4. All patients showed typical imaging findings on MRI except 1 (case 941-3) who underwent only x-ray that showed bilateral flattened femoral head. The most commonly affected joints and bones were the hip joint (44%), the knee joint (25%), and the femur (13%). Three patients (19%) exhibited multiple lesions. Nine (56%) continued to receive the planned steroid therapy despite the diagnosis of ON, whereas the doses were decreased or withdrawn in 7 (44%). ON management varied for each patient depending on the physician discretion. Most patients (75%) received supportive care only and were advised to avoid lifting heavy weights (grade 2 according to Common Terminology Criteria for Adverse Event version 4.0). Three patients (19%) underwent surgical intervention (grade 3) and 1 was treated with oral bisphosphonates (grade 2). With the median follow-up times of 33 months (range, 4 to 194), the clinical outcomes of ON were as follows: 12 with amelioration of ON and 3 with stable disease, except 1 who suffered a relapse of leukemia. TABLE 4 Affected Joints and Clinical Course of Patients With ON DISCUSSION In the 3 most recent JCCLSG ALL studies, we found that a significant number of patients developed ON during or after treatment. ALL2004 study was conducted to evaluate the efficacy of DEX usage as a corticosteroid in the context of intensification of reinduction phase, comparing with the preceding 2 studies wherein PSL was the only corticosteroid adopted. This strategy also enabled us to compare the DEX toxicity with that of PSL. The results clearly demonstrated the higher incidence of ON in ALL2004, indicating DEX exposure was the risk for ON in ALL chemotherapy.

reinduction phase, comparing with the preceding 2 studies wherein PSL was the only corticosteroid adopted. This strategy also enabled us to compare the DEX toxicity with that of PSL. The results clearly demonstrated the higher incidence of ON in ALL2004, indicating DEX exposure was the risk for ON in ALL chemotherapy. The overall incidence of ON was 1.5% (16/1095), which was comparable with that in a previous study by the Japan Association of Childhood Leukemia Study (JACLS) (2.4%, Hiroki H, Yasushi I, Teruaki H, Makoto Y, Megumi O, Tooru K, Shinichiro N, Junichi H, Keizo H, Keiko Y, and Tatsutoshi N; unpublished data). In studies from Europe and the United states, the ON incidence was highly variable (1% to 2% up to 9%) and dependent on patient characteristics and treatment intensity.5–7 Furthermore, the detection methods of ON have significantly affected the incidence. Recent report from St Jude Total XV study showed that 17.6% of patients had the symptomatic ON, whereas the asymptomatic ON was detected in >50% of patients by the prospective screening with MRI test.15 With regard to the effects of race, the incidence of ON is reportedly higher in whites than in patients of African descent.7 Although it remains unclear whether the Asian race is related to an increased risk of ON, our results showed that the incidence of ON in Japanese children seemed to be comparable with that in European and American children. However, it should be taken into account the limitation of the present assessment: the possible missing of asymptomatic cases and the diagnosis partly depending on physician’s discretion.

ur results showed that the incidence of ON in Japanese children seemed to be comparable with that in European and American children. However, it should be taken into account the limitation of the present assessment: the possible missing of asymptomatic cases and the diagnosis partly depending on physician’s discretion. In this study, female sex, age 10 years and above, and the use of DEX as a corticosteroid were significant risk factors for ON. Of the 33 female patients aged over 10 years who received DEX, 7 developed ON (cumulative incidence, 25.6%). This was the extremely higher incidence of ON comparing with the rest of patients. Although females were found to be at a higher risk of developing ON in the Children’s Cancer Study Group (CCG) and Italian studies,5,7 there was no such correlation in studies performed in the UK and Germany and at the Dana Farber Cancer Institute (Boston, MA).6,16,17 In addition, a Japanese study conducted by the JACLS failed to show a significant female predominance (male to female ratio, 7:9). Therefore, the effects of sex on ON pathogenesis remain unclear.

no such correlation in studies performed in the UK and Germany and at the Dana Farber Cancer Institute (Boston, MA).6,16,17 In addition, a Japanese study conducted by the JACLS failed to show a significant female predominance (male to female ratio, 7:9). Therefore, the effects of sex on ON pathogenesis remain unclear. A significant contribution of age to ON onset has been robustly documented by most retrospective and prospective studies.5–7,9,16,18–21 Among children aged 10 years and above, those aged 16 to 20 years were at the highest risk of ON. The eligible patient age was 1 to 15 years in ALL941/2000 and 1 to 18 years in ALL2004; therefore, we may have underestimated the incidence of ON. Further monitoring is necessary when ALL treatment protocols designed for children are extended to adolescence and young adulthood.

20 years were at the highest risk of ON. The eligible patient age was 1 to 15 years in ALL941/2000 and 1 to 18 years in ALL2004; therefore, we may have underestimated the incidence of ON. Further monitoring is necessary when ALL treatment protocols designed for children are extended to adolescence and young adulthood. The potential effect of DEX on ALL is 6.5 times that of PSL, resulting in an increase in the use of DEX for ALL treatment. Because DEX is more toxic to bone tissues,14,22 a higher incidence of ON has been a major concern in the design of treatment protocols. In ALL2004, DEX was incorporated only in the reinduction phase because an increased incidence of ON and mortality was reported with the use of DEX in the induction phase.23 Nonetheless, our data revealed a higher cumulative incidence of ON associated with DEX administration; this finding was comparable with the results of the Dana Farber Consortium study DFCI 00-01, wherein DEX was used in postremission intensification therapy and/or in the maintenance phase.24 Although the total corticosteroid dose (analyzed as PSL equivalents) at therapy completion were slightly lower in ALL2004 than in ALL941/2000 (Table 2), ON was most frequent in patients who had received only DEX in the HR group in ALL2004. These results suggest that DEX administration at any dose (as PSL equivalents) and in any treatment phase affects the incidence of ON. A recent report from the CCG found that DEX administration could influence the risk of ON21 and that alternate-week DEX administration during delayed intensification therapy decreased ON incidence compared with continuous DEX. In our ALL2004 protocol, DEX was administered continuously for 2 weeks, and it would have been beneficial to modify the DEX schedule from continuous administration to alternate-week administration.

ernate-week DEX administration during delayed intensification therapy decreased ON incidence compared with continuous DEX. In our ALL2004 protocol, DEX was administered continuously for 2 weeks, and it would have been beneficial to modify the DEX schedule from continuous administration to alternate-week administration. Recently, biological and genetical basis for ON development has been extensively investigated. Children’s Oncology Group tested 12 polymorphisms of candidate genes and identified children with PAI-1 GA/AA genotypes were significantly associated with ON.25 Another study from St Jude Children’s Research Hospital showed polymorphisms of ACP1 were associated with risk of symptomatic ON as well as with lower serum albumin and higher cholesterol levels.15 These results suggest that some patients are prone to develop ON and individualized therapy should be needed in the future ALL studies.

from St Jude Children’s Research Hospital showed polymorphisms of ACP1 were associated with risk of symptomatic ON as well as with lower serum albumin and higher cholesterol levels.15 These results suggest that some patients are prone to develop ON and individualized therapy should be needed in the future ALL studies. In the present report, cases with ON were retrospectively collected by the questionnaire, and most of the ON patients were identified by symptoms and confirmed with imaging studies (x-ray/MRI) without central review. Despite such limitations, the clinical features of all 16 ON patients in our study were virtually comparable with those of patients in previous studies.6,7,16 Weight-bearing joints were commonly affected, whereas asymptomatic lesions might have been overlooked.15 Once ON is confirmed, the physician must decide whether steroids should be withheld or continued, considering that no consensus guideline is available thus far. Most of our patients were prescribed a planned dose of steroids without compromising functional outcomes after ON development. We believe that it may not be necessary to withhold steroids at the risk of leukemia relapse.

ids should be withheld or continued, considering that no consensus guideline is available thus far. Most of our patients were prescribed a planned dose of steroids without compromising functional outcomes after ON development. We believe that it may not be necessary to withhold steroids at the risk of leukemia relapse. Bisphosphonates, which are structurally similar to pyrophosphates, inhibit osteoclast activity and bone turnover, thus exerting beneficial effects on bone mineralization.26 Alendronate, a third-generation bisphosphonate, is reportedly effective in the prevention of femoral head collapse in ON patients.27 Wiernikowski et al28 showed that alendronate-induced changes in bone mineral metabolism/homeostasis benefited bone mineralization in children with ALL or non-Hodgkin lymphoma with steroid-induced osteopenia. Another bisphosphonate, pamidronate, was shown to be effective in the management of pain and motor function recovery in symptomatic ON occurring in children with ALL.29 In the present study, alendronate was administered to 1 patient with symptomatic ON of the bilateral hip and knee joints; this resulted in no further deterioration of functional outcome and no treatment-induced side effects. However, further studies are required to clarify the potential benefits of concomitant bisphosphonate and steroid use for ON treatment.

ministered to 1 patient with symptomatic ON of the bilateral hip and knee joints; this resulted in no further deterioration of functional outcome and no treatment-induced side effects. However, further studies are required to clarify the potential benefits of concomitant bisphosphonate and steroid use for ON treatment. In summary, the overall incidence of ON was 1.5% in the JCCLSG ALL studies, which was comparable with that reported in previous studies conducted in the Unites States and Europe. The known risk factors of age above 10 years, female sex, and DEX use were all significantly associated with an increase in the cumulative incidence of ON. In our future studies, we are intending to routinely screen for ON development with MRI test, especially those incorporating DEX in the treatment protocol. Although an ON management regimen remains to be established, steroids should not be withheld at the risk of ALL relapse. Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.jpho-online.com. The authors declare no conflict of interest. Reprints: Nobuyuki Hyakuna, MD, Center of Bone Marrow Transplantation, Hospital of University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan (e-mail: hyakunan@med.u-ryukyu.ac.jp).

Medulloblastoma is the most common malignant brain tumor of childhood. Surgery and chemotherapy are integral parts of treatment, with craniospinal radiotherapy also utilized in patients over 3 years of age. Although an increase in the intensity of therapy over time has led to an improved overall survival, this has conversely led to a higher frequency of short-term and long-term complications of therapy.

motherapy are integral parts of treatment, with craniospinal radiotherapy also utilized in patients over 3 years of age. Although an increase in the intensity of therapy over time has led to an improved overall survival, this has conversely led to a higher frequency of short-term and long-term complications of therapy. Hepatic sinusoidal obstruction syndrome (HSOS), also known as veno-occlusive disease, is a clinical syndrome characterized by tender hepatomegaly, hyperbilirubinemia, and weight gain due to fluid retention. The underlying pathogenesis is complex and thought to involve toxic injury to sinusoidal endothelial cells.1 Histologic confirmation with liver biopsy is the gold standard for diagnosis; however, due to the procedural risk and invasive nature of a biopsy the majority of cases are diagnosed clinically. Diagnosis is based on either the McDonald (modified Seattle) or Jones (Baltimore) criteria. The McDonald criteria require the presence of 2 features from hepatomegaly with right upper quadrant pain, total bilirubin of 34.2 μmol/L or more (normal range <20 μmol/L), and ascites or unexplained weight gain >2% baseline.2 The Jones criteria require a total bilirubin of 34.2 μmol/L or more and presence of at least 2 of the following: hepatomegaly, ascites, and weight gain >5% baseline.3 These criteria allow a diagnosis of HSOS with good specificity and negative predictive value,4 but they have a relatively low sensitivity.5 Additional characteristic features that may assist in diagnosis include thrombocytopenia refractory to platelet transfusions and an increase in fibrinolytic parameters, particularly plasminogen activator inhibitor-1 antigen in combination with elevated d-dimer and bilirubin.6 Doppler-ultrasonographic findings can help to support the diagnosis of HSOS and may include hepatomegaly, splenomegaly, ascites, gallbladder wall thickening, elevation of the hepatic artery resistive index, and reversal of flow in the portal vein.7

nhibitor-1 antigen in combination with elevated d-dimer and bilirubin.6 Doppler-ultrasonographic findings can help to support the diagnosis of HSOS and may include hepatomegaly, splenomegaly, ascites, gallbladder wall thickening, elevation of the hepatic artery resistive index, and reversal of flow in the portal vein.7 HSOS is most frequently reported as a complication of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and in patients receiving chemotherapy for Wilms tumor and rhabdomyosarcoma. However, HSOS is a rare occurrence in the context of conventional chemotherapy for other childhood malignancies. We report a 5-year-old girl who developed severe HSOS during therapy for high-risk anaplastic medulloblastoma and was treated using defibrotide with resolution of the HSOS. Consent for publication of the case report was obtained from the parents of the patient.

onventional chemotherapy for other childhood malignancies. We report a 5-year-old girl who developed severe HSOS during therapy for high-risk anaplastic medulloblastoma and was treated using defibrotide with resolution of the HSOS. Consent for publication of the case report was obtained from the parents of the patient. CASE REPORT A 5-year-old Aboriginal girl presented with a 2-week history of ataxia and speech disturbance. Magnetic resonance imaging of the brain and spine showed a localized 46 mm×37 mm mass present within the posterior fossa comprising of a solid and cystic component with no evidence of drop metastases or leptomeningeal spread. Gross total resection was performed with operative findings consisting of a right-sided cerebellar tumor extending through the foramen of Luschka into the cerebello-pontine angle. Secondary hydrocephalus was present due to compression of the fourth ventricle. Histologic examination revealed a diffusely anaplastic medulloblastoma, with no C-MYC or N-MYC amplification detected by fluorescence in situ hybridization.

ar tumor extending through the foramen of Luschka into the cerebello-pontine angle. Secondary hydrocephalus was present due to compression of the fourth ventricle. Histologic examination revealed a diffusely anaplastic medulloblastoma, with no C-MYC or N-MYC amplification detected by fluorescence in situ hybridization. She was treated according to the Children’s Oncology Group high-risk medulloblastoma protocol ACNS0332. She received 36 Gy craniospinal radiation therapy with a 19.8 Gy boost to the posterior fossa along with weekly vincristine (1.5 mg/m2 per dose). After a 6-week rest period, maintenance chemotherapy was started comprising six 29-day cycles, each consisting of cisplatin (75 mg/m2) on day 1, vincristine (1.5 mg/m2) on day 1 and 8, and cyclophosphamide (1000 mg/m2) on day 2 and 3. Pegylated human granulocyte colony stimulating factor was administered on day 4 of each cycle. The first cycle of chemotherapy was well tolerated. Three days into her second cycle she developed low-grade fever and was commenced on intravenous antibiotics as part of our institutional protocol. The following day she was noted to have developed thrombocytopenia (7×109/L, normal range 150 to 400×109/L) and parainfluenza virus type 1 was isolated on nasopharyngeal aspirate. At this stage her liver function tests were normal. By day 6 of her second cycle she was noted to have a mildly enlarged and tender liver. There was no significant weight gain and no evidence of HSOS on abdominal ultrasound. However, by day 8 she had an 11% increase in weight, a tense, tender and distended abdomen, ascites, hepatomegaly, and persistent thrombocytopenia refractory to platelet transfusions. Laboratory investigations revealed hyperbilirubinemia (40 μmol/L, normal range <20 μmol/L), low hemoglobin (77 g/L, normal range 115 to 155 g/dL), elevated liver enzymes alanine transaminase (ALT) (152 U/L, normal range <30 U/L), aspartate transaminase (AST) (422 U/L, normal range <50 U/L), and hypoalbuminemia (25 g/L, normal range 32 to 48 g/L). A large right-sided pleural effusion was present on chest x-ray. A repeat abdominal ultrasound scan and Doppler study demonstrated reversal in portal vein flow and increased peak velocity in the hepatic arteries (450 cm/s). HSOS was diagnosed and she was started on defibrotide (25 mg/kg/d) and ursodeoxycholic acid (20 mg/kg/d).

ight-sided pleural effusion was present on chest x-ray. A repeat abdominal ultrasound scan and Doppler study demonstrated reversal in portal vein flow and increased peak velocity in the hepatic arteries (450 cm/s). HSOS was diagnosed and she was started on defibrotide (25 mg/kg/d) and ursodeoxycholic acid (20 mg/kg/d). Vincristine was withheld and she was transferred to the pediatric intensive care unit for supportive care, which included strict regulation of fluid balance and maintenance of urine output with frusemide and dopamine infusions, preservation of oncotic pressure with human albumin infusions, correction of coagulopathy using fresh-frozen plasma, supplementary oxygen, and broad spectrum intravenous antimicrobials for fever. Peak laboratory values included an AST of 3290 U/L, ALT of 1370 U/L, and a total bilirubin of 310 μmol/L, occurring 7, 8, and 12 days after the first manifestation of HSOS (thrombocytopenia), respectively. Her symptoms resolved over 19 days following supportive care and 2 weeks of defibrotide, with normalization of her liver function tests. Graphs 1 and 2 show the liver transaminase and bilirubin profiles during the second cycle of maintenance chemotherapy. GRAPH 1 Liver transaminase profile during second cycle of maintenance chemotherapy. Days on which defibrotide was started and stopped are shown on x-axis. GRAPH 2 Bilirubin profile during second cycle of maintenance chemotherapy. Days on which defibrotide was started and stopped are shown on x-axis.

Vincristine was withheld and she was transferred to the pediatric intensive care unit for supportive care, which included strict regulation of fluid balance and maintenance of urine output with frusemide and dopamine infusions, preservation of oncotic pressure with human albumin infusions, correction of coagulopathy using fresh-frozen plasma, supplementary oxygen, and broad spectrum intravenous antimicrobials for fever. Peak laboratory values included an AST of 3290 U/L, ALT of 1370 U/L, and a total bilirubin of 310 μmol/L, occurring 7, 8, and 12 days after the first manifestation of HSOS (thrombocytopenia), respectively. Her symptoms resolved over 19 days following supportive care and 2 weeks of defibrotide, with normalization of her liver function tests. Graphs 1 and 2 show the liver transaminase and bilirubin profiles during the second cycle of maintenance chemotherapy. GRAPH 1 Liver transaminase profile during second cycle of maintenance chemotherapy. Days on which defibrotide was started and stopped are shown on x-axis. GRAPH 2 Bilirubin profile during second cycle of maintenance chemotherapy. Days on which defibrotide was started and stopped are shown on x-axis. The patient was subsequently able to complete her remaining 4 cycles of chemotherapy. Audiometric evaluation revealed grade 2 ototoxicity, resulting in a 50% reduction in the cisplatin dose for her third cycle. In addition, there was a 25% dose reduction of cyclophosphamide due to delayed hematopoietic recovery and vincristine was omitted due to persistent hyperbilirubinemia (40 μmol/L). Grade 3 ototoxicity resulted in a 75% dose reduction of cisplatin for the fourth cycle with full-dose administration of cyclophosphamide and vincristine. Prophylactic defibrotide (25 mg/kg/d) was administered during the third and fourth maintenance cycles and ursodeoxycholic acid was continued throughout the duration of therapy. During cycles 5 and 6, cisplatin was completely omitted due to grade 4 ototoxicity and cyclophosphamide and vincristine were administered at full dose. There was no further recurrence of HSOS. The patient remained in clinical and radiologic remission for 8 months following the completion of therapy. Subsequent surveillance magnetic resonance imaging revealed disease recurrence in the right ventricular frontal horn.

amide and vincristine were administered at full dose. There was no further recurrence of HSOS. The patient remained in clinical and radiologic remission for 8 months following the completion of therapy. Subsequent surveillance magnetic resonance imaging revealed disease recurrence in the right ventricular frontal horn. DISCUSSION The incidence of HSOS after HSCT in children has been reported by a number of groups and ranges between 11% and 60%, with a mean incidence of 25%.8 Risk factors in HSCT recipients include younger age (<6.7 y), previous liver irradiation, previous Gemtuzumab ozogamicin, prior hepatic disease, busulfan-containing conditioning regimens, and total body irradiation, with a higher risk for the latter 2 when administered in conjunction with cyclophosphamide. An increased risk is seen after HSCT for neuroblastoma, familial hemophagocytic lymphohistiocytosis, and osteopetrosis.8 The incidence of HSOS after therapy for Wilms tumor ranges from 1.2% to 8%9 with actinomycin-D identified as the primary etiological agent. Additional risk factors in this group include young age, previous abdominal radiotherapy, right-sided tumors, and lower body weight.10–12 HSOS has also been reported during treatment for childhood rhabdomyosarcoma9,13–19 with an incidence of 1.2% to 5.3%.14,18 Identified risk factors in this population include VAC (vincristine, actinomycin-D, and cyclophosphamide) chemotherapy and age <36 months. There is a single report of HSOS occurring in a 2-year-old boy with recurrent yolk sac tumor receiving VAC chemotherapy20 and a report of HSOS after 2 doses of actinomycin-D in a 14-year-old girl with a paraspinal primitive neuroectodermal tumor who had received prior combination chemotherapy with etoposide, ifosfamide, cyclophosphamide, doxorubicin, vincristine, and external beam radiation partly involving the liver.20 In addition, HSOS is a well-reported complication of oral 6-thioguanine during acute lymphoblastic leukemia (ALL) therapy,21,22 while there have been anecdotal reports of HSOS during induction therapy for pediatric ALL23,24 and induction therapy for primary CNS lymphoma.25

l beam radiation partly involving the liver.20 In addition, HSOS is a well-reported complication of oral 6-thioguanine during acute lymphoblastic leukemia (ALL) therapy,21,22 while there have been anecdotal reports of HSOS during induction therapy for pediatric ALL23,24 and induction therapy for primary CNS lymphoma.25 Although the incidence of HSOS in children undergoing HSCT, treatment for Wilms tumor, rhabdomyosarcoma, and ALL, has been established within large co-operative group trials, there are no reports within such trials of HSOS in children undergoing therapy for medulloblastoma.26–29 Outside of the large co-operative group trials, there have only been 2 prior case reports of HSOS following standard-dose chemotherapy for medulloblastoma.30,31 One patient was a 19-month-old girl who developed fatal HSOS after 8 days of a vincristine, carboplatin, and lomustine based regimen, despite supportive measures and treatment with low-molecular weight heparin.30 The second was a 14-year-old boy who initially received 36 Gy craniospinal radiation with a 20 Gy boost to the posterior fossa with concurrent weekly vincristine and developed severe HSOS 4 days after completion of the first cycle of a vincristine, carboplatin, and cyclophosphamide based regimen. He was treated with 1 week of oral ursodeoxycholic acid and 3 days of parenteral N-acetylcysteine, with resolution of the HSOS. To prevent recurrence, the patient subsequently received a reduction in the frequency of cyclophosphamide, with administration every second cycle.31 Our case further highlights the occurrence of HSOS outside the traditional settings discussed above. HSOS may have been precipitated in our patient by a number of factors including cyclophosphamide, young age, and scatter to the liver from her prior craniospinal radiotherapy. Notably, the only feature common to all 3 cases was the administration of vincristine, which also forms part of therapy for Wilms tumor, rhabdomyosarcoma, and ALL. This suggests that vincristine may not be an innocent bystander, but could have a potentiating role in the development of HSOS.

r craniospinal radiotherapy. Notably, the only feature common to all 3 cases was the administration of vincristine, which also forms part of therapy for Wilms tumor, rhabdomyosarcoma, and ALL. This suggests that vincristine may not be an innocent bystander, but could have a potentiating role in the development of HSOS. Severity of HSOS is one of the key factors influencing the outcome of patients, with mortality in excess of 85% in severe disease,5 however, assessment of severity is difficult as it is generally retrospectively defined. Three grades of increasing severity have been classified: mild (resolution of symptoms, decrease of serum bilirubin <34.2 μmol/L, with or without specific treatment), moderate (clinical signs and symptoms of progressive disease, including ascites and/or pleural effusion but no multiorgan failure), and severe (multiorgan failure needing oxygen or mechanical ventilation and/or renal failure and/or encephalopathy).1 In addition to supportive care measures, a number of therapeutic strategies have attempted to improve outcome including N-acetylcysteine,24,31,32 high-dose methylprednisolone,16,33 recombinant human tissue plasminogen activator alone34,35 and in combination with heparin,9,36,37 antithrombin III,23,38 and prostaglandin E1 in combination with heparin39 and thrombomodulin,40 although the data for their use is limited. There is increasing evidence, however, for the use of defibrotide, a polydisperse oligonucleotide with local antithrombotic, anti-ischemic, and anti-inflammatory activity. The therapeutic potential of defibrotide has largely been studied in patients who developed HSOS after HSCT. Our case reports successful treatment with defibrotide for HSOS after standard-dose chemotherapy for childhood medulloblastoma, with the dose of 25 mg/kg/d based on the recommendations from the outcome of a recent dose-finding trial.41

l of defibrotide has largely been studied in patients who developed HSOS after HSCT. Our case reports successful treatment with defibrotide for HSOS after standard-dose chemotherapy for childhood medulloblastoma, with the dose of 25 mg/kg/d based on the recommendations from the outcome of a recent dose-finding trial.41 In summary, our case highlights the occurrence of HSOS in a child receiving standard-dose chemotherapy for medulloblastoma after craniospinal radiotherapy. A diagnosis of HSOS should not be excluded based on the absence of classic risk factors and disease settings, with early consideration given in the presence of premature, unexplained thrombocytopenia after chemotherapy. Defibrotide was an effective therapeutic strategy in our case. R.S.K. is supported by the Whiteman Paediatric Oncology/Haematology Fellowship and the Princess Margaret Hospital Foundation. N.G.G. is supported by the John Lillie Cancer Research Fellowship. The authors declare no conflict of interest. Reprints: Rishi S. Kotecha, MB ChB(Hons), MRCPCH, FRACP, Department of Haematology and Oncology, Princess Margaret Hospital for Children, GPO Box D184, Perth 6840, WA, Australia (e-mail: rishi_k28@hotmail.com).

Despite ongoing improvements in outcomes for children with acute leukemias, relapses continue to occur and the prognosis for these patients remains poor. Thus, new treatments are needed for patients whose leukemia progresses or recurs following established therapies. Bendamustine is a bifunctional mechlorethamine derivative containing a benzimidazole heterocyclic ring. Mechlorethamine and its derivatives are alkylating agents that induce DNA damage, resulting in apoptosis.1 Although the exact mechanism of action of bendamustine and the role of the benzimidazole ring have not been fully defined, bendamustine is active against both quiescent and dividing cells, and treatment of tumor cells with bendamustine results in a large number of DNA double-strand breaks, consistent with its classification as an alkylating agent.2 However, bendamustine is distinct from other alkylating agents in that it possesses additional antitumor activity because of its unique composition.3 Bendamustine has demonstrated activity in adults with chronic lymphocytic leukemia and rituximab-refractory non-Hodgkin lymphoma (NHL).4,5 However, no data with regard to the use of bendamustine in children or in childhood acute leukemia have been reported.

itional antitumor activity because of its unique composition.3 Bendamustine has demonstrated activity in adults with chronic lymphocytic leukemia and rituximab-refractory non-Hodgkin lymphoma (NHL).4,5 However, no data with regard to the use of bendamustine in children or in childhood acute leukemia have been reported. In vitro, bendamustine has been shown to have activity against 2 pediatric acute lymphoblastic leukemia (ALL) T-cell (CCRF-CEM) and B-cell (CCRF-SB) lines at concentrations of 18 and 20 µM, respectively (data on file, Teva Branded Pharmaceutical Products R&D Inc.). Bendamustine has also demonstrated cytotoxic activity in the acute myeloid leukemia (AML) cell line HL-60, albeit to a lesser extent than cell lines of lymphoid origin.6 These results suggest that bendamustine may provide a benefit in acute childhood leukemia that warrants studying in the pediatric population. The present study is an open-label, single-arm, phase I/II, dose-escalation study to determine the recommended phase II dose (RP2D), pharmacokinetics, and tolerability profile, as well as efficacy of bendamustine as a single agent in heavily pretreated pediatric patients with relapsed or refractory acute leukemia.

In vitro, bendamustine has been shown to have activity against 2 pediatric acute lymphoblastic leukemia (ALL) T-cell (CCRF-CEM) and B-cell (CCRF-SB) lines at concentrations of 18 and 20 µM, respectively (data on file, Teva Branded Pharmaceutical Products R&D Inc.). Bendamustine has also demonstrated cytotoxic activity in the acute myeloid leukemia (AML) cell line HL-60, albeit to a lesser extent than cell lines of lymphoid origin.6 These results suggest that bendamustine may provide a benefit in acute childhood leukemia that warrants studying in the pediatric population. The present study is an open-label, single-arm, phase I/II, dose-escalation study to determine the recommended phase II dose (RP2D), pharmacokinetics, and tolerability profile, as well as efficacy of bendamustine as a single agent in heavily pretreated pediatric patients with relapsed or refractory acute leukemia. MATERIALS AND METHODS Study Design This international study was conducted in accordance with the Good Clinical Practice: Consolidated Guidance approved by the International Conference on Harmonisation and all applicable national and local laws and regulations. Patients received bendamustine infused intravenously over 60 minutes on days 1 and 2 of each 21-day cycle. Delays of up to 2 weeks were allowed for neutrophil and platelet recovery, for a maximum cycle duration of 35 days. Clinical response and hematologic recovery were assessed using International Working Group 2003 criteria (morphologic leukemia-free state) with bone marrow evaluation (Fig. 1).7

nd 2 of each 21-day cycle. Delays of up to 2 weeks were allowed for neutrophil and platelet recovery, for a maximum cycle duration of 35 days. Clinical response and hematologic recovery were assessed using International Working Group 2003 criteria (morphologic leukemia-free state) with bone marrow evaluation (Fig. 1).7 FIGURE 1 Treatment and evaluation plan. *All subsequent cycles require clinical determination of patient benefit. †Count recovery for induction defined as neutrophil count ≥1.0×109/L and platelet count ≥100×109/L. ‡Disease evaluation may include peripheral laboratory testing and/or bone marrow evaluation at the discretion of the treating physician. Count recovery for subsequent cycles defined as platelet count not requiring transfusion and neutrophil count ≥500. §Clinical discretion for additional cycles of therapy is required based on patient’s clinical need; maximum of 35 days for recovery, minimum neutrophil count is 500, and platelet count may be supported with transfusion. CR indicates complete response; CRp, complete response without platelet recovery; PR, partial response; SD, stable disease.

r additional cycles of therapy is required based on patient’s clinical need; maximum of 35 days for recovery, minimum neutrophil count is 500, and platelet count may be supported with transfusion. CR indicates complete response; CRp, complete response without platelet recovery; PR, partial response; SD, stable disease. The study consisted of 2 phases. In phase I (dose-escalating), a starting dose of bendamustine 90 mg/m2 was used in the first cohort of 3 patients, with planned escalation to 120 mg/m2 and potentially to 150 mg/m2 in subsequent cohorts of patients. Clearance in adults aged 31 to 84 years does not vary by patient’s age,8 and it was expected that the clearance will be similar among children. Therefore, the initial dose was selected on the basis of the metabolic profile of bendamustine in adults.

mg/m2 and potentially to 150 mg/m2 in subsequent cohorts of patients. Clearance in adults aged 31 to 84 years does not vary by patient’s age,8 and it was expected that the clearance will be similar among children. Therefore, the initial dose was selected on the basis of the metabolic profile of bendamustine in adults. In this 3+3 study design, each cohort could be expanded to up to 6 patients based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 toxicity.9 Dose-limiting toxicity (DLT) was defined as any nonhematologic NCI CTCAE grade 4 toxicity or any grade 3 skin rash or allergic reaction (hematologic events were not considered DLTs because of the nature of patients’ cancers). Bendamustine 150 mg/m2 was to be implemented only if the 120 mg/m2 dose was acceptably tolerated but resulted in subtherapeutic plasma levels when compared with data obtained from adults. In the event that ≥2 patients in the 90 mg/m2 cohort experienced a DLT, the bendamustine dosage was to be reduced to 60 mg/m2. The RP2D was defined as the dose one step below the dose at which ≥2 patients experienced a DLT. In phase II, additional patients were enrolled at the RP2D determined in phase I. Patients were followed until disease progression, withdrawal due to intolerability or other reasons, loss to follow-up, or completion of maximum of 12 cycles of therapy. After the end of treatment, patients were evaluated every 3 months for 12 months after the last dose, or until progression, death, or start of new cancer treatment.

followed until disease progression, withdrawal due to intolerability or other reasons, loss to follow-up, or completion of maximum of 12 cycles of therapy. After the end of treatment, patients were evaluated every 3 months for 12 months after the last dose, or until progression, death, or start of new cancer treatment. Patients Children aged 1 to 20 years with histologically proven (>5% blasts on morphologic assessment) ALL or AML with ≥1 relapse or refractory to the prior treatment and without the opportunity for potentially curative therapy were eligible to participate in this study. Nonhematologic toxic effects of prior therapy (ended ≥2 wk before first dose of study drug) were required to have resolved to grade 0 to 2 according to the NCI CTCAE version 4.0. Inclusion criteria included adequate liver function (bilirubin ≤1.5 times the upper limit of normal [ULN] and aspartate aminotransferase [AST] and alanine aminotransferase [ALT] ≤5 times the age-appropriate ULN) and renal function (serum creatinine <2 times the ULN) and Karnofsky or Lansky performance status ≥60. Previous hematopoietic stem cell transplant (HSCT) was allowed, but a patient’s last myelosuppressive therapy had to have ended at least 2 weeks before the first dose of the study drug. Patients with child-bearing potential were required to agree to practice birth control during the duration of the study and for 30 days after the end of treatment. Written and dated informed consent was obtained from patients or the parent(s) or guardian(s) of minor patients, or as required by local regulations. Patients were excluded if they were currently receiving any other systemic cancer treatment; had any active, uncontrolled systemic infection; severe concurrent disease; symptomatic untreated central nervous system involvement (concurrent systemic treatments were not allowed during the study); active graft-versus-host disease; known human immunodeficiency virus or active hepatitis B or C infection; pregnancy or lactation; or any serious uncontrolled medical or psychologic disorder that would impair the ability of the patient to receive the study drug. Patients treated at any dose of bendamustine were included in the evaluation for safety and efficacy, and pharmacokinetic analysis included all patients eligible for safety analysis who had valid pharmacokinetic data.

edical or psychologic disorder that would impair the ability of the patient to receive the study drug. Patients treated at any dose of bendamustine were included in the evaluation for safety and efficacy, and pharmacokinetic analysis included all patients eligible for safety analysis who had valid pharmacokinetic data. Assessments The primary objective of phase I was to establish the RP2D; efficacy was a secondary assessment. Efficacy was also measured for patients who participated in phase II. The primary efficacy measure was overall response rate (ORR), defined as a composite of complete response (CR) and CR without platelet recovery (CRp) among patients enrolled at the RP2D in both phases at any cycle. The best response was assessed through the study (cycle 1: days 21 [% blasts on complete blood count] and 35 [bone marrow morphologic assessment]; subsequent cycles: complete blood count and differential unless prolonged aplasia required repeat bone marrow examination). CR required no evidence of circulating blasts or extramedullary disease, an M1 marrow (≤5% bone marrow blasts), absolute neutrophil count of 1.0×109/L or greater, and platelet count of 100×109/L or greater.7 Additional efficacy outcomes included ORR for phase II only, duration of response (DOR) in patients who achieved CR or CRp, and biologic activity (at least a partial response [PR], defined as complete disappearance of circulating blasts, an M2 marrow [5% to 25% bone marrow blasts], or appearance of normal progenitor cells or an M1 marrow that did not qualify for CR or CRp) in patients enrolled in either phase. Tolerability assessments included adverse events, clinical laboratory values, concomitant medication throughout treatment, and vital signs.

sts, an M2 marrow [5% to 25% bone marrow blasts], or appearance of normal progenitor cells or an M1 marrow that did not qualify for CR or CRp) in patients enrolled in either phase. Tolerability assessments included adverse events, clinical laboratory values, concomitant medication throughout treatment, and vital signs. Pharmacokinetics Pharmacokinetic samples to determine the plasma concentrations of bendamustine were obtained from all patients in phase I or II during cycle 1 only. Samples were obtained before bendamustine infusion and at preselected time points at 3, 6, 10 (±2), and 24 hours. The 24-hour postinfusion sample was obtained before the start of the infusion on day 2. Further pharmacokinetic sampling was not done after 24 hours because, with the short half-life and low plasma concentrations observed by 12 hours after bendamustine infusion in adults, plasma concentrations were expected to be nonquantifiable or negligible beyond 24 hours in this pediatric population.8 After collection, blood samples were inverted 5 to 10 times to mix the contents, and the tubes were immediately placed into an ice bath. Plasma was harvested by low-speed centrifugation (2000 rpm, ∼10 min, 4°C) within approximately 30 minutes of sampling. Samples were then shipped on dry ice to BASi, West Lafayette, IN, for analysis. Concentrations of bendamustine were determined by BASi using a validated high-performance liquid chromatography method with tandem mass spectrometric detection. The quantifiable range of the assay was from 0.10 to 100.00 ng/mL for bendamustine.

les were then shipped on dry ice to BASi, West Lafayette, IN, for analysis. Concentrations of bendamustine were determined by BASi using a validated high-performance liquid chromatography method with tandem mass spectrometric detection. The quantifiable range of the assay was from 0.10 to 100.00 ng/mL for bendamustine. Pharmacokinetic parameters calculated for bendamustine and its metabolites included maximum observed plasma drug concentration (Cmax), time to maximum drug concentration (tmax), area under the plasma drug concentration by time curve from time 0 until the last measurable plasma concentration (AUC0-t), and area under the plasma drug concentration by time curve from time 0 until 24 hours after study drug administration (AUC0-24). Pharmacokinetic data from adults were used for comparison.

(tmax), area under the plasma drug concentration by time curve from time 0 until the last measurable plasma concentration (AUC0-t), and area under the plasma drug concentration by time curve from time 0 until 24 hours after study drug administration (AUC0-24). Pharmacokinetic data from adults were used for comparison. Statistics The primary analysis group for efficacy included all patients who received the RP2D in phases I and II; efficacy was also measured for other doses in phase I. There was no prespecified enrollment in phase I. In phase II, additional patients were enrolled at the RP2D identified in phase I until a total planned sample of 26 patients was exposed to the RP2D. There was no planned minimum number of patients for either ALL or AML. The safety analysis set included all patients exposed to any dose of bendamustine, whereas the pharmacokinetic analysis set included all patients who received study drug and who had valid pharmacokinetic data. For the primary efficacy measure (ORR), a 1-sided 95% confidence interval was calculated based on the binomial distribution. If the lower boundary of this confidence interval was >5%, the null hypothesis (no worthwhile effect of bendamustine) of response rate of ≤5% was rejected. Summary statistics were provided only for the observed data; missing data were not imputed. Median DOR was assessed using the Kaplan-Meier method.

ial distribution. If the lower boundary of this confidence interval was >5%, the null hypothesis (no worthwhile effect of bendamustine) of response rate of ≤5% was rejected. Summary statistics were provided only for the observed data; missing data were not imputed. Median DOR was assessed using the Kaplan-Meier method. RESULTS Patient Disposition and Characteristics From August 2010 to July 2011, 11 patients were treated in the dose-escalation (phase I) portion of the study and 32 patients were treated in the efficacy and tolerability (phase II) portion (Fig. 2). The 2 phase I cohorts were expanded to replace individuals who were nonevaluable because of early disease progression. Overall, 27 patients with ALL and 16 patients with AML were enrolled from 24 centers. All patients had received multiple prior therapies, including 26 patients with >3 chemotherapy regimens and 21 patients with prior HSCT (Table 1). All 43 patients were eligible for efficacy and tolerability analyses. FIGURE 2 Patient disposition. TABLE 1 Patient Demographics and Baseline Clinical Characteristics

RESULTS Patient Disposition and Characteristics From August 2010 to July 2011, 11 patients were treated in the dose-escalation (phase I) portion of the study and 32 patients were treated in the efficacy and tolerability (phase II) portion (Fig. 2). The 2 phase I cohorts were expanded to replace individuals who were nonevaluable because of early disease progression. Overall, 27 patients with ALL and 16 patients with AML were enrolled from 24 centers. All patients had received multiple prior therapies, including 26 patients with >3 chemotherapy regimens and 21 patients with prior HSCT (Table 1). All 43 patients were eligible for efficacy and tolerability analyses. FIGURE 2 Patient disposition. TABLE 1 Patient Demographics and Baseline Clinical Characteristics RP2D In phase I, 5 patients received bendamustine 90 mg/m2 and 6 received bendamustine 120 mg/m2. No DLTs were observed with either dose. Among the 6 patients receiving 120 mg/m2, Cmax ranged from 3494 to 9137 ng/mL, AUC0-24 from 5322 to 14,039 ng h/mL, and AUC0-t from 5322 to 14,039 ng h/mL. Analysis of pharmacokinetic data from patients during phase I, along with a review of available preliminary safety and efficacy data, showed that the plasma concentrations attained from these pediatric patients were within the therapeutic range previously determined for adults.8 For this reason, escalation to the 150 mg/m2 dose level did not occur during phase I per protocol, and 120 mg/m2 was determined to be the RP2D. In phase II, all 32 patients received bendamustine 120 mg/m2. Overall, a total of 38 patients received the RP2D of bendamustine 120 mg/m2.

ge previously determined for adults.8 For this reason, escalation to the 150 mg/m2 dose level did not occur during phase I per protocol, and 120 mg/m2 was determined to be the RP2D. In phase II, all 32 patients received bendamustine 120 mg/m2. Overall, a total of 38 patients received the RP2D of bendamustine 120 mg/m2. Study Drug Exposure Among the 5 patients treated with bendamustine 90 mg/m2, 3 received 1 cycle, 1 received 2 cycles, and 1 received 8 cycles; the median total dose received was 182.0 mg/m2 (range, 177.0 to 1436.0 mg/m2). Among the 38 patients treated with bendamustine 120 mg/m2, 31 received 1 cycle and 7 received 2 cycles; the median total dose received was 241.5 mg/m2 (range, 233.0 to 498.0 mg/m2). Overall median dose intensity was 98.5% in the 90 mg/m2 group (range, 77.2% to 101%) and 99.8% in the 120 mg/m2 group (range, 56% to 123%). Efficacy In phase I, 2 patients in the bendamustine 90 mg/m2 group (both with ALL) experienced a CR. Among the 38 patients who received bendamustine 120 mg/m2 in either phase, no patients experienced a CR or CRp (Table 2); thus, the primary efficacy measure was not achieved. In this group, 2 (5%) patients experienced a PR (both with ALL) and 7 (18%) had stable disease (4 with ALL and 3 with AML). The median DOR was not calculated for patients receiving 120 mg/m2 because of lack of complete responders; among patients in the phase I 90 mg/m2 dose group, 2 achieved CR, with 1 patient still in remission at last follow-up after unrelated HSCT. The details of the 2 patients who achieved a CR are outlined in Table 3.

The median DOR was not calculated for patients receiving 120 mg/m2 because of lack of complete responders; among patients in the phase I 90 mg/m2 dose group, 2 achieved CR, with 1 patient still in remission at last follow-up after unrelated HSCT. The details of the 2 patients who achieved a CR are outlined in Table 3. TABLE 2 Summary of Responses TABLE 3 Efficacy Results of Patients Who Achieved CR Following Bendamustine 90 mg/m2 Tolerability In these heavily pretreated patients, among those who received bendamustine 120 mg/m2, 3 patients experienced a dose delay beyond the standard 21-day cycle, because of thrombocytopenia (second cycle delayed until study day 43), febrile neutropenia (second cycle delayed until study day 39), and multiple skin rash (second dose of second cycle delayed 7 days) in 1 patient each. In addition, 1 patient in the 90 mg/m2 group experienced a dose delay (second cycle delayed until study day 30) because of tenderness at the port site. Doses could be delayed up to 2 weeks (for a maximum cycle length of 35 days) if a patient’s hematologic values did not recover and platelet counts could be supported by transfusion. In the second and final cycle for 1 of these patients, the dose was also reduced (to 90 mg/m2, per protocol) because of thrombocytopenia. Overall, the median length of cycle 1 was 21 days in both groups (90 mg/m2, n=5; 120 mg/m2, n=38), whereas cycle 2 was a median of 23.5 days in the 90 mg/m2 group (n=2) and 21 days in the 120 mg/m2 group (n=7).

e for 1 of these patients, the dose was also reduced (to 90 mg/m2, per protocol) because of thrombocytopenia. Overall, the median length of cycle 1 was 21 days in both groups (90 mg/m2, n=5; 120 mg/m2, n=38), whereas cycle 2 was a median of 23.5 days in the 90 mg/m2 group (n=2) and 21 days in the 120 mg/m2 group (n=7). Two deaths due to progressive disease occurred among patients receiving 90 mg/m2, with 1 death occurring ≤30 days (T-cell precursor acute leukemia) after the last dose and 1 death occurring >30 days after the last dose (B-ALL). Neither was associated with treatment. Among the 120 mg/m2 patients, 15 deaths occurred (14 from progressive disease and 1 with pancytopenia who died at home after further active medical treatment was declined); 6 occurred ≤30 days after the last dose; and 9 occurred >30 days after the last dose. None of the deaths were considered to be associated with treatment.

the 120 mg/m2 patients, 15 deaths occurred (14 from progressive disease and 1 with pancytopenia who died at home after further active medical treatment was declined); 6 occurred ≤30 days after the last dose; and 9 occurred >30 days after the last dose. None of the deaths were considered to be associated with treatment. Overall, 33 patients (77%) experienced at least 1 serious adverse event; the most common serious adverse events were febrile neutropenia and infection. Among all 43 patients in the tolerability analysis, the most common adverse events (all grades) were anemia (65%), pyrexia (49%), nausea (47%), febrile neutropenia (35%), vomiting (35%), diarrhea (33%), and thrombocytopenia (33%). The most common grade 3/4 hematologic toxicities were decreased lymphocytes, platelets, and absolute neutrophil counts (Table 4). Forty-two patients experienced at least 1 patient-reported or investigator-reported nonhematologic grade 3 to 5 adverse event (any cause), and hypokalemia was the most common grade 3/4 adverse event (Table 4); 2 of the cases were considered related to treatment, both in the 12- to 20-year age group. One patient who received 120 mg/m2 withdrew from the study because of progressive disease (AML); based on study definitions, this qualified as significant worsening (change in nature, severity, or frequency) of the disease under study and was considered an adverse event. TABLE 4 Grade 3/4 Events

Overall, 33 patients (77%) experienced at least 1 serious adverse event; the most common serious adverse events were febrile neutropenia and infection. Among all 43 patients in the tolerability analysis, the most common adverse events (all grades) were anemia (65%), pyrexia (49%), nausea (47%), febrile neutropenia (35%), vomiting (35%), diarrhea (33%), and thrombocytopenia (33%). The most common grade 3/4 hematologic toxicities were decreased lymphocytes, platelets, and absolute neutrophil counts (Table 4). Forty-two patients experienced at least 1 patient-reported or investigator-reported nonhematologic grade 3 to 5 adverse event (any cause), and hypokalemia was the most common grade 3/4 adverse event (Table 4); 2 of the cases were considered related to treatment, both in the 12- to 20-year age group. One patient who received 120 mg/m2 withdrew from the study because of progressive disease (AML); based on study definitions, this qualified as significant worsening (change in nature, severity, or frequency) of the disease under study and was considered an adverse event. TABLE 4 Grade 3/4 Events Pharmacokinetic Data Results Among the entire cohort of patients receiving bendamustine at either 90 or 120 mg/m2, median tmax for the parent drug and its active metabolites was 1.1 hours, which was just after the end of the 1-hour infusion. Systemic exposure for all patients receiving bendamustine 120 mg/m2 was similar to that of the phase 1 group, with a mean Cmax of 7490 ng/mL and mean AUC0-t was 13,208 ng h/mL—again, the results were consistent with those reported for adults with indolent NHL8 (see Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JPHO/A56, for a summary of the pharmacokinetic results for bendamustine and its metabolites in pediatric patients).

ng/mL and mean AUC0-t was 13,208 ng h/mL—again, the results were consistent with those reported for adults with indolent NHL8 (see Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JPHO/A56, for a summary of the pharmacokinetic results for bendamustine and its metabolites in pediatric patients). DISCUSSION Bendamustine has potential utility in relapsed or refractory lymphoid malignancies because of its lack of cross-reactivity with other alkylating agents. In vitro data suggest bendamustine to be more active in lymphoid versus myeloid disease (data on file, Teva Branded Pharmaceutical Products R&D Inc.). In general, relapsed ALL cells acquire resistance to all or most of the chemotherapeutic agents to which they are exposed.10 However, as demonstrated in the NCI-60 cell-line panel, bendamustine demonstrates limited cross-resistance with other alkylating agents (data on file, Teva Branded Pharmaceutical Products R&D Inc.). This has been borne out in clinical studies on adults, in which bendamustine showed superior activity compared with chlorambucil in patients with chronic lymphocytic leukemia4 and significant clinical activity despite prior alkylating therapy in patients with NHL.5 Thus, there is a theoretical basis for investigating whether bendamustine might have a role in the treatment of relapsed ALL or leukemia that is resistant to other chemotherapeutic agents, including alkylating agents, or as part of up-front therapy for patients who demonstrate resistant disease, such as those with minimal residual disease after induction therapy.

igating whether bendamustine might have a role in the treatment of relapsed ALL or leukemia that is resistant to other chemotherapeutic agents, including alkylating agents, or as part of up-front therapy for patients who demonstrate resistant disease, such as those with minimal residual disease after induction therapy. In vitro analysis of bendamustine in the AML cell line HL-60 indicated modest cytotoxicity6; however, hematologic response was not achieved in a pilot study of bendamustine in adult patients (median age, 69 y) with high-risk AML.11 Although this is a different AML population, these results are consistent with the lack of bendamustine activity observed in childhood AML patients in the present study. The lack of efficacy in this trial may limit future use of bendamustine monotherapy in heavily pretreated AML. However, bendamustine activity cannot be completely ruled out in untreated AML or possibly in conjunction with other therapy or at higher doses.

ctivity observed in childhood AML patients in the present study. The lack of efficacy in this trial may limit future use of bendamustine monotherapy in heavily pretreated AML. However, bendamustine activity cannot be completely ruled out in untreated AML or possibly in conjunction with other therapy or at higher doses. There are few data on the utility of bendamustine in the pediatric population. Indeed, to the best of our knowledge, this is the first study to demonstrate bendamustine activity in childhood relapsed or refractory ALL. The results of the present study suggest that bendamustine has an acceptable tolerability profile in pediatric patients with multiple relapsed or refractory ALL or AML. The most common serious grade ≥3 adverse event in this pediatric population with acute leukemia was hypokalemia, which is less common among adults with indolent lymphoma, but overall adverse events were similar to those reported for adult patients with indolent NHL.5,12 The RP2D was established as 120 mg/m2, which is identical to the single-agent dose that is used to treat adults with rituximab-refractory NHL. In addition, the pharmacokinetic results were consistent with those obtained from adult patients with indolent NHL.8 As these findings suggested that bendamustine dosed at 120 mg/m2 resulted in therapeutic bendamustine plasma levels, no patients were escalated to 150 mg/m2, as per protocol. The decision to rely on plasma concentration levels shown to be therapeutic in older patients with a different disease but yet hematologic, and thereby avoid subjecting patients in this study to potentially serious DLTs by increasing the dose, may have resulted in failure to administer a clinically effective dose of bendamustine in this patient population. Alternatively, it is not known if this heavily pretreated population including transplantation before the trial might have had a bone marrow reserve to recover adequately after a dose toxicity. A benefit-risk ratio assessment was carried out as well to move forward to RP2D based on current safety and pharmacokinetic results.

atively, it is not known if this heavily pretreated population including transplantation before the trial might have had a bone marrow reserve to recover adequately after a dose toxicity. A benefit-risk ratio assessment was carried out as well to move forward to RP2D based on current safety and pharmacokinetic results. In this study, the primary efficacy measure was not achieved, as only 2 patients achieved an overall response. Interestingly, both patients were in the group receiving 90 mg/m2, which was not the RP2D. Furthermore, both responders had ALL; no activity was demonstrated in patients with AML. This is not entirely surprising given the in vitro data suggesting that bendamustine is more active in lymphoid rather than myeloid malignancies. However, the limited activity of bendamustine observed in this study is not unexpected for a single agent in such a very heavily pretreated population. As with other agents, bendamustine may result in a higher response rate when it is combined with other agents; thus, it should be further explored in combination regimens in pediatric patients with acute leukemias including ALL.13,14 Overall, the response data for this study population suggest that bendamustine is tolerable and has some activity in heavily pretreated patients with relapsed or refractory ALL but not in relapsed or refractory AML. Further studies are required to evaluate the role of bendamustine in combination with regimens that are the backbone of current leukemia therapy in children.

suggest that bendamustine is tolerable and has some activity in heavily pretreated patients with relapsed or refractory ALL but not in relapsed or refractory AML. Further studies are required to evaluate the role of bendamustine in combination with regimens that are the backbone of current leukemia therapy in children. ACKNOWLEDGMENTS The authors thank the sites and staff and patients and families who helped with and participated in the study. They also thank Philmore Robertson Jr., PhD, and Mary Bond, MS, MBA, from Teva Branded Pharmaceutical Products R&D Inc., who assisted with the pharmacokinetic schedule and data interpretation. Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.jpho-online.com. Presented as a poster at the 53rd Annual Meeting of the American Society of Hematology, San Diego, CA, December 10–13, 2011. This research was sponsored and conducted by Teva Branded Pharmaceutical Products R&D Inc., Frazer, PA. Funding for editorial, design, and production support was provided by Teva Branded Pharmaceutical Products R&D Inc., to The Curry Rockefeller Group LLC, Tarrytown, NY. C.F. received funding from Teva to present this work at ASH in 2011.

onsored and conducted by Teva Branded Pharmaceutical Products R&D Inc., Frazer, PA. Funding for editorial, design, and production support was provided by Teva Branded Pharmaceutical Products R&D Inc., to The Curry Rockefeller Group LLC, Tarrytown, NY. C.F. received funding from Teva to present this work at ASH in 2011. L.F. is employed by PPD-PharmacoVigilance (contractual relationship with Teva Branded Pharmaceutical Products R&D Inc.). D.B.K., M.M., and J.W. were employed by Teva Branded Pharmaceutical Products R&D Inc. at the time the study was conducted. The remaining authors declare no conflict of interest.

Hematopoietic stem cell transplantation is a radical, curative treatment for hematological malignancies, such as leukemia and malignant lymphoma. Total body irradiation (TBI), as well as chemotherapy, is widely performed as a preconditioning regimen, with the aim of inhibiting graft-versus-host disease (GVHD) after transplantation and to eradicate remaining tumor cells. However, these preconditioning regimens result in delayed secondary sex characteristics in childhood and adolescence, and failure of normal ovarian function recovery in women, leading to a high incidence of amenorrhea and infertility. CASE REPORT In April 1997, an 11-year-old girl visited a pediatric clinic for evaluation of repeated febrile episodes and was found to have a low white blood cell count of 2800/μL. Her bone marrow biopsy showed that blasts accounted for 94.1% of white blood cells, peroxidase staining was negative, and the cells were positive for CD10, CD19, and HLA-DR. She was subsequently diagnosed with acute lymphocytic leukemia (ALL; French-American-British Class L1, common ALL). On the basis of the high-risk protocol of the Children’s Cancer & Leukemia Study Group (CCLSG; ALL 941 Protocol Study; ages 10 to 19 y),1 she was administered tetrahydropyranyl adriamycin, vincristine, prednisone, and 6-mercaptopurine and achieved complete remission. Thereafter, she received intensification therapy using the same drug regimen.

Acute lymphoblastic leukemia (ALL) is one of the common malignancies of childhood. In developed countries, the 5-year event-free survival (EFS) of pediatric ALL has reached 83% and overall survival (OS) is nearly up to 90%.1 However, 5-year EFS rate in low and middle income countries has been reported to be <35%.2 The resultant survival gap has been attributed to delayed presentation, greater treatment-related mortality, and a higher rate of relapse.3–5 Another important yet often overlooked cause is abandonment of therapy, the failure to start or complete potentially curative therapy.6 In China, although 5-year EFS and OS of children with ALL has improved greatly, abandonment rate (AR) still accounts for a large percentage of deaths.7,8 Suzhou is a city with above GDP per capita in China. In 2005, Suzhou government instituted funds for protection against catastrophic diseases expenditure (PACDE), including childhood cancer. In 2011, Chinese government instituted funds for protection against ALL of low and intermediate risk in children living in the countryside. Although the authors found several recent reports describing abandonment of ALL treatment in China, we found limited data regarding the impact of government assistance on AR. Therefore, we retrospectively analyzed the AR by dividing the time period into 2002 to 2005, 2005 to 2010, and 2011 to 2012, so as to evaluate the impact of medical policies on the abandoned treatment.

bing abandonment of ALL treatment in China, we found limited data regarding the impact of government assistance on AR. Therefore, we retrospectively analyzed the AR by dividing the time period into 2002 to 2005, 2005 to 2010, and 2011 to 2012, so as to evaluate the impact of medical policies on the abandoned treatment. MATERIALS AND METHODS We collected and retrospectively analyzed information on 1151 patients diagnosed with ALL at the Children’s Hospital of Soochow University from 2002 to 2012, which included general data (name, sex, age, home address, and telephone number), medical payment, final diagnosis, MICM (morphology, immunology, cytogenetics, and molecular biology) test results, treatment response, the stage, and reason for abandonment. In addition, we surveyed parents of inpatients by phone and noted their current living condition and treatment after hospital discharge, especially aiming at those with abandoned treatment. Treatment abandonment is defined as failure to start or complete curative treatment (except in situations when such treatment is contraindicated for medical reasons, eg, if the patient is critically ill).9 Treatment abandonment after recurrence is defined as failure to continue treatment or just receiving palliative cure after recurrence. Lost to follow-up is defined as losing contact because of change of telephone number or address.

reatment is contraindicated for medical reasons, eg, if the patient is critically ill).9 Treatment abandonment after recurrence is defined as failure to continue treatment or just receiving palliative cure after recurrence. Lost to follow-up is defined as losing contact because of change of telephone number or address. Patients were classified into urban, town, and rural population according to their addresses. On the basis of 5 administrative levels in China, including state, provincial, prefectural, county, and township level, people living in provincial or prefectural city who do not live on agriculture are considered as urban population. People living in county or town who do not mainly live on agriculture are classified into town population, and people living in countryside who mainly live on agriculture are viewed as rural population. PACDE was carried out on Suzhou children since 2005. New rural cooperative medical system (NRCMS) was carried out in some parts of rural areas since 2011. The family annual income is relatively lower for rural people, generally highest for urban people, and intermediate for town people. Statistics Frequency distributions, median, mean, and SDs were assessed for each variable. SPSS 18.0 version was employed for data analysis. ARs were compared using the χ2 test. P<0.05 was considered statistically significant.

Patients were classified into urban, town, and rural population according to their addresses. On the basis of 5 administrative levels in China, including state, provincial, prefectural, county, and township level, people living in provincial or prefectural city who do not live on agriculture are considered as urban population. People living in county or town who do not mainly live on agriculture are classified into town population, and people living in countryside who mainly live on agriculture are viewed as rural population. PACDE was carried out on Suzhou children since 2005. New rural cooperative medical system (NRCMS) was carried out in some parts of rural areas since 2011. The family annual income is relatively lower for rural people, generally highest for urban people, and intermediate for town people. Statistics Frequency distributions, median, mean, and SDs were assessed for each variable. SPSS 18.0 version was employed for data analysis. ARs were compared using the χ2 test. P<0.05 was considered statistically significant. RESULTS General Information According to the policies carried out, the time period studied was divided into 3 phases: year 2002 to 2004, 2005 to 2010, and 2011 to 2012. The numbers of diagnosed patients were 258, 511, and 382 for each phase. Of the 1151 ALL diagnosed cases, 731 cases were boys and 420 cases were girls (boy:girl=1.74:1). The median age was 4.85 years ranging from 1 month to 16 years. Fifty-two cases abandoned as outpatients and 7 cases of inpatients were excluded in further analysis of abandonment reasons as unknown address. The number of inpatients from urban, town, and rural area were 250, 224, and 618, respectively. Families bearing 1 child accounted for 820 cases and 193 for >1 child. Of the 1099 ALL inpatients, 114 cases were Suzhou citizen and 90 cases benefited from the project of PACDE since 2005, and 132 cases as rural population benefited from the NRCMS during 2011 and 2012. Patients covered by these 2 health insurance programs usually paid 500 to 2000 RMB, roughly equal to monthly salary of people working in Suzhou city, for each course of chemotherapy and their economic burdens were largely reduced. Others had to pay the full cost of the treatment in the hospital and could be partially imbursed (<50% of the hospital fee) from the local government within 2 to 12 months after being discharged. The interruption of financial support somehow caused difficulty for the family to continue the following course of chemotherapy on time. We found 316 cases of abandoned treatment after diagnosis and 92 cases abandoning treatment after recurrence. Furthermore information is provided in Table 1.

s after being discharged. The interruption of financial support somehow caused difficulty for the family to continue the following course of chemotherapy on time. We found 316 cases of abandoned treatment after diagnosis and 92 cases abandoning treatment after recurrence. Furthermore information is provided in Table 1. TABLE 1 Demography and Features of ALL Patients Abandoning Therapy

s after being discharged. The interruption of financial support somehow caused difficulty for the family to continue the following course of chemotherapy on time. We found 316 cases of abandoned treatment after diagnosis and 92 cases abandoning treatment after recurrence. Furthermore information is provided in Table 1. TABLE 1 Demography and Features of ALL Patients Abandoning Therapy Patients Abandoning Treatment A total of 316 patients were found to abandon treatment from 2002 to 2012, of which, 52 cases abandoned at their diagnosis of ALL, and only 264 cases were hospitalized (Fig. 1). A total of 178 patients abandoned after starting the chemotherapy <7 days, 61 cases abandoned after completing induction remission therapy, 14 cases abandoned on consolidation therapy, and 11 cases abandoned on intensification therapy. A total of 105 cases suffered a severe complication such as fatal infection or bleeding before abandonment. A significant difference in AR was found among those with various factors: boy>girl, children below 1 year>children above 1 year, self-paid>medical insurance covered, rural>town>urban, family with more than 1 child>family with only single child. Furthermore, the AR of patients in Suzhou from 2002 to 2005 was much higher than that from 2006 to 2012 (8/16 vs. 15/75, P=0.05) (Fig. 2). As a rapidly developed city, the population of Suzhou increased strikingly since 2006 by attracting more people nationwide. Most notably, as NRCMS covered all the rural districts of Suzhou during recent years, AR of patients residing in the countryside in 2011 and 2012 remarkably reduced compared with the AR from 2002 to 2010 (4/128 vs. 70/81, P<0.001) (Fig. 3).

of Suzhou increased strikingly since 2006 by attracting more people nationwide. Most notably, as NRCMS covered all the rural districts of Suzhou during recent years, AR of patients residing in the countryside in 2011 and 2012 remarkably reduced compared with the AR from 2002 to 2010 (4/128 vs. 70/81, P<0.001) (Fig. 3). FIGURE 1 The number of abandonment for the newly diagnosed pediatric ALL at different stage of chemotherapy. A total of 230 cases of abandonment at diagnosis included 2 parts: one part was 52 outpatients and another part was 178 inpatients who abandoned within 1 week after starting induction remission therapy. FIGURE 2 Comparison of the number of patients and ARs before and after PACDE funding in Suzhou City. The first period from 2002 to 2005 was the duration before the project was carried out. The second period from 2006 to 2012 was the time after the project. The ratio of completing treatment to abandonment in the second period is much higher than that in first period (A, P=0.05). ARs decreased rapidly since 2006 and kept a stable level about 15% since then (B) coinciding with the new policy.

s carried out. The second period from 2006 to 2012 was the time after the project. The ratio of completing treatment to abandonment in the second period is much higher than that in first period (A, P=0.05). ARs decreased rapidly since 2006 and kept a stable level about 15% since then (B) coinciding with the new policy. FIGURE 3 Comparison of the number of patients and ARs in remote areas before and after NRCMS (the new rural cooperative medical care system) began. The first period from 2002 to 2010 was the duration before NRCMS was carried out in rural regions. The second period from 2011 to 2012 was the time after NRCMS was carried out. The ratio of completing treatment to abandonment in the second period is much higher than that in first period, which reached a significant difference (A, P<0.001). ARs decreased steadily since 2006 and reached a very lower level of 5% in 2012 (B). From the telephone survey information collected from 263 patients, we determined that financial difficulty involved was the primary reason for abandonment (206/263, 78.3%) (Table 2). TABLE 2 Reasons for Abandoning Treatment (Interviewed, n=263)

FIGURE 3 Comparison of the number of patients and ARs in remote areas before and after NRCMS (the new rural cooperative medical care system) began. The first period from 2002 to 2010 was the duration before NRCMS was carried out in rural regions. The second period from 2011 to 2012 was the time after NRCMS was carried out. The ratio of completing treatment to abandonment in the second period is much higher than that in first period, which reached a significant difference (A, P<0.001). ARs decreased steadily since 2006 and reached a very lower level of 5% in 2012 (B). From the telephone survey information collected from 263 patients, we determined that financial difficulty involved was the primary reason for abandonment (206/263, 78.3%) (Table 2). TABLE 2 Reasons for Abandoning Treatment (Interviewed, n=263) Patients Abandoning Treatment After Recurrence A total of 92 patients abandoned treatment after recurrence who accounted for 41.26% of all the relapsed patients in our hospital from 2002 to 2012. To analyze the relevant factors influencing the AR, we evaluated the effect of relapse site, sex, medical insurance, and duration of first remission. Our data revealed that abandoned patients with bone marrow relapse accounted for 62 cases, testicular relapse 10, CNS relapse 11, and combined relapse 9. The ratio of abandoned male to female was 1.71 (58/34), which was similar to the ratio of patients receiving treatment (1.91, 86/45). Relapse patients abandoned who were covered by medical insurance accounted for 35.48% (11/31) and self-paid accounted for 42.19% (81/192). No significant difference could be found between them. The number of relapse patients at very early stage, early stage, and late stage was 109, 42, and 35, respectively, and abandoned patients were 47 (43.11%), 17 (40.48%), and 11 (31.43%) correspondingly. No statistic differences were calculated among them.

(81/192). No significant difference could be found between them. The number of relapse patients at very early stage, early stage, and late stage was 109, 42, and 35, respectively, and abandoned patients were 47 (43.11%), 17 (40.48%), and 11 (31.43%) correspondingly. No statistic differences were calculated among them. DISCUSSION Treatment abandonment is one of the most important reasons for the short survival of patients with ALL in developing countries.10 It is important to determine what factors contribute to abandonment. Analyzing 316 cases of treatment abandonment, we found that children below 1 year of age represent the highest percentage (P<0.001). As the infant leukemia has a relatively unfavorable prognosis,11 and the parents are always informed with the poor prognosis, further more, most infants with leukemia are not covered by medical insurance which cause the desperate parents to abandon the treatment. Therefore, more researches on infant leukemia are urgently needed and the medical care system emphasizing on infants should decrease infants’ AR.

ys informed with the poor prognosis, further more, most infants with leukemia are not covered by medical insurance which cause the desperate parents to abandon the treatment. Therefore, more researches on infant leukemia are urgently needed and the medical care system emphasizing on infants should decrease infants’ AR. The AR of patients from rural areas is remarkably higher than that from urban and town regions (P<0.001). In China, parents from rural areas usually have a relatively lower income and poorer educational background. Furthermore, they also regard leukemia as an incurable and high expense bearing disease. From our statistical analysis, we found that AR is also higher in the self-paid patients and families with >1 child. ALL relapse is an index of unfavorable prognosis.12 The recurrence rate was 20.2% (223/1099) and the AR was 41.3% (92/223) in our hospital from 2002 to 2012. Although the AR of self-paid relapsed patients is higher than those covered by medical insurance, there is no statistical difference among them. From these information gathered we determined that financial burden is the major factor in treatment abandonment. We believe that revision and improvement of the policy of China’s medical care system, enhancement of the awareness of the inevitable ALL relapse, and more close communication and mutual understanding between doctors and parents to impart medical knowledge are necessarily important for reducing AR and improving OS of ALL.

believe that revision and improvement of the policy of China’s medical care system, enhancement of the awareness of the inevitable ALL relapse, and more close communication and mutual understanding between doctors and parents to impart medical knowledge are necessarily important for reducing AR and improving OS of ALL. Patients with ALL, especially those from undeveloped regions, can greatly benefit from the new policy, which is demonstrated in our statistical data (Table 1). Since 2006, AR in Suzhou has dramatically dropped to only 4 children from the countryside in 2011 and 2012. As Ribeiro13 pointed out that every cancer child could receive corresponding treatment of higher quality by expanding government’s medical care support, within the time period studied, we can see a clear picture demonstrating the decrease in the AR after the incorporation of new policies. Therefore, we conclude that the improvement of our medical care system can relieve economical burden, reduce AR, and improve OS of patients with ALL to a great extent. In summary, financial crisis was the main reason for abandoning treatment in pediatric ALL patients. Government-funded health care expenditure programs reduced families’ economic burden and thereby reduced the AR with resultant increased OS.

Patients with ALL, especially those from undeveloped regions, can greatly benefit from the new policy, which is demonstrated in our statistical data (Table 1). Since 2006, AR in Suzhou has dramatically dropped to only 4 children from the countryside in 2011 and 2012. As Ribeiro13 pointed out that every cancer child could receive corresponding treatment of higher quality by expanding government’s medical care support, within the time period studied, we can see a clear picture demonstrating the decrease in the AR after the incorporation of new policies. Therefore, we conclude that the improvement of our medical care system can relieve economical burden, reduce AR, and improve OS of patients with ALL to a great extent. In summary, financial crisis was the main reason for abandoning treatment in pediatric ALL patients. Government-funded health care expenditure programs reduced families’ economic burden and thereby reduced the AR with resultant increased OS. Supported by the grants from Jiangsu Province’s Clinical Medical Science and Technology Projects (Grant No. BL2013014), National Natural Science Foundation of China (No. 81100371 and No. 81370627), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and National clinical key subject construction project. The authors declare no conflict of interest.

Invasive fungal infections are an important cause of morbidity and mortality in immunocompromised children.1,2 Candida and Aspergillus species are the most common fungal pathogens in pediatric patients, and are associated with mortality rates of 19% to 31% and 68% to 77%, respectively.2–4 Micafungin, an echinocandin antifungal agent, specifically inhibits the synthesis of 1,3-β-d-glucan, a major component of the fungal cell wall, and exerts potent antifungal activity against Candida and Aspergillus species in vitro.1,5–7 This antifungal agent is effective against candidemia, candidiasis, and aspergillosis, and is generally well tolerated in adult patients,1,7–11 and is approved for the treatment of patients with invasive fungal infections, such as candidiasis or aspergillosis, in many countries worldwide.

rgillus species in vitro.1,5–7 This antifungal agent is effective against candidemia, candidiasis, and aspergillosis, and is generally well tolerated in adult patients,1,7–11 and is approved for the treatment of patients with invasive fungal infections, such as candidiasis or aspergillosis, in many countries worldwide. Although micafungin has been licensed for children in Japan since April 2006, in Europe since April 2008, and then in the United States since June 2013, there are limited data available regarding the efficacy and safety of micafungin in children, largely because the primary objectives in some of these studies were pharmacokinetic analyses.1,12–15 Recently, safety data from the pooled micafungin pediatric clinical trials (phase I, II, and III), conducted before 2006, were summarized.3 However, prospective studies in larger numbers of patients are needed to clarify the role of micafungin for the management of fungal infections in children. Thus, the first postmarketing surveillance study was conducted in 201 patients to confirm the safety and effectiveness of micafungin in clinical practice.

arized.3 However, prospective studies in larger numbers of patients are needed to clarify the role of micafungin for the management of fungal infections in children. Thus, the first postmarketing surveillance study was conducted in 201 patients to confirm the safety and effectiveness of micafungin in clinical practice. MATERIALS AND METHODS This prospective multicenter observational surveillance study was conducted between October 2006 and September 2008. Pediatric patients aged under 16 years who were receiving micafungin (Astellas Pharma Inc., Japan) for deep mycosis caused by Candida or Aspergillus were registered prospectively at a central office within 6 days of the initiation of micafungin treatment. This was a postmarketing study conducted in compliance with the ministerial ordinance on Good Postmarketing Study Practice, which was authorized by Ministry of Health, Labour and Welfare in Japan, Ordinance No. 171 dated December 20, 2004.

ely at a central office within 6 days of the initiation of micafungin treatment. This was a postmarketing study conducted in compliance with the ministerial ordinance on Good Postmarketing Study Practice, which was authorized by Ministry of Health, Labour and Welfare in Japan, Ordinance No. 171 dated December 20, 2004. Data on the following were obtained: sex, age, body weight, diagnosis of fungal infection, complications, baseline hepatic and renal function, daily dose and duration of micafungin treatment, concomitant drugs, clinical response, and adverse events. Diagnosis of fungal infection, clinical response, and adverse events were determined by the physician in charge. The severity of hepatic or renal impairment at the start of micafungin treatment was assessed according to the grade classification, which was issued by the Ministry of Health, Labour and Welfare, Ordinance No. 80 dated June 29, 1992, and the Common Terminology Criteria for Adverse Events (CTCAE) version 4. The protocol of this surveillance study was reviewed and approved by the Institutional Review Board of each participating institution in accordance with the rules of each institution. Study Drug and Dosage Regimen Micafungin was administered once daily by intravenous infusion over at least 60 minutes at a dose of 1 mg/kg of body weight for candidiasis and 1 to 3 mg/kg for aspergillosis, which are the approved dosages in Japan, with the option of increasing the dose, if required, up to 6 mg/kg daily.

Study Drug and Dosage Regimen Micafungin was administered once daily by intravenous infusion over at least 60 minutes at a dose of 1 mg/kg of body weight for candidiasis and 1 to 3 mg/kg for aspergillosis, which are the approved dosages in Japan, with the option of increasing the dose, if required, up to 6 mg/kg daily. Assessment of Safety All remarks regarding adverse events, including abnormal changes in laboratory test findings, were recorded during micafungin treatment. Adverse drug reactions were classified based on the Japanese version of the Medical Dictionary for Regulatory Activities (MedDRA/J, version 13.1). In cases where symptoms and findings defined as adverse drug reactions were ongoing at the end of the administration of micafungin, cases were to be followed up, in principle, until symptoms and findings resolved, and the outcome (eg, recovery, remission) was reported by the primary investigators at each site. The degree of seriousness was classified as serious or nonserious by the principal investigators at each site, in accordance with the classification of adverse events, which is provided in the Pharmaceutical Affairs Law, Enforcement Regulations. The causal relationship of each event with micafungin was assessed on a 4-point scale: probable, possible, not related, or unknown. All adverse events except for those deemed to be “not related” were defined as adverse drug reactions, in accordance with the ICH E2A guideline (International Conference on Harmonisation, Clinical Safety Data Management: Definitions and Standards for Expedited Reporting).

e: probable, possible, not related, or unknown. All adverse events except for those deemed to be “not related” were defined as adverse drug reactions, in accordance with the ICH E2A guideline (International Conference on Harmonisation, Clinical Safety Data Management: Definitions and Standards for Expedited Reporting). Assessment of Efficacy Patients who received >5 days’ micafungin were evaluated by primary physicians for improvement in clinical symptoms and findings, imaging findings, mycological tests, and serological tests at the end of treatment, scoring results on a 3-point scale: response, no response, or indeterminate.9 Statistical Analyses The χ2 test was used, and the Cochran-Armitage test was used when category order had to be considered. The level of significance was set at <5% 2-sided. All statistical analyses were performed using JMP 9 software (SAS Institute Inc., Cary, NC). RESULTS Study Population A total of 201 patients were enrolled in the study from 59 medical institutions around Japan (Fig. 1). Of these, 11 patients were excluded from the safety analysis for reasons such as enrollment outside the registration period and insufficient safety data for assessment. Of the remaining 190 patients, 99 were excluded from the efficacy analysis for reasons such as insufficient efficacy data for assessment, giving a total of 91 patients ultimately included in the efficacy analysis. FIGURE 1 Number of patients enrolled and included in the safety and efficacy analysis.

RESULTS Study Population A total of 201 patients were enrolled in the study from 59 medical institutions around Japan (Fig. 1). Of these, 11 patients were excluded from the safety analysis for reasons such as enrollment outside the registration period and insufficient safety data for assessment. Of the remaining 190 patients, 99 were excluded from the efficacy analysis for reasons such as insufficient efficacy data for assessment, giving a total of 91 patients ultimately included in the efficacy analysis. FIGURE 1 Number of patients enrolled and included in the safety and efficacy analysis. The patient characteristics of both all 190 and the 91 patients evaluated for safety and efficacy are summarized in Table 1. Of the 190 patients, 103 were males (54.2%) and 87 were females (45.8%), and of the 91 patients evaluated for safety and efficacy there were 51 males (56.0%) and 40 females (44.0%). The median ages were 5.5 and 5.0 years, respectively, and 18 neonates under 4 weeks of age were included. Of the 190 patients, common underlying conditions were hematopoietic stem cell and other transplantations (61.1%), solid tumor (15.8%), and low birth weight (6.3%). Micafungin was used for candidiasis in 49 patients (25.8%), aspergillosis in 15 (7.9%), empiric therapy in 42 (22.1%), febrile neutropenia in 42 (22.1%), and prophylaxis of fungal infection in 30 patients (15.8%). Eighty-four patients (44.2%) were neutropenic at baseline (absolute neutrophil count <500 cells/μL). Hepatic and renal dysfunctions were reported in 23.2% and 7.4% of patients, respectively.

%), empiric therapy in 42 (22.1%), febrile neutropenia in 42 (22.1%), and prophylaxis of fungal infection in 30 patients (15.8%). Eighty-four patients (44.2%) were neutropenic at baseline (absolute neutrophil count <500 cells/μL). Hepatic and renal dysfunctions were reported in 23.2% and 7.4% of patients, respectively. TABLE 1 Baseline Characteristics of Pediatric Patients Participating in Postmarketing Surveillance of Micafungin Micafungin Exposure The mean maximal daily micafungin dose (ie, the maximal dose used at any point during the entire course of observation) for the assessment of safety was 3.4 and for efficacy was 3.6 mg/kg, and the mean treatment duration was 19.7 days (range, 1 to 168 d) for the safety evaluation and 20.5 days (range, 5 to 91 d) for the efficacy evaluation, and these were similar when analyzed by diagnosis (Table 2). Of the 190 patients, 41 patients (21.6%) received concomitant antifungal therapy. TABLE 2 Exposure of Micafungin and Other Antifungals Safety Fifty-five adverse drug reactions were reported in 42 of 190 patients evaluated for safety (22.1%, Table 3). The most frequently reported adverse drug reactions were hepatobiliary disorders, with an incidence of 13.7% (27 episodes in 26 patients). Nineteen episodes (10.0%) of laboratory abnormalities, including increases in alanine aminotransferase (2.1%) and aspartate aminotransferase (2.6%), were reported in 19 patients. TABLE 3 Adverse Drug Reactions (n=190)

Safety Fifty-five adverse drug reactions were reported in 42 of 190 patients evaluated for safety (22.1%, Table 3). The most frequently reported adverse drug reactions were hepatobiliary disorders, with an incidence of 13.7% (27 episodes in 26 patients). Nineteen episodes (10.0%) of laboratory abnormalities, including increases in alanine aminotransferase (2.1%) and aspartate aminotransferase (2.6%), were reported in 19 patients. TABLE 3 Adverse Drug Reactions (n=190) Among 42 patients who had adverse drug reactions, recovery or remission was observed in 13 patients after cessation of micafungin treatment, and in 23 patients without discontinuation of micafungin. One patient who had hepatic function abnormal died due to exacerbation of the primary disease (acute lymphocytic leukemia). One patient who had increased blood bilirubin, aspartate aminotransferase, and alanine aminotransferase died due to exacerbation of sepsis that developed during the treatment of the primary disease (acute lymphocytic leukemia). One patient died of aggravated trichosporonosis that developed during micafungin administration. There were 3 cases where follow-up reporting from the primary investigator was not completed. Seven serious adverse drug reactions (1 event each of Trichosporon infection, liver disorder, rash, alanine aminotransferase increased, aspartate aminotransferase increased, blood bilirubin increased, and hepatic enzyme increased) were observed in 5 patients. Except for Trichosporon infection, the outcome of these events was recovered or remitted.

actions (1 event each of Trichosporon infection, liver disorder, rash, alanine aminotransferase increased, aspartate aminotransferase increased, blood bilirubin increased, and hepatic enzyme increased) were observed in 5 patients. Except for Trichosporon infection, the outcome of these events was recovered or remitted. The incidence of adverse drug reactions was analyzed by baseline patient characteristics and micafungin exposure (Table 4). No adverse drug reactions were reported in the 18 neonates. When analyzed by diagnosis, the incidence of adverse drug reactions in the 42 patients with febrile neutropenia (35.7%) seemed to be slightly higher than in patients with other diagnoses. No adverse drug reactions were observed in patients with renal dysfunction, and hepatic dysfunction did not affect the incidence of adverse drug reactions. Three of 9 patients treated with micafungin at a dose of >6 mg/kg experienced adverse drug reactions of nonserious hepatobiliary disorders. There were no relationships between the incidence of adverse drug reaction and micafungin daily dose or treatment duration, or with concomitant antifungal agents. TABLE 4 Incidence of Adverse Drug Reactions by Baseline Patient Characteristics and Exposure of Micafungin and Other Antifungal Agents

The incidence of adverse drug reactions was analyzed by baseline patient characteristics and micafungin exposure (Table 4). No adverse drug reactions were reported in the 18 neonates. When analyzed by diagnosis, the incidence of adverse drug reactions in the 42 patients with febrile neutropenia (35.7%) seemed to be slightly higher than in patients with other diagnoses. No adverse drug reactions were observed in patients with renal dysfunction, and hepatic dysfunction did not affect the incidence of adverse drug reactions. Three of 9 patients treated with micafungin at a dose of >6 mg/kg experienced adverse drug reactions of nonserious hepatobiliary disorders. There were no relationships between the incidence of adverse drug reaction and micafungin daily dose or treatment duration, or with concomitant antifungal agents. TABLE 4 Incidence of Adverse Drug Reactions by Baseline Patient Characteristics and Exposure of Micafungin and Other Antifungal Agents Effectiveness The overall clinical response rate in 91 patients analyzed for efficacy was 86.8% (Table 5). The response rate in neonates (below 4 wk of age) was 90.0% (9/10 patients), and there were no differences in the response rate by age. When analyzed by diagnosis, the response rate was 88.2% (30/34) among patients with candidiasis, 75.0% (9/12) among patients with aspergillosis, 91.3% (21/23) among patients receiving empiric therapy, and 89.5% (17/19) among patients receiving treatment for febrile neutropenia. The response rate among patients with neutropenia, <500/μL throughout the micafungin treatment, was 92.9% (13/14 patients, Table 6). No marked differences in response rates were observed in terms of patient characteristics (sex or body weight), duration of micafungin treatment, daily dose of micafungin, use of previous medication with antifungals, or concomitant antifungals.

L throughout the micafungin treatment, was 92.9% (13/14 patients, Table 6). No marked differences in response rates were observed in terms of patient characteristics (sex or body weight), duration of micafungin treatment, daily dose of micafungin, use of previous medication with antifungals, or concomitant antifungals. TABLE 5 Efficacy by Clinical Characteristics and Study Treatment TABLE 6 Efficacy by Change in Neutrophil Count

L throughout the micafungin treatment, was 92.9% (13/14 patients, Table 6). No marked differences in response rates were observed in terms of patient characteristics (sex or body weight), duration of micafungin treatment, daily dose of micafungin, use of previous medication with antifungals, or concomitant antifungals. TABLE 5 Efficacy by Clinical Characteristics and Study Treatment TABLE 6 Efficacy by Change in Neutrophil Count DISCUSSION In the present postmarketing surveillance study, the incidence of adverse drug reactions was 22.1% (42/190 patients), and hepatobiliary disorders were the most common adverse drug reaction (13.7%). The incidence of adverse drug reactions in this study was similar to that seen in the data from the pooled micafungin pediatric clinical trials (phase I, II, and III) conducted before 2006 in Europe, the Americas, and South or Southeast Asia (26.7%, 79/296 patients).3 The incidence of adverse drug reactions observed in this postmarketing surveillance study (22.1%, 42/190 patients) was comparable with or lower than the incidence of treatment-related adverse events (ie, adverse drug reactions) in pediatric patients with invasive candidiasis in a randomized double-blind trial (36.5%, 19/52 patients).14 In addition, in this study, the incidences of adverse drug reactions and hepatobiliary disorders including investigations for such (19.4%, 37/190 patients) in these pediatric patients were similar to those in a Japanese postmarketing surveillance study in adult patients (adverse drug reactions: 28.5%, 306/1074 patients; hepatobiliary disorders: 16.8%).9 Causality between hepatobiliary disorders and micafungin treatment was assessed as possibly related or not related in most instances, and several other causative factors—such as concomitant medications and underlying diseases—were reported by physicians.

ions: 28.5%, 306/1074 patients; hepatobiliary disorders: 16.8%).9 Causality between hepatobiliary disorders and micafungin treatment was assessed as possibly related or not related in most instances, and several other causative factors—such as concomitant medications and underlying diseases—were reported by physicians. In the present surveillance study, 1 Trichosporon infection was observed in a patient treated with 100 mg/d micafungin for 63 days; the patient died of aggravated trichosporonosis, although voriconazole was administered for trichosporonosis after the discontinuation of micafungin treatment. Although it is possible that micafungin treatment may cause breakthrough trichosporonosis in patients with hematological diseases,16,17 it is unlikely that micafungin directly causes the breakthrough of trichosporonosis because other strong confounders are more plausible explanations for the relationship with trichosporonosis in reported cases.

at micafungin treatment may cause breakthrough trichosporonosis in patients with hematological diseases,16,17 it is unlikely that micafungin directly causes the breakthrough of trichosporonosis because other strong confounders are more plausible explanations for the relationship with trichosporonosis in reported cases. In this surveillance study, the incidence of adverse drug reactions was slightly higher in patients with febrile neutropenia than in patients with other diagnoses. It might be possible to speculate that several antibiotics and antifungals, which were concomitantly used for the treatment of febrile neutropenia, may affect the incidence of adverse events. The dosage of micafungin can be increased up to 6 mg/kg based on the patient’s condition for severe or refractory candidiasis/aspergillosis. Although adverse drug reactions of nonserious hepatobiliary disorders were recorded in 3 of 9 patients treated with micafungin at a dose of >6 mg/kg, their causal relationship with micafungin was not assessed as probable because 2 cases developed after discontinuation of micafungin, and the remaining 1 case recovered during micafungin treatment. Thus, the data suggest that the micafungin dose has little influence on safety. In addition, no adverse drug reactions were reported in 18 neonates. Other factors including age, duration of treatment, and baseline hepatic/renal function were not found to have any impact on the incidence of adverse drug reactions.

ment. Thus, the data suggest that the micafungin dose has little influence on safety. In addition, no adverse drug reactions were reported in 18 neonates. Other factors including age, duration of treatment, and baseline hepatic/renal function were not found to have any impact on the incidence of adverse drug reactions. It has been demonstrated that micafungin can be effective against candidemia, candidiasis, and aspergillosis in pediatric and adult patients.1,11–15 For instance, treatment success was observed in 72.9% of pediatric patients with invasive candidiasis treated with micafungin (2 mg/kg) in a randomized double-blind trial.14 In a Japanese surveillance study, the clinical response rates for adult patients with candidiasis and aspergillosis were 86.3% and 70.8%, respectively, and the overall response rate was 83.0%.9 Thus, the response rates in candidiasis and aspergillosis in the presented surveillance study in pediatric patients (88.2% and 75.0%, respectively; overall response, 86.8%) are comparable with those in previous reports in both pediatrics and adults.9,14 In addition, it is notable that the response rate—even in the persistent neutropenic patients, whose neutrophil count was <500/μL throughout micafungin treatment—was 92.9% (13/14). However, this study is limited by its small sample size, as well as by the fact that more than half of all patients were excluded from efficacy assessment because of lack of data for analysis. Furthermore, about 60% of patients evaluated for efficacy did not have confirmed invasive fungal infections; among these patients, micafungin was administered empirically or prophylactically. Therefore, verification of the findings in this study will require further studies with an increased number of patients with confirmed invasive fungal infection.

s evaluated for efficacy did not have confirmed invasive fungal infections; among these patients, micafungin was administered empirically or prophylactically. Therefore, verification of the findings in this study will require further studies with an increased number of patients with confirmed invasive fungal infection. In conclusion, we evaluated the safety and effectiveness of micafungin in real clinical practice using data obtained from the first postmarketing surveillance study, which enrolled 201 Japanese pediatric patients. It is suggested that micafungin has sufficient safety and effectiveness when used for candidiasis, aspergillosis, empiric therapy, febrile neutropenia, or prophylaxis of fungal infection in pediatric patients with various backgrounds, although this finding needs to be further validated in additional large-scale, prospective studies.

d that micafungin has sufficient safety and effectiveness when used for candidiasis, aspergillosis, empiric therapy, febrile neutropenia, or prophylaxis of fungal infection in pediatric patients with various backgrounds, although this finding needs to be further validated in additional large-scale, prospective studies. ACKNOWLEDGMENTS The authors gratefully acknowledge all the investigators in the following institutes for their cooperation with the postmarketing surveillance of micafungin and for providing valuable data: Chiba Children’s Hospital, Chiba University Hospital, Dokkyo Medical University Hospital, Ehime University Hospital, Fujita Health University Hospital, Gifu University Hospital, Gunma Children’s Medical Center, Hamamatsu University School of Medicine, University Hospital, Hiroshima Red Cross Hospital & Atomic-bomb Survivors Hospital, Hokkaido University Hospital, Hyogo Prefectural Kobe Children’s Hospital, Japanese Red Cross Medical Center, Japanese Red Cross Otsu Hospital, Japanese Red Cross Society Himeji Hospital, Juntendo University Hospital, Kagawa National Children’s Hospital, Kagawa University Hospital, Kagoshima City Hospital, Kagoshima University Medical and Dental Hospital, Kakogawa City Hospital, Kanazawa University Hospital, Kansai Medical University Hirakata Hospital, Kawaguchi Municipal Medical Center, Kawasaki Medical School Hospital, Kinki University Hospital, Kitasato University Hospital, Kiyose Children’s Hospital, Kyoto University Hospital, Kyoto-Katsura Hospital, Mie University Hospital, Miyagi Children’s Hospital, Nagasaki University Hospital, Nagoya City University Hospital, Nagoya Daini Red Cross Hospital, Nakadori General Hospital, National Hospital Organization Kumamoto Medical Center, National Hospital Organization Kyushu Cancer Center, Nihon University Itabashi Hospital, Nippon Medical School Hospital, Okazaki City Hospital, Osaka City Sumiyoshi Hospital, Saitama Children’s Medical Center, Saitama Medical University Hospital, Saitama Medical University International Medical Center, Sapporo Hokuyu Hospital, Sapporo Medical University Hospital, Shiga University of Medical Science Hospital, Shinshu University Hospital, Shizuoka Children’s Hospital, St Luke’s International Hospital, Teikyo University Chiba Medical Hospital, The University of Tokyo Hospital, Tokushima University Hospital, Tokyo Medical and Dental University Hospital, Tottori University Hospital, Toyama University Hospital, University Hospital, Kyoto Prefectural University

zuoka Children’s Hospital, St Luke’s International Hospital, Teikyo University Chiba Medical Hospital, The University of Tokyo Hospital, Tokushima University Hospital, Tokyo Medical and Dental University Hospital, Tottori University Hospital, Toyama University Hospital, University Hospital, Kyoto Prefectural University of Medicine, University of Fukui Hospital, and Yamagata University Hospital. Supported by Astellas Pharma Inc., Tokyo, Japan. At the time of study C.K., T.H., T.N., T.Y., M.S., and K.M. were employed by Astellas Pharma Inc. and received a wage.

risk protocol of the Children’s Cancer & Leukemia Study Group (CCLSG; ALL 941 Protocol Study; ages 10 to 19 y),1 she was administered tetrahydropyranyl adriamycin, vincristine, prednisone, and 6-mercaptopurine and achieved complete remission. Thereafter, she received intensification therapy using the same drug regimen. Systemic chemotherapy is usually ineffective as a central nervous system treatment because of the inability to cross the blood-brain barrier. To prevent central nerve recurrence, the high-risk protocol recommends prophylactic cranial irradiation (PCI). Thus, in October of the same year, the patient received PCI of 18 Gy in 12 fractions. From March to September 1998, she remained in remission and was administered maintenance therapy consisting of cyclophosphamide (CY), cytarabine, and 6-mercaptopurine. In October, the patient was admitted to receive bone marrow transplant from her brother who was a complete HLA match. As preconditioning chemotherapy, she received etoposide (60 mg/kg) for 24 hours, followed by the administration of CY (60 mg/kg) for 2 days. Thereafter, at 13 years of age, she received TBI with uterine and ovarian shielding; a total dose of 12 Gy with a fraction dose of 3 Gy, once a day, for 4 days. The following day (day 0), she received 6.37×107/kg of bone marrow from her HLA-matched brother. On day 30, graft survival was confirmed by bone marrow biopsy. Methotrexate (short-MTX) and cyclosporine A were administered for GVHD prevention. As the patient had neither GVHD nor other complications, she was discharged on day 41.

wing day (day 0), she received 6.37×107/kg of bone marrow from her HLA-matched brother. On day 30, graft survival was confirmed by bone marrow biopsy. Methotrexate (short-MTX) and cyclosporine A were administered for GVHD prevention. As the patient had neither GVHD nor other complications, she was discharged on day 41. The patient was treated with radiotherapy using 2 anteroposterior opposed fields with 10 MV x-rays from a linear accelerator (PRIMUS; Toshiba Medical Systems Corp., Tokyo, Japan). A dose of 3 Gy per fraction was delivered once daily 4 times during the 4-day period (total: 12 Gy), and the dose rate was 10 cGy/min. For ovarian shielding, pelvic computed tomography was performed before TBI to identify the locations of the uterus and ovaries. Next, pelvic radiographs were obtained and using pelvic computed tomography images for reference, the locations of the uterus and ovaries were identified and shielding was performed with an 80 mm thick lead block (Fig. 1). The attenuation rate of the lead block was measured using a water-equivalent phantom corresponding to the size measured in the patient; it was one eighth. Accordingly, the ovarian dose was reduced to approximately 1.5 Gy. FIGURE 1 A, Radiograph for confirmation of the uterine and ovarian shielding location. The box shows the location of the lead block. B, A portal image obtained at the location of the uterine and ovarian shielding in TBI. TBI indicates total body irradiation.

The patient was treated with radiotherapy using 2 anteroposterior opposed fields with 10 MV x-rays from a linear accelerator (PRIMUS; Toshiba Medical Systems Corp., Tokyo, Japan). A dose of 3 Gy per fraction was delivered once daily 4 times during the 4-day period (total: 12 Gy), and the dose rate was 10 cGy/min. For ovarian shielding, pelvic computed tomography was performed before TBI to identify the locations of the uterus and ovaries. Next, pelvic radiographs were obtained and using pelvic computed tomography images for reference, the locations of the uterus and ovaries were identified and shielding was performed with an 80 mm thick lead block (Fig. 1). The attenuation rate of the lead block was measured using a water-equivalent phantom corresponding to the size measured in the patient; it was one eighth. Accordingly, the ovarian dose was reduced to approximately 1.5 Gy. FIGURE 1 A, Radiograph for confirmation of the uterine and ovarian shielding location. The box shows the location of the lead block. B, A portal image obtained at the location of the uterine and ovarian shielding in TBI. TBI indicates total body irradiation. Ovarian function before and after bone marrow transplantation was evaluated using the luteinizing hormone-releasing hormone (LH-RH) loading test. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels at baseline before transplantation were 8.6 and 6.2 mIU/mL, respectively, both of which were normal premenarche levels. Levels 30 minutes after LH-RH administration were 12.2 and 20.6 mIU/mL for FSH and LH, respectively, which represent normal responses. FSH and LH levels at baseline 2 months after transplantation were 114.8 and 25.2 mIU/mL, respectively. As only the FSH level was abnormally high, secondary ovarian dysfunction was suspected (Fig. 2). Thirty minutes after LH-RH administration, FSH and LH levels were 143.7 and 25.2 mIU/mL, respectively, which represent normal responses. The patient remained in remission and experienced no chronic GVHD or other complications. In March 2000, menarche began at 14 years of age. Thereafter, she had a slightly irregular menstrual cycle. However, in January 2003 her FSH was 6.3 mIU/mL, LH 7.7 mIU/mL, and estradiol (E2) 73.4 pg/mL, which were all normal. In July 2009, she was confirmed pregnant at 23 years of age and had a favorable course thereafter. In April 2010, at 24 years of age, she naturally delivered a baby girl weighing 3182 g at 40 weeks and 3 days gestation. The mother and baby have been well ever since, with no apparent abnormalities nor complications.

al. In July 2009, she was confirmed pregnant at 23 years of age and had a favorable course thereafter. In April 2010, at 24 years of age, she naturally delivered a baby girl weighing 3182 g at 40 weeks and 3 days gestation. The mother and baby have been well ever since, with no apparent abnormalities nor complications. FIGURE 2 Ovarian function evaluated using luteinizing hormone-releasing hormone (LH-RH) before and after TBI. Month zero is the day of bone marrow transplantation. FSH indicates follicle-stimulating hormone; LH, luteinizing hormone; TBI, total body irradiation.

al. In July 2009, she was confirmed pregnant at 23 years of age and had a favorable course thereafter. In April 2010, at 24 years of age, she naturally delivered a baby girl weighing 3182 g at 40 weeks and 3 days gestation. The mother and baby have been well ever since, with no apparent abnormalities nor complications. FIGURE 2 Ovarian function evaluated using luteinizing hormone-releasing hormone (LH-RH) before and after TBI. Month zero is the day of bone marrow transplantation. FSH indicates follicle-stimulating hormone; LH, luteinizing hormone; TBI, total body irradiation. DISCUSSION TBI and CY are often administered as preconditioning regimens for bone marrow transplantation to achieve effective immunosuppression and to eradicate tumor cells. In girls, however, such preconditioning regimens are highly likely to result in ovarian dysfunction and infertility. When CY alone is used for preconditioning, ovarian function recovers at a high rate even if the CY concentration was high.2 Meanwhile, ovarian function recovery is only observed in 6.25% of patients treated with CY plus TBI.3 An irradiation dose of 3 Gy reportedly causes ovarian complications in 5% of patients,4 and the recovery rate in patients who receive TBI without ovarian shielding is no more than 15%.5 Moreover, TBI can cause uterine hypoplasia and disturb blood flow.6 Thus, even if patients become pregnant, they may experience miscarriages or preterm delivery. However, fertility may be preserved if the uterus and ovaries are shielded during TBI. From previous reports, ovarian function recovery was reported in 6 of 8 patients undergoing TBI with ovarian shielding, and 2 of the 6 ultimately gave birth to a baby.7,8 Table 1 shows a summary of the reports in which pregnancies were obtained after TBI with ovarian shielding. Among the patients who underwent TBI with ovarian shielding, ours was the youngest at only 13 years old and in premenarche. Ovarian function recovery is influenced by CY dose and irradiation dose. The most important factor is reportedly age, with better results obtained in younger patients.9 As our patient showed transiently elevated FSH levels 2 months after bone marrow transplantation, ovarian dysfunction was suspected (Fig. 2). This was attributed to preconditioning, because ovarian function immediately before transplantation had been normal. As the CY dose for preconditioning regimen was a total of 120 mg, and therefore not especially high, the FSH elevation might have been due to TBI as a preconditioning agent.

ction was suspected (Fig. 2). This was attributed to preconditioning, because ovarian function immediately before transplantation had been normal. As the CY dose for preconditioning regimen was a total of 120 mg, and therefore not especially high, the FSH elevation might have been due to TBI as a preconditioning agent. A past report on TBI with ovarian shielding described a patient with high FSH level and transient amenorrhea.7 The irradiation dose to the ovary in our patient was approximately 1.5 Gy with 8 fractioning times for 4 days due to performing TBI with ovarian shielding, but transient ovarian dysfunction still occurred. This is the first report of a patient giving birth to a baby after receiving PCI plus TBI, as she underwent an 18 Gy dose of PCI before TBI. When cranial plus spinal radiotherapy is performed to treat leukemia, the risk of miscarriage is approximately doubled owing to the effect of direct spinal cord irradiation or scattered rays, though the risk reportedly increases by 1.4-fold with cranial radiotherapy alone.10 Our patient had normal menarche at 14 years of age, normal menstruation. There is a model predicting the age of ovarian failure after radiotherapy.11 In this model, at the age of 13 years our patient received TBI, when ovarian dose is 12 Gy, the predicted age at ovarian failure is 13.6 to 21.4. Our patient had an uneventful pregnancy and delivery. The mother and baby have been healthy since with no leukemia recurrence or GVHD. Therefore, we consider uterine and ovarian shielding with TBI to be effective for preserving fertility. Recently, technologies in delivery of external beam of radiotherapy advance and intensity-modulated radiotherapy is used to concentrate dose into target tumors with sparing normal tissue. Total marrow irradiation using intensity-modulated radiotherapy to reduce doses to normal organs is reported12 and this technique may be used to TBI instead of ovarian shielding. Although advances in the field of reproductive endocrinology have made it possible to perform in vitro fertilization and to store frozen embryos, the clinical application of these procedures to children is difficult. In addition, it may delay treatment owing to ovarian stimulation and there is a high risk that collected cells may contain tumor cells.13 Uterine and ovarian shielding in TBI is a useful technique and can be performed relatively easily.

zen embryos, the clinical application of these procedures to children is difficult. In addition, it may delay treatment owing to ovarian stimulation and there is a high risk that collected cells may contain tumor cells.13 Uterine and ovarian shielding in TBI is a useful technique and can be performed relatively easily. However, as uterine and ovarian shielding may result in shielding tumor cells present in the pelvic bone, it should only be performed when patients and their family request fertility preservation and provide sufficient informed consent. TABLE 1 A Summary of Reports Presenting Pregnancies After TBI With Ovarian Shielding The authors declare no conflict of interest.

Myeloablative hematopoietic stem cell transplantation (HSCT) is the treatment of choice for certain very high-risk, relapsed, or refractory hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), myelodysplastic syndrome, chronic leukemias, and lymphomas. Total body irradiation (TBI) is often used in ablative transplant preparative regimens. Early studies of a variety of both lymphoid and myeloid malignancies, including both pediatric and adult age groups, showed that regimens using TBI had superior survival rates to those using chemotherapy alone.1–6 The role of TBI in treating hematopoietic malignancies is evolving. TBI-based regimens remain a preferred treatment for lymphoid malignancies, but they are no longer preferred for AML. The use of intravenous (IV) busulfan, which has replaced the more toxic and less efficacious oral preparation used in early comparative studies, yields better survival in this population.7 The frequent use of TBI, however, warrants its continued investigation.

oid malignancies, but they are no longer preferred for AML. The use of intravenous (IV) busulfan, which has replaced the more toxic and less efficacious oral preparation used in early comparative studies, yields better survival in this population.7 The frequent use of TBI, however, warrants its continued investigation. TBI-based regimens typically consist of a total dose of ≥1000 cGy, fractionated total body irradiation (FTBI), and delivered over several days according to various schemas at a dose rate of 7 to 19 cGy/min. TBI is associated with significant multiorgan toxicities, both acute and chronic. Acute toxicities include interstitial pneumonitis and severe mucositis; chronic toxicities include restrictive pulmonary disease, gonadal dysfunction, hypothyroidism, bone abnormalities such as osteochondroma and avascular necrosis (AVN), cataracts, secondary malignancies, and of particular concern in children, growth hormone deficiency, linear growth deceleration, and neurocognitive dysfunction.8–15

icities include restrictive pulmonary disease, gonadal dysfunction, hypothyroidism, bone abnormalities such as osteochondroma and avascular necrosis (AVN), cataracts, secondary malignancies, and of particular concern in children, growth hormone deficiency, linear growth deceleration, and neurocognitive dysfunction.8–15 Our institution explored whether the TBI toxicity profile could be improved without compromising outcomes. A novel regimen was developed in which a lower total amount of TBI was administered in a single fraction of 550 cGy (SFTBI), but administered at a high-dose rate of 30 cGy/min to achieve myeloablation.16 This approach, based on preclinical models as well as a single human-based feasibility study by Fyles et al,17 yielded similar efficacy but with lower toxicity relative to regimens using a higher total dose and a lower rate of delivery.18,19 A SFTBI regimen was developed for children by Druley et al,20 and demonstrated a 1-year overall survival (OS) of 60% and event-free survival (EFS) of 47%, which was similar to that seen with FTBI in both children and adults, with less acute toxicity. The utility of this treatment regimen in the pediatric population, however, is contingent not only on effective disease control, but also on the magnitude of long-term toxicities on children’s growth and development. The objective of this study is to examine the long-term effects in children >2 years following SFTBI-based HSCT.

ty of this treatment regimen in the pediatric population, however, is contingent not only on effective disease control, but also on the magnitude of long-term toxicities on children’s growth and development. The objective of this study is to examine the long-term effects in children >2 years following SFTBI-based HSCT. METHODS Patients and Assessments Sixty-one consecutive patients between the ages of 1 and 21 years with hematopoietic malignancies underwent transplant while enrolled on an institution-based study at St Louis Children’s Hospital using SFTBI and cyclophosphamide between March 1998 and May 2006. This was a heterogenous population of high-risk patients who had been exposed to a variety of prior treatments. Cyclophosphamide (60 mg/kg IV) was given on days −3 and −2 and SFTBI (550 cGy) was given on day −1. The protocol allows for additional radiation for CNS or local disease before or as part of the conditioning regimen. Details of TBI administration and stem cell dose and administration were described previously by Druley et al.20

phamide (60 mg/kg IV) was given on days −3 and −2 and SFTBI (550 cGy) was given on day −1. The protocol allows for additional radiation for CNS or local disease before or as part of the conditioning regimen. Details of TBI administration and stem cell dose and administration were described previously by Druley et al.20 An objective of this institution-based study was to explore toxicity of the novel preparative regimen described above. Early toxicities were described by Druley and colleagues. This work explores the late toxicities experienced by patients enrolled on this clinical trial, which completed accrual in May 2006. Patients included in this analysis met the following criteria: (1) alive at least 2 years following transplant with chart available for review; (2) in remission at the beginning of the late effects period (defined as beginning at 2 y following transplant); and (3) no additional radiation used in subsequent (posttransplant) treatment, that is, for relapse, before late effects period. Approval for this retrospective chart review was granted by the Washington University School of Medicine Institutional Review Board, with waiver of consent.

ning at 2 y following transplant); and (3) no additional radiation used in subsequent (posttransplant) treatment, that is, for relapse, before late effects period. Approval for this retrospective chart review was granted by the Washington University School of Medicine Institutional Review Board, with waiver of consent. Treatment-related toxicities were graded by a single investigator using the CTCAE v4.0 (http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf). Patients underwent a standard evaluation annually, which included height and weight measurements, Tanner staging, metabolic panel, and TSH and T4 levels. Pulmonary function tests were performed when developmentally appropriate at frequent intervals in the first year posttransplant (minimally at days +100, +180, 1 y) then annually for 5 years, and as clinically indicated. Echocardiogram and audiology studies were performed 2 years posttransplant and then repeated if clinically indicated. Ophthalmology studies were performed every 6 months posttransplant. Measurement of sex hormone levels (FSH, LH, testosterone, estradiol) of patients who were entering puberty (Tanner stage II), were in puberty (Tanner stages III to IV), or had completed puberty was offered beginning at 2 years posttransplant. Gonadal failure was defined as failure to begin puberty at expected age (standard Tanner criteria: 13 y for girls, 14 y for boys), regression of pubertal development, or depressed testosterone and estradiol levels. Growth hormone (GH) level was measured at the discretion of the treating physician.

years posttransplant. Gonadal failure was defined as failure to begin puberty at expected age (standard Tanner criteria: 13 y for girls, 14 y for boys), regression of pubertal development, or depressed testosterone and estradiol levels. Growth hormone (GH) level was measured at the discretion of the treating physician. Linear growth disturbance was assessed using Centers for Disease Control and Prevention (CDC) standard pediatric growth curves, comparing pretransplant and posttransplant growth velocity for each patient. Growth suppression is defined by the CTCAE as a decrease in expected growth velocity, and is classified as grade 1 (decrease of 10% to 29%; equivalent to 1 line crossed in a CDC standard growth curve), grade 2 (30% to 49%; equivalent to 2 lines crossed), or grade 3 (≥50%; equivalent to ≥3 lines crossed). Patients were tested at the discretion of the treating physician for osteopenia using dual-energy x-ray absorptiometry scan. Magnetic resonance imaging and plain film XR were used to evaluate AVN or osteochondroma if indicated by bone pain, joint signs or symptoms. Graft-versus-host disease (GVHD) was classified according to the NIH Consensus Development Project on Criteria for Clinical Trials in Chronic GVHD working group report.21

ry scan. Magnetic resonance imaging and plain film XR were used to evaluate AVN or osteochondroma if indicated by bone pain, joint signs or symptoms. Graft-versus-host disease (GVHD) was classified according to the NIH Consensus Development Project on Criteria for Clinical Trials in Chronic GVHD working group report.21 Neurocognitive testing was performed by the Pediatric Psychology Department at St Louis Children’s Hospital using age-appropriate standard test batteries pretransplant and typically 1 to 2 years posttransplant. Additional testing may have been performed at the request of the treating physician or family. A change in full-scale intelligence quotient (FSIQ) of 15 points (1 SD) was considered meaningful. Statistical Analysis IBM SPSS Statistics version 21 was used for analysis. The χ2 analysis was performed for categorical variables. The level of significance was specified as P<0.05.

Neurocognitive testing was performed by the Pediatric Psychology Department at St Louis Children’s Hospital using age-appropriate standard test batteries pretransplant and typically 1 to 2 years posttransplant. Additional testing may have been performed at the request of the treating physician or family. A change in full-scale intelligence quotient (FSIQ) of 15 points (1 SD) was considered meaningful. Statistical Analysis IBM SPSS Statistics version 21 was used for analysis. The χ2 analysis was performed for categorical variables. The level of significance was specified as P<0.05. RESULTS Patient characteristics, including indication for transplant, are summarized in Table 1. Eighty-one percent of patients were male. Fifty-two percent of patients had pre-B ALL, 9.5% T-cell ALL, 14% AML, 4.8% had biphenotypic leukemia, and 19% had NHL. Forty-eight percent were in CR1 and 52% in CR2 at transplant. Median follow-up time posttransplant was 6.8 years (range, 2.0 to 11.1 y). Median age at transplant was 13.2 years (range, 1.0 to 20.5 y) and median age at follow-up was 16.3 years (range, 9.1 to 30.5 y). Six patients (28%) received additional radiation therapy before or as part of the preparative regimen: 1 patient received radiation therapy to abdomen, liver, testes at relapse of Burkitt lymphoma; 1 received testicular and craniospinal (CS) radiation for extramedullary relapse of T-cell lymphoblastic lymphoma; 2 received CS radiation for isolated CNS relapse of pre-B ALL and previously unradiated CNS-negative T-cell ALL; 1 received CS radiation for marrow plus CNS relapse of pre-B ALL; and 1 received CS radiation for T-cell ALL in CR1 (induction failure).

radiation for extramedullary relapse of T-cell lymphoblastic lymphoma; 2 received CS radiation for isolated CNS relapse of pre-B ALL and previously unradiated CNS-negative T-cell ALL; 1 received CS radiation for marrow plus CNS relapse of pre-B ALL; and 1 received CS radiation for T-cell ALL in CR1 (induction failure). TABLE 1 Patient Characteristics Survival and follow-up status of the original cohort is shown in Figure 1. Sixty-one patients were transplanted on this regimen between March 1998 and May 2006; 55 patients had records available for evaluation of survival demonstrating a 2-year OS of 49% and 2-year EFS of 44%. FIGURE 1 Mortality and cohort eligibility. Schema of mortality, survival, and eligibility for late effects cohort. EFS indicates event-free survival; GVHD, graft-versus-host disease; LE, late effects; LTF, lost to follow-up; MOF, multiorgan failure; OS, overall survival.

Survival and follow-up status of the original cohort is shown in Figure 1. Sixty-one patients were transplanted on this regimen between March 1998 and May 2006; 55 patients had records available for evaluation of survival demonstrating a 2-year OS of 49% and 2-year EFS of 44%. FIGURE 1 Mortality and cohort eligibility. Schema of mortality, survival, and eligibility for late effects cohort. EFS indicates event-free survival; GVHD, graft-versus-host disease; LE, late effects; LTF, lost to follow-up; MOF, multiorgan failure; OS, overall survival. Twenty-one patients from the original cohort met the criteria for analysis of long-term effects. Six patients (9.8%) were lost to follow-up before 2 years posttransplant, 2 (3.3%) had insufficient records for review during the long-term effects period. Three patients relapsed just before the late effects period and were excluded. One patient with multiple local relapses underwent further radiation and was excluded from analysis. Five deaths occurred in the late effects cohort, resulting in a 76% survival for this group. One patient had a late relapse of ALL 5.5 years posttransplant, received salvage chemotherapy, and is alive and in remission >4 years after relapse and is included. One child had a relapse of his Philadelphia chromosome-positive ALL and underwent a second myeloablative HSCT with chemotherapy preparative regimen 1 year following the first HSCT with SFTBI. This patient entered the cohort 2 years following the first transplant with SFTBI.

ssion >4 years after relapse and is included. One child had a relapse of his Philadelphia chromosome-positive ALL and underwent a second myeloablative HSCT with chemotherapy preparative regimen 1 year following the first HSCT with SFTBI. This patient entered the cohort 2 years following the first transplant with SFTBI. Posttransplant Complications Generalized grading criteria are described in Table 2. Posttransplant complications are shown in Table 3. Because of the inevitable confounding of GVHD, its treatment complications, and TBI-related toxicity, toxicities in the subset of patients with GVHD are shown in Table 4 to clarify the toxicity profile of this group. TABLE 2 General Guideline of CTCAE V4.0 Grading TABLE 3 Complications Posttransplant TABLE 4 GVHD-associated Medical Complications in Patients With cGVHD

Posttransplant Complications Generalized grading criteria are described in Table 2. Posttransplant complications are shown in Table 3. Because of the inevitable confounding of GVHD, its treatment complications, and TBI-related toxicity, toxicities in the subset of patients with GVHD are shown in Table 4 to clarify the toxicity profile of this group. TABLE 2 General Guideline of CTCAE V4.0 Grading TABLE 3 Complications Posttransplant TABLE 4 GVHD-associated Medical Complications in Patients With cGVHD Endocrine Toxicity One child had panhypopituitarism as a complication of prior brain surgery during infancy and is not included in the analysis of endocrine toxicity and 1 had no records for endocrine follow-up. Thyroid: 4 of 19 evaluable patients (21%) had hypothyroidism identified with screening labs. Only 1 of 19 (5.2%) had overt (symptomatic) hypothyroidism. Gonadal function: documentation of gonadal dysfunction by history, Tanner stage, and/or menarche was available for 13 of the 15 patients, who were of pubertal age or greater at or after HSCT. By history and physical examination, 3 of the 13 (23%), 2 males and 1 female, had gonadal failure, which was confirmed by laboratory testing. Both males had received testicular radiation, 1 of the males and 1 female received CS radiation. Sex hormone levels were measured in 9 of the 15 patients (Table 5), yielding a 33% rate of hypogonadism in this group. All were treated successfully with hormone replacement. No patients had pubertal regression. One 3-year-old female patient was found to have precocious puberty on physical examination and was found to have elevated FSH and LH levels. GH deficiency is discussed below.

, yielding a 33% rate of hypogonadism in this group. All were treated successfully with hormone replacement. No patients had pubertal regression. One 3-year-old female patient was found to have precocious puberty on physical examination and was found to have elevated FSH and LH levels. GH deficiency is discussed below. TABLE 5 Laboratory Measurement of Gonadal Failure Linear Growth Toxicity Fourteen patients had linear growth potential at the time of the transplant, and 7 of the 14 (50%) had growth disturbance. One of the 14 (7.1%) had mild (grade 1) failure and history of steroid treatment for GVHD, and his subsequent growth tracked consistently along a new curve. Five of the 14 (36%) had moderate (grade 2) disturbance: 2 had steroid treatment for GVHD, 2 others had CS radiation, and 1 had preexisting panhypopituitarism. One child had severe (grade 3) growth failure and history of CS radiation. GH level was evaluated in 4 of the 6 patients with grade 2 or 3 linear growth impairment, and was found to be abnormally low in 1 patient with grade 2 impairment and history of CS radiation. Musculoskeletal Toxicity One patient in the 21-patient cohort (4.8%) had AVN and 4 of 21 patients (19%) had osteopenia. Two of the 21 patients (9.5%) had diagnosis of arthritis without diagnosis of AVN, although arthritis was attributed to steroid treatment in 1 of the 2. Two of 21 (9.5%) had osteochondroma.

Linear Growth Toxicity Fourteen patients had linear growth potential at the time of the transplant, and 7 of the 14 (50%) had growth disturbance. One of the 14 (7.1%) had mild (grade 1) failure and history of steroid treatment for GVHD, and his subsequent growth tracked consistently along a new curve. Five of the 14 (36%) had moderate (grade 2) disturbance: 2 had steroid treatment for GVHD, 2 others had CS radiation, and 1 had preexisting panhypopituitarism. One child had severe (grade 3) growth failure and history of CS radiation. GH level was evaluated in 4 of the 6 patients with grade 2 or 3 linear growth impairment, and was found to be abnormally low in 1 patient with grade 2 impairment and history of CS radiation. Musculoskeletal Toxicity One patient in the 21-patient cohort (4.8%) had AVN and 4 of 21 patients (19%) had osteopenia. Two of the 21 patients (9.5%) had diagnosis of arthritis without diagnosis of AVN, although arthritis was attributed to steroid treatment in 1 of the 2. Two of 21 (9.5%) had osteochondroma. Pulmonary, Cardiac, and Renal Toxicity Fifteen patients had the capability to undergo pulmonary function tests and records available. Four of the 15 (27%) had evidence of lung disease: 2 (13%) had grade 2 obstructive plus restrictive disease and 2 (13%) had grade 1 (asymptomatic) restrictive disease only. Three of the 4 had history of GVHD. There were no long-term cardiac toxicities. One patient of the 21 evaluated (4.8%) on prolonged calcineurin inhibitor therapy had grade 1 renal insufficiency as evidenced by mildly elevated creatinine.

strictive disease and 2 (13%) had grade 1 (asymptomatic) restrictive disease only. Three of the 4 had history of GVHD. There were no long-term cardiac toxicities. One patient of the 21 evaluated (4.8%) on prolonged calcineurin inhibitor therapy had grade 1 renal insufficiency as evidenced by mildly elevated creatinine. Ophthalmology and Audiology Results of ophthalmological evaluations were available for 20 patients. Five of 20 (25%) had cataracts: 4 (20%) were asymptomatic (grade 1) and 1 (5.0%) had overt disease, requiring surgery (grade 3). Audiology records were available for 17 patients. There was no transplant-associated hearing impairment. GVHD, Immune Dysfunction, Recurrence, and Secondary Malignancy Six patients (28%) had active cGVHD ≥2 years following transplant. Four patients had mild cGVHD (3 with skin disease and 1 with liver and sicca syndrome), 1 had moderate cGVHD (skin), and 1 had severe cGVHD (lung). Four patients were on systemic immune suppression at ≥2 years posttransplant. Six of the 21 patients (29%) had moderate to severe single or multiple infections. Five of the 6 had current active, or history of, cGVHD. Two (9.5%) experienced fatal infections with history of prolonged immune suppression for cGVHD. One patient (4.8%) on prolonged immune suppression experienced posttransplant lymphoproliferative disorder. There were no regimen-associated secondary malignancies. The association between cGVHD and growth disturbance, infection rate, endocrinopathy, pulmonary toxicity, and mortality was not statistically significant.

Six of the 21 patients (29%) had moderate to severe single or multiple infections. Five of the 6 had current active, or history of, cGVHD. Two (9.5%) experienced fatal infections with history of prolonged immune suppression for cGVHD. One patient (4.8%) on prolonged immune suppression experienced posttransplant lymphoproliferative disorder. There were no regimen-associated secondary malignancies. The association between cGVHD and growth disturbance, infection rate, endocrinopathy, pulmonary toxicity, and mortality was not statistically significant. Neurocognitive Effects and Performance Status Neurocognitive data were available for 9 of the 21 (43%) patients (Table 6). Three patients had follow-up testing at <2 years after transplant. FSIQ, verbal (VIQ), and performance or nonverbal (PIQ) IQs were compared. There was meaningful change in FSIQ in only 1 patient. This patient had stable IQ following transplant with SFTBI. He subsequently underwent a second myeloablative transplant 1 year after the first, and had a decline of 16 points after the second transplant. TABLE 6 Neurocognitive Testing Results Performance status was assessed in all patients. The majority of patients (17 of 21, 81%) had Lansky or Karnofsky performance status of 100%. Two of 21 (9.5%) had performance of 90% and 2 (9.5%) of 80%.

Neurocognitive Effects and Performance Status Neurocognitive data were available for 9 of the 21 (43%) patients (Table 6). Three patients had follow-up testing at <2 years after transplant. FSIQ, verbal (VIQ), and performance or nonverbal (PIQ) IQs were compared. There was meaningful change in FSIQ in only 1 patient. This patient had stable IQ following transplant with SFTBI. He subsequently underwent a second myeloablative transplant 1 year after the first, and had a decline of 16 points after the second transplant. TABLE 6 Neurocognitive Testing Results Performance status was assessed in all patients. The majority of patients (17 of 21, 81%) had Lansky or Karnofsky performance status of 100%. Two of 21 (9.5%) had performance of 90% and 2 (9.5%) of 80%. DISCUSSION Here we report the late toxicities in a cohort of 21 patients followed for ≥2 years after receiving a SFTBI-based HSCT. There were a total of 41 toxicities, the majority of which (71%) were grade ≤2. Known complications of cGVHD and corticosteroid treatment overlap with those of TBI and therefore confound the analysis. These include growth failure, endocrinopathy, infection, bone disease, and restrictive and obstructive lung disease.22 Although we observed that some of the most severe overlapping toxicities occurred in patients with GVHD, χ2 analysis revealed that this association was not statistically significant. It is possible that this finding was due to the small numbers in this cohort, and warrants further study.

and obstructive lung disease.22 Although we observed that some of the most severe overlapping toxicities occurred in patients with GVHD, χ2 analysis revealed that this association was not statistically significant. It is possible that this finding was due to the small numbers in this cohort, and warrants further study. Despite the challenges of comparing survival data across studies with variation with respect to disease profile, remission number, age range, and specific preparative and radiation regimens, we feel OS using this SFTBI-based regimen at 2 years remains comparable with that of reasonably similar patient populations undergoing transplant with FTBI-based preparative regimens.23–26 OS and EFS need to be interpreted with the caveat that in the current transplant era, which is subsequent to transplant in this cohort, myeloid malignancies are no longer treated with TBI-based regimens, as discussed above.

bly similar patient populations undergoing transplant with FTBI-based preparative regimens.23–26 OS and EFS need to be interpreted with the caveat that in the current transplant era, which is subsequent to transplant in this cohort, myeloid malignancies are no longer treated with TBI-based regimens, as discussed above. Endocrine late effects are among the most common seen following any form of TBI, with hypothyroidism, gonadal failure, and growth hormone deficiency particularly common in this population. We observed a rate of hypothyroidism comparable with the 12% to 21% reported for patients exposed to FTBI, with similar rates of compensated and overt hypothyroidism, and a gonadal failure rate (23% by clinical evaluation) similar to the 22% to 43% reported for patients exposed to FTBI.10,23,27–30 Linear growth disturbance is one of the most common toxicities of TBI and strongly impacts quality of life.31 The etiology of growth disturbance is multifactorial, and includes growth hormone deficiency, sex hormone deficiency, and severe hypothyroidism, as well as nonendocrine causes such as radiation damage to bone and growth plates and protein catabolism due to steroid toxicity. Patients receiving FTBI are reported to have growth disturbances between 24% and 100%, with many patients requiring growth hormone replacement.10,23,30 Our rate (50%) was consistent with these findings. Interestingly, all patients in our cohort with growth disturbance had either a history of prolonged steroid treatment, additional CS radiation, or a preexisting condition (panhypopituitarism). Only 4 of the 7 patients with growth disturbance had GH levels measured, but as these data are limited, it is encouraging that only 1 patient was found to have GH deficiency.

growth disturbance had either a history of prolonged steroid treatment, additional CS radiation, or a preexisting condition (panhypopituitarism). Only 4 of the 7 patients with growth disturbance had GH levels measured, but as these data are limited, it is encouraging that only 1 patient was found to have GH deficiency. Pulmonary toxicities, including interstitial pneumonitis and pulmonary fibrosis leading predominantly to restrictive lung disease, are among the most serious late effects of TBI-based regimens. Pulmonary disease in the postallogeneic transplant population is commonly confounded by GVHD, which can present both a restrictive and obstructive picture. Although some authors report minimal lung disease in patients receiving FTBI,23 restrictive disease can occur in up to 74% of patients, severe disease in up to 12%, and death in up to 6% in patients receiving FTBI.28,30,32 We saw very little restrictive disease and there was no severe lung disease. Only 1 patient had pulmonary disease (restrictive) outside the setting of history of GVHD. Although not a common toxicity, cardiomyopathies have been reported in patients who have received TBI.23,33 To date, we have not observed any patients with cardiomyopathies in our cohort. Cataracts are a common complication of TBI, with rates in FTBI ranging from 22% to 78%.23,27,28,30,34 Our rate was similar to the lowest rates reported, with overt cases (5%) consistent with the lowest rates reported for FTBI.

Pulmonary toxicities, including interstitial pneumonitis and pulmonary fibrosis leading predominantly to restrictive lung disease, are among the most serious late effects of TBI-based regimens. Pulmonary disease in the postallogeneic transplant population is commonly confounded by GVHD, which can present both a restrictive and obstructive picture. Although some authors report minimal lung disease in patients receiving FTBI,23 restrictive disease can occur in up to 74% of patients, severe disease in up to 12%, and death in up to 6% in patients receiving FTBI.28,30,32 We saw very little restrictive disease and there was no severe lung disease. Only 1 patient had pulmonary disease (restrictive) outside the setting of history of GVHD. Although not a common toxicity, cardiomyopathies have been reported in patients who have received TBI.23,33 To date, we have not observed any patients with cardiomyopathies in our cohort. Cataracts are a common complication of TBI, with rates in FTBI ranging from 22% to 78%.23,27,28,30,34 Our rate was similar to the lowest rates reported, with overt cases (5%) consistent with the lowest rates reported for FTBI. Both steroid use and TBI are risk factors for AVN. In studies including children and adults receiving either a single large dose or FTBI, incidences range from 4% to about 20%, with incidences increasing with increasing doses of radiation.11,27,35–37 Our findings are similar to the lowest levels reported. We saw less osteochondroma than the 24% to 36% reported for FTBI.15,28,38 Our rate of cGVHD at 2 years (28%) was comparable with that seen in a large study by Zecca et al39 at 3 years (27%).

with incidences increasing with increasing doses of radiation.11,27,35–37 Our findings are similar to the lowest levels reported. We saw less osteochondroma than the 24% to 36% reported for FTBI.15,28,38 Our rate of cGVHD at 2 years (28%) was comparable with that seen in a large study by Zecca et al39 at 3 years (27%). Secondary malignancies are complications of both TBI and chemotherapy, and can occur in up to 20% of patients.13,23,27,28,30 Although none were observed in our cohort, we recognize that longer follow-up is needed for a more meaningful assessment, and most reports of secondary malignancy occur at least 10 years from radiation. Neurocognitive function is vulnerable, especially in the most formative years of brain growth and development (age below 3), to any form of radiation including TBI, with very young patients exhibiting the most chronic deficits.9,14,40,41 Reducing radiation to the developing brain was a motivation for developing a reduced radiation regimen. Although data are limited, it is reassuring that deficits in our cohort, as measured by IQ, were limited in both quantity and degree.

ding TBI, with very young patients exhibiting the most chronic deficits.9,14,40,41 Reducing radiation to the developing brain was a motivation for developing a reduced radiation regimen. Although data are limited, it is reassuring that deficits in our cohort, as measured by IQ, were limited in both quantity and degree. Limitations of this study include the small number of patients, a variety of underlying diagnoses, and large age range of patients. Chart review design and inconsistent reporting, particularly with respect to neurocognitive function, gonadal function, and GH levels, were also limiting, as was the total of 13% loss to follow-up or incomplete records. Neurocognitive testing posttransplant was performed in only a fraction of patients, and in some cases <2 years from transplant, at a time that may miss the most serious deficits, particularly as younger patients mature. Given scant reporting of neurocognitive effects in the literature, we felt results were worth reporting. We recognize that several toxicities, particularly secondary malignancies, cataracts, and bone toxicity almost certainly require more time than was allotted here to develop. However, this is the first long-term toxicity data reported on patients receiving this novel preparative regimen, and the only report on an exclusively pediatric population. Bearing in mind the limitations, the results are nonetheless encouraging in that some of the more common toxicities of TBI are found to be at worst similar to, and in some cases less than, those seen with FTBI. Study in a larger cohort would be beneficial in exploring these observations further.

y pediatric population. Bearing in mind the limitations, the results are nonetheless encouraging in that some of the more common toxicities of TBI are found to be at worst similar to, and in some cases less than, those seen with FTBI. Study in a larger cohort would be beneficial in exploring these observations further. In summary, SFTBI as part of an ablative transplant regimen delivers less total radiation than a FTBI regimen. This study was limited by the small number of patients and a large range of ages and underlying diagnoses, hence comparison with FTBI regimen is challenging. Further study directly comparing SFTBI to a FTBI regimen is warranted. We feel this study does demonstrate the feasibility of this preparative regimen, and clinicians might consider this therapy as an alternative to FTBI if committed to a TBI-based regimen. ACKNOWLEDGMENTS The authors thank Dr Nicole Cruz for the help in interpreting neurocognitive testing results and Susan Hayashi for the help in interpreting audiology data. The authors declare no conflict of interest.

This manuscript presents 2 types of systemic lymphatic malformations described in the recently revised ISSVA classification approved in Melbourne in April 2014.1 Generalized lymphatic anomaly (GLA) is characterized by multifocal lymphatic malformation affecting the skin and superficial soft tissues, viscera of the abdominal and thoracic cavities, and bone. The bone disease in GLA usually spares the bone cortex, and does not progress with time. GLA can present with acute or persistent pericardial, pleural, or peritoneal effusions. Although the lymphatic malformation in Gorham-Stout disease (GSD) is also characterized by lymphatic malformation affecting a single or multiple bones and adjacent soft tissues, the osteolysis is progressive and invades the bone cortex. It was originally described in the orthopedic literature as disappearing or vanishing bone disease.2–4 The progression of GSD often includes visceral progression with thoracic and abdominal involvement leading to effusions and ascites. Pathologic fractures occur in both GLA and GSD, even though they are more frequent in GSD. The older literature can be confusing as clinicians often describe involvement of soft tissues and visceral organs by lymphatics as “congenital lymphangiomatosis,” “disseminated lymphangiomatosis,” or “multifocal lymphangiomatosis.” These terms are better to be avoided because they are not very helpful for prognostication or in guiding future medical management.

nicians often describe involvement of soft tissues and visceral organs by lymphatics as “congenital lymphangiomatosis,” “disseminated lymphangiomatosis,” or “multifocal lymphangiomatosis.” These terms are better to be avoided because they are not very helpful for prognostication or in guiding future medical management. In addition to the skeleton, systemic lymphatic disease often includes aggressive unremitting proliferation of lymphatic vessels in lungs, spleen, and other visceral organs.5–7 This aggressive disease benefits from early intervention. In contrast, many patients with GLA can be safely observed, and timely management depends on correct diagnosis. At least at present, there are no molecular markers or radiologic features that can identify the patient at risk for progression. Some clinicians feel that multiple lesions with distinct sclerotic edges may suggest disease that will remain stable for years, and that the presence of a soft tissue component and/or pleural effusion may identify disease more likely to progress,7 but even for experts, defining an incipient progression is difficult. Notable are also the differences in the character of the disease across the life span. Commonly, a previously quiescent disease is exacerbated by puberty,8,9 suggesting an anabolic effect of sex-specific steroids on the lymphatic vasculature. Although in early childhood most GLA/GSD diagnoses are incidental findings of a lytic lesion during a trauma evaluation, the diagnoses of GSD/GLA during puberty/adolescence are usually due to symptoms associated with disease progression.9,10

ting an anabolic effect of sex-specific steroids on the lymphatic vasculature. Although in early childhood most GLA/GSD diagnoses are incidental findings of a lytic lesion during a trauma evaluation, the diagnoses of GSD/GLA during puberty/adolescence are usually due to symptoms associated with disease progression.9,10 For those pediatricians working without the help of a vascular anomalies expert, it is often difficult to recognize an acute exacerbation of systemic lymphatic lesions such as GLA/GSD and recommend the most appropriate early intervention. GLA, because of its resemblance to metastatic lesions is often referred to oncology, whereas GSD is usually evaluated by orthopedics. The referral to a vascular anomalies center is often delayed and the window of opportunity is often missed until the disease gathers an unremitting course, involving multiple organs leading eventual cardiovascular collapse. Even though there are no standard therapies for GLA, early case reports indicate that stabilization of the disease and/or remission can be achieved. As is the case with many rare diseases, a large spectrum of interventions has been used and includes: glucocorticoids, radiation, bisphosphonates, combination of radiation and bisphosphonates, pegylated-interferon, regular interferon and heparin, bevacizumab, or thalidomide.

he disease and/or remission can be achieved. As is the case with many rare diseases, a large spectrum of interventions has been used and includes: glucocorticoids, radiation, bisphosphonates, combination of radiation and bisphosphonates, pegylated-interferon, regular interferon and heparin, bevacizumab, or thalidomide. The treatment varies widely. A retrospective analysis of 67 cases (64 published in Chinese and 3 in English) from 54 publications were analyzed,11 and the treatment varied from surgery (n=27, 40.3%), radiation therapy (n=6, 9.0%), surgery combined with radiation therapy (n=2, 3.0%), and medical therapy (n=7, 10.4%), such as interferon (n=2), bisphosphonates (n=1), calcium/vitamin D (n=1), cyclophosphamide/5FU (n=2), and calcitonin/alendronate (n=1). The few available larger case series10,12 also included cases before the emergence of molecular markers and the efficacy of the used oncological therapies could not be assessed. Recently, a successful use of bevacizumab, an antibody against vascular endothelial growth factor (VEGF), in a Gorham-Stout disease patient was reported.13

able larger case series10,12 also included cases before the emergence of molecular markers and the efficacy of the used oncological therapies could not be assessed. Recently, a successful use of bevacizumab, an antibody against vascular endothelial growth factor (VEGF), in a Gorham-Stout disease patient was reported.13 As oncology embraces molecularly based therapeutics, many promising antiangiogenic agents and combinations that may be useful in GLA/GSD are being accepted, and sharing these strategies is extremely important. In absence of evidence-based, disease-modifying, curative therapy for GLA/GSD, this manuscript provides a rational approach to individualizing the treatments of patients with GLA/GSD on the basis known biology, and their tissue-specific molecular markers. We used a synergistic combination of sunitinib, a direct inhibitor of angiogenesis, and low-dose metronomic schedule14 of taxol to minimize toxicity in this nonmalignant disease. CASE REPORTS The patients and their guardians were informed of the risks, benefits, and therapeutic alternatives before treatment on an individualized protocol. It was stressed that their lesions were not amenable to more standard therapeutic approaches such as surgery or sclerotherapy, and they understood that the goal of the therapy was disease stabilization rather than cure.

of the risks, benefits, and therapeutic alternatives before treatment on an individualized protocol. It was stressed that their lesions were not amenable to more standard therapeutic approaches such as surgery or sclerotherapy, and they understood that the goal of the therapy was disease stabilization rather than cure. The first patient was initially diagnosed with GLA at the age of 7 years. He presented with recurrent coughing leading to the diagnosis of a left-sided chylothorax requiring multiple drains. The effusion did not recede despite a medium chain triglyceride (MCT) diet. The child’s pulmonary disease was exacerbated as he entered puberty. By 13 years, he had severe pulmonary insufficiency, and large persistent bilateral chylous pleural effusions due to the presence of bilateral paravertebral lymphatic malformation extending from third to seventh thoracic vertebrae. He had undergone external drainage of the pleural effusion followed by pleurodesis, with minimal improvement in symptoms. Immunohistochemistry of this pleural biopsy showed D2-40 and CD31+ endothelium with expression of PDGFR and VEGFR1 (Fig. 1). At the time of this child’s diagnosis, there was no clinically applicable antibody for VEGFR2, but there was sufficient overlap between VEGFR1 and 2 and 3 using the ZYTOMED Systems (GmbH, Berlin) antibody to justify its use. The choice of PDGFR was based on previously published data on its expression in Gorham disease.15 The child ultimately required tracheostomy for long-term airway management, and a gastroduodenostomy to facilitate low fat, MCT diet. It was at this late point of the disease progression that he was referred to the oncologist. He was initially treated with sarcoma-like therapy with 2 cycles of cyclophosphamide (1500 mg/m2) and vincristine (1.5 mg/m2). Despite severe side effects such as neutropenia, sepsis, and candidiasis, this regimen provided no benefit. After 2 months on mechanical ventilation, and no standard therapeutic options, we reviewed the tissue markers with hope to develop new approaches for this child. Although it is not always possible to do colocalization studies to establish that lymphatic endothelium is positive for tyrosine kinase receptors such as VEGFR and PDGFR in clinical setting, the pleural biopsy showed D2-40+ and CD31+ endothelium, with PDGFR+ and VEGFR-1/2+ expression in the same vascular pattern (Fig. 1) providing some reassurance about the histologic configuration.

o establish that lymphatic endothelium is positive for tyrosine kinase receptors such as VEGFR and PDGFR in clinical setting, the pleural biopsy showed D2-40+ and CD31+ endothelium, with PDGFR+ and VEGFR-1/2+ expression in the same vascular pattern (Fig. 1) providing some reassurance about the histologic configuration. The goals of the therapy, namely disease stabilization and extubation, were clearly identified to parents, and consents obtained before initiation of treatment. The child’s therapy was then changed to sunitinib 12.5 mg daily and taxol 10 mg/m2/once weekly. The dose of taxol was increased every 4 weeks up to 60 mg/m2/once weekly, and was tittered to avoid myelosuppression. There were no significant toxicities with this regimen, no peripheral neuropathy, no neutropenia, and no increase in infections. Over the subsequent 12 months, he was weaned off mechanical ventilation, extubated, and weaned from 8 to 10 L of O2 to room air (supplemental data, Table 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A111). He had both clinical and radiologic regression of the pulmonary infiltration, as well as of the soft tissue lesions (Fig. 2). Unfortunately, the patient later relapsed on therapy, required reintubation, and ultimately succumbed to the disease.

a, Table 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A111). He had both clinical and radiologic regression of the pulmonary infiltration, as well as of the soft tissue lesions (Fig. 2). Unfortunately, the patient later relapsed on therapy, required reintubation, and ultimately succumbed to the disease. FIGURE 1 Immunhistochemistry of pleural biopsy in case 1 (A), and of left ileosacral soft tissue in case 2 (B). Formalin-fixed, paraffin-embedded archival tissues were processed using standard IHC methods and stained with anti-VEGFR-1, anti-PDGFR, and anti-podoplanin (D2-40) antibodies (all from ZYTOMED Systems; GmbH) at 1:20 dilution. In both cases the CD34+ endothelium was also positive for D2-40 confirming its lymphatic origin. Further support for the therapy was the positive staining for PDGFR and VEGFR-1 tyrosine kinases, both targets of sunitinib. All images were taken at ×20 magnification with the exception of PDGFR and VEGFR in case B (ileosacral soft tissue), which was captured with ×40 lens. VEGFR indicates vascular endothelial growth factor receptor. FIGURE 2 Radiologic findings. For both patients T2-weighted magnetic resonance imaging at baseline (before initiation of therapy), at 6 and 18 months on therapy. In both, the thorax images for case 1 (A), and the pelvis images in case 2 (B) there is a marked reduction of the pathologic lymphatic tissue burden, as well as of the accompanying edema.

For both patients T2-weighted magnetic resonance imaging at baseline (before initiation of therapy), at 6 and 18 months on therapy. In both, the thorax images for case 1 (A), and the pelvis images in case 2 (B) there is a marked reduction of the pathologic lymphatic tissue burden, as well as of the accompanying edema. The second patient was diagnosed with GSD at the age of 14 years, after an episode of severe pain and swelling in the lower back after a football injury. The magnetic resonance imaging revealed a diffuse osteolytic lesion in the left ileosacral articulation, ileum, and sacrum, as well as in several vertebral bodies. The difficulty with histologic diagnosis necessitated 3 consecutive biopsies, leading to a recalcitrant lymphatic leak for over 3 weeks. This child also received MCT diet. The ileosacral articulation was stabilized by a supportive corset, which also provided some degree of vascular compression. As the lesion continued to progress, the decision was made to escalate therapy, but no standard therapeutic options were available. We again discussed with parents and clearly identified the goal of the potential targeted therapy, namely disease stabilization. We summarized this in a consent that included the risk, benefits, and goals of the various therapies used in the past. We then initiated a combination therapy with sunitinib 12.5 mg PO daily and intravenous taxol 10 mg/m2/once weekly. The child completed 12 months of therapy, and experienced gradual improvement in both clinical and radiologic measures (Fig. 2). There were no significant toxicities with this regimen, no peripheral neuropathy, no neutropenia, and no increase in infections. The therapy was stopped after a year, and he has remained stable for over 4 years.

12 months of therapy, and experienced gradual improvement in both clinical and radiologic measures (Fig. 2). There were no significant toxicities with this regimen, no peripheral neuropathy, no neutropenia, and no increase in infections. The therapy was stopped after a year, and he has remained stable for over 4 years. DISCUSSION The classification of lymphatic lesions has recently been updated.1 By this new ISSVA classification, case 1 carries the diagnosis of “GLA” because pulmonary effusion was the first pathologic finding, and the second case with a large aggressively osteolytic lesion in the sacrum is a “lymphatic malformation in Gorham-Stout disease.” The purpose of this manuscript is to introduce new therapeutic options for GLA/GSD rather than review classification, and we refer the reader to recent publications discussing the differences between the 2 entities.7 Whether one views GSD/GLA as a continuum ranging from a single lymphatic malformation of the bone (monostotic), multiple but nonprogressive lytic lesions of the bone (polyostotic), to disseminated lymphangiomatosis with pulmonary compromise,6,16 the diagnostic distinction is important for management. Recognizing which lesion is likely to progress is more likely to lead to a timely referral to an experienced multidisciplinary team familiar with the disorder, and to institution of a timely and effective therapy.

d lymphangiomatosis with pulmonary compromise,6,16 the diagnostic distinction is important for management. Recognizing which lesion is likely to progress is more likely to lead to a timely referral to an experienced multidisciplinary team familiar with the disorder, and to institution of a timely and effective therapy. The etiology of GLA/GSD remains unclear, even though the triggering effect of inflammation, puberty, and trauma is quite established. There are early indications that at least some of these osteolytic bone lesions are due to genetic mutations.17 The V-MAF—musculoaponeurotic fibrosarcoma oncogene family protein B was described in 11 cases of multicentric carpotarsal osteolysis syndrome by the Australian group of Zankl and Duncan,18 the lymphoedema-distichiasis syndrome described in 74 patients with FOXC2 mutation,19 the Nonne-Milroy syndrome in patients with FLT4/VEGFR3 mutation,20 hypotrichosis-lymphedema-telangiectasia in patients with SOX18,21,22 and primary GLA (Hennekam lymphangiectasia-lymphedema syndrome) in patients with CCBE1 mutation.23 It is very likely that with time additional genetic mutations will further clarify the spectrum of lymphatic malformations of the bone. Although the genetic alterations may be both germinal and/or somatic, neither GLA nor GSD are presently associated with a risk of malignant transformation.7

ents with CCBE1 mutation.23 It is very likely that with time additional genetic mutations will further clarify the spectrum of lymphatic malformations of the bone. Although the genetic alterations may be both germinal and/or somatic, neither GLA nor GSD are presently associated with a risk of malignant transformation.7 The pathogenesis of GLA/GSD is also unclear. Although some investigators believe that there is minimal cell turnover in the pathologic lesions of GLA or GSD, the disease is in fact characterized by an active proliferation of lymphatic endothelium,24 by platelet derived growth factor pathway activation,15 and by upregulation of a number of lymphangiogenesis stimulating pathways.5 A recent analysis of the cellular and humoral mechanisms underlying GSD25 compared the numbers and sensitivity to osteoclastogenic factors between age/sex-matched controls and a GSD patient. They found that even though the number of circulating osteoclast progenitors was not increased, the osteoclast precursors showed an increased sensitivity to IL-1β, IL-6/sIL-6R, and TNFα, leading to increased osteoclastic activity.

nsitivity to osteoclastogenic factors between age/sex-matched controls and a GSD patient. They found that even though the number of circulating osteoclast progenitors was not increased, the osteoclast precursors showed an increased sensitivity to IL-1β, IL-6/sIL-6R, and TNFα, leading to increased osteoclastic activity. It is unclear whether the lymphatic vessel proliferation in GLA/GSD is a secondary or a primary event. Although physiologically normal, healthy lymphatic endothelium serves to decompress tissues by returning lymphatic fluid to venous circulation, its pathologic counterpart looses this directional flow and transforms into a leaky, invasive, and proliferative vascular meshwork. These types of leaky lymphatic endothelial cells have been well described in other lesions with pathologic lymphangiogenesis such as in Klippel-Trenaunay syndrome and kaposiform lymphangiomatosis, as well as in many malignancies. The invasive nature and bone destruction within these lesions can be surmised from the increased acid phosphatase activity in mononuclear phagocytes, multinuclear osteoclasts, and vascular endothelium.26 Furthermore, because lymphatic vessels are not present in healthy intact bones,27 lesions in both GLA and GSD are likely to represent a manifestation of systemic disease with either primary or secondary bone and soft tissue activation. In either way, activated lymphatic endothelium can be inhibited by a combination of metronomic therapy and antiangiogenic agent.14 The inhibition of activated stroma using this approach may be further amplified by inhibition of alternative pathways such as the PI3K/Akt/mTOR pathway by sirolimus. The efficacy of sirolimus in complex vascular anomalies such as kaposiform hemangioendothelioma and diffuse microcystic lymphangiogenesis has recently been published,28 but its efficacy had not been established at the time we needed intervention for our 2 patients. It should be pointed out here that the inhibition of the ras/raf/MAPK pathway by sunitinib had equivalent outcome to the inhibition of PI3K/Akt/mTOR pathway (extubation and stabilization of disease), suggesting that these 2 pathways are alternatives and inhibition of both would have synergistic effect.

our 2 patients. It should be pointed out here that the inhibition of the ras/raf/MAPK pathway by sunitinib had equivalent outcome to the inhibition of PI3K/Akt/mTOR pathway (extubation and stabilization of disease), suggesting that these 2 pathways are alternatives and inhibition of both would have synergistic effect. The lack of understanding of the etiology and pathogenesis of GLA/GSD has been the biggest hindrance in developing effective therapeutic approaches. Although gene therapy may be possible one day for those sybtypes of systemic lymphatic malformations where a single genetic defect has been identified single gene mutations, many GLA/GSD patients will need therapy before then. Because both GLA and GSD are difficult to treat by surgery alone, systemic therapies need to be directed to minimizing the process of lymphangiogenesis, stromal invasion, and at maximizing bone restoration. Even though these lesions are not cancers, or even tumors, they grow and progress. Thus, even though they have not been historically thought to be proliferative, and are therefore presumed not to respond to traditional high-dose chemotherapy, they do grow and proliferate24 and are highly dependent on growth factors15 and can therefore be treated with biological agents. Because the target of metronomic chemotherapy is the tumor stroma rather than the cancer cell, the combination of low-dose metronomic chemotherapy and a direct angiogenesis inhibitor provides a good, minimally toxic therapeutic alternative for lymphatic malformations. The strategy has shown efficacy in preclinical and clinical settings.29 The rationale of using this particular combination of a tubulin inhibitor and direct anti-angiogenic agent was supported by the extreme sensitivity of endothelial cells (both lymphatic and microvascular) to tubulin inhibitors,30 and on the presence of increased levels of PDGFR and VEGFR1/2 in the patient tissues (Fig. 1). Unlike tumor cells, endothelial cells rely on cytoskeleton for both adhesion and polarization, and cannot grow in anchorage independent manner.

of endothelial cells (both lymphatic and microvascular) to tubulin inhibitors,30 and on the presence of increased levels of PDGFR and VEGFR1/2 in the patient tissues (Fig. 1). Unlike tumor cells, endothelial cells rely on cytoskeleton for both adhesion and polarization, and cannot grow in anchorage independent manner. Endothelial cells are also exquisitely dependent on continuous supply of growth factors and the combination of a direct angiogenesis inhibitor with low-dose tubulin inhibitor has been shown to lead to vascular collapse.14 The combination is particularly attractive in cases of histologically benign, but aggressively invasive disease such as GLA, because a long-term minimally toxic treatment is usually needed and because the likelihood of causing secondary malignancies with this regimen is extremely low.

wn to lead to vascular collapse.14 The combination is particularly attractive in cases of histologically benign, but aggressively invasive disease such as GLA, because a long-term minimally toxic treatment is usually needed and because the likelihood of causing secondary malignancies with this regimen is extremely low. The radiologic and clinical responses observed in the 2 patients presented in this manuscript support the applicability of this approach in GLA and severe progressive GSD. In our first case, an argument can be made that an earlier application of this therapeutic combination may have prevented the end-organ damage resulting from a long-standing pathologic lymphatic infiltration of pulmonary vasculature. He was diagnosed at 7 years of age, and as expected, his disease was exacerbated by puberty leading to irreversible respiratory compromise. Despite the chronicity of the pulmonary disease, he experienced a marked radiologic and clinical improvement on the combination regimen. It should also be noted that he required long-term therapy. Because long-term anti-angiogenic therapy has been shown to lead to tolerance,31 one is left to wonder if a drug holiday, or a change to an alternative regimen may have resulted in a better outcome for this child. Even though the damage caused by many years of chronic disease would not have been reversed, it is possible that a long-term quiescence may have ensued with changing the regimen.

rance,31 one is left to wonder if a drug holiday, or a change to an alternative regimen may have resulted in a better outcome for this child. Even though the damage caused by many years of chronic disease would not have been reversed, it is possible that a long-term quiescence may have ensued with changing the regimen. Both of the cases presented in this manuscript showed with disease exacerbation during puberty. Although many experts in the vascular anomalies field have made this observation, there is paucity of studies on the topic. The frequency of disease exacerbation during puberty, pregnancy, and other conditions involving hormonally unstable states is consistent with the preclinical evidence of testosterone and estrogen having anabolic effect on the vasculature.32 The second inciting stimulus often observed in clinic is trauma. In the case of the second child, the inflammation accompanying an acute injury may have contributed to an exacerbation of otherwise quiescent disease. The inflammation associated with injury tends to disturb the balance of stimulators and inhibitors of lymphangiogenesis and can lead to progressive disease. A reinduction of disease quiescence and adequate control of the lymphatic endothelium proliferation using a combination of sunitinib and low-dose taxol, can reinstitute disease stability. Indeed, after only a year of therapy, the child has remained stable for over 3 years.

iogenesis and can lead to progressive disease. A reinduction of disease quiescence and adequate control of the lymphatic endothelium proliferation using a combination of sunitinib and low-dose taxol, can reinstitute disease stability. Indeed, after only a year of therapy, the child has remained stable for over 3 years. In conclusion, a timely referral to vascular anomalies centers with expertise in diagnosis and management of GLA and GSD, leads to timely therapy. In these rare diseases, tailoring the therapy to the underlying biology represents a very rational alternative. Because large randomized double blind clinical trials would be difficult, it will be essential to share both positive and negative experiences with specific molecularly based therapies, and record the outcomes obtained with specific individualized protocols. As more and more angiogenesis inhibitors and biological response modifiers are becoming available in an “off label” setting, collaborative consortia should be used to share these strategies and define the best future approaches. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.jpho-online.com. G.L.K. was funded by NIH RO1 GM93050, and by philanthropic funding of Newman Lakka Cancer Foundation. The authors declare no conflict of interest.

Mortality from childhood malignancies has declined significantly over the past 40 years, largely due to advances in pediatric cancer treatments.1 Although childhood cancers remain one of the leading causes of death for children 1 to 14 years old in the United States,2 their origin is only partly understood.3 However, a growing body of literature has implicated environmental hazards in the etiology of certain childhood cancers. The President’s Cancer Panel states in their 2008 to 2009 Annual Report that, “the true burden of environmentally induced cancer has been grossly underestimated.”4 Exposure to ionizing radiation from nuclear accidents, x-rays, or radiation therapy is associated with an increased risk of childhood leukemia5–7 and solid tumors.8–10 Exposures to solvents and ambient air pollutants, including benzene, may also contribute to an increased risk of childhood leukemia.11–14 Evidence suggests a link between parental, prenatal, and childhood exposures to pesticides and childhood leukemia in both residential and occupational settings.15–21 In utero exposure to household insecticides and indoor pesticides is linked to increased risk of childhood leukemia.22 Finally, numerous studies from around the world have consistently identified associations between pesticide exposures and risk of lymphomas, brain tumors, and other solid tumors.19,23

al settings.15–21 In utero exposure to household insecticides and indoor pesticides is linked to increased risk of childhood leukemia.22 Finally, numerous studies from around the world have consistently identified associations between pesticide exposures and risk of lymphomas, brain tumors, and other solid tumors.19,23 The US Surgeon General and the State of California have reported prenatal and postnatal exposures to environmental tobacco smoke to have a suggestive association with childhood leukemia, lymphomas, and brain tumors.24,25 The International Agency for Research on Cancer has classified tobacco smoke as carcinogenic to smokers’ children with sufficient evidence for hepatoblastoma and limited evidence for childhood leukemia.26 Paternal smoking, in particular, before conception has also been linked to an increased risk of childhood acute lymphoblastic leukemia.27

cy for Research on Cancer has classified tobacco smoke as carcinogenic to smokers’ children with sufficient evidence for hepatoblastoma and limited evidence for childhood leukemia.26 Paternal smoking, in particular, before conception has also been linked to an increased risk of childhood acute lymphoblastic leukemia.27 Many of the above studies find odds ratios >2 (including meta-analyses) for overall risk or specific exposure strata. The evidence from case-control studies (and meta-analyses based on them) is strengthened by additional studies finding polychlorinated biphenyls and polybrominated diphenyl ethers, fire retardant chemicals in house dust that are associated with elevated risk of childhood leukemia. These studies provide an objective measure of chemical exposure and eliminate recall bias.15,28 Because of the rarity of childhood cancer, the ability to form prospective studies is limited. Research collaborations such as the Childhood Leukemia International Consortium (CLIC) and the International Childhood Cancer Cohort Consortium (I4C) provide hope for the future, as pooled data and biospecimens from large-scale studies will help identify more robust findings regarding childhood cancer causation.29,30

. Research collaborations such as the Childhood Leukemia International Consortium (CLIC) and the International Childhood Cancer Cohort Consortium (I4C) provide hope for the future, as pooled data and biospecimens from large-scale studies will help identify more robust findings regarding childhood cancer causation.29,30 Greaves31 has proposed that a delay in a child’s exposure to common childhood infections may result in an improperly modulated immune system and a subsequent risk of aberrantly high levels of lymphoblastic cell proliferation following the barrage of infections when the child enters day care or preschool. This delayed infection hypothesis has been supported by subsequent studies and meta-analyses showing that children exposed to common infections early in life by social contact (such as day care attendance), are at reduced risk of acute lymphoblastic leukemia.32,33

infections when the child enters day care or preschool. This delayed infection hypothesis has been supported by subsequent studies and meta-analyses showing that children exposed to common infections early in life by social contact (such as day care attendance), are at reduced risk of acute lymphoblastic leukemia.32,33 Despite the growing insight into potentially modifiable risk factors for childhood cancer, there is little evidence that this knowledge is being translated to clinical practice. Surveys conducted among general pediatricians show that, while these physicians attach considerable importance to the impact of environmental exposures on children’s health, they spend little time discussing this information with families.34,35 A literature review found consistent gaps in knowledge of environmental hazards and confidence in addressing these issues among pediatric health care providers in a variety of geographic regions.35 Despite Institute of Medicine recommendations in 1988 that called for the integration of environmental health concepts into all levels of nursing and medical education, relatively little progress has been made.36,37 Few medical schools, pediatric residencies, or nurse practitioner training programs devote substantial training time to environmental contributors to disease.38–40 Nevertheless, initiatives have begun in both the nursing and medical communities to bridge this gap.41,42

on, relatively little progress has been made.36,37 Few medical schools, pediatric residencies, or nurse practitioner training programs devote substantial training time to environmental contributors to disease.38–40 Nevertheless, initiatives have begun in both the nursing and medical communities to bridge this gap.41,42 We conducted a survey to learn whether practicing pediatric hematologists and oncologists encounter barriers to integrating environmental research findings into practice similar to those reported by general pediatricians. Specifically, we sought to assess their level of knowledge and attitudes related to potential environmental contributions to childhood cancers and their history-taking practices.

cologists encounter barriers to integrating environmental research findings into practice similar to those reported by general pediatricians. Specifically, we sought to assess their level of knowledge and attitudes related to potential environmental contributions to childhood cancers and their history-taking practices. MATERIALS AND METHODS An online survey was sent from June to October 2012 to 20 clinical sites: 18 pediatric cancer treatment centers in California, plus the Dana-Farber Cancer Institute (Boston, MA) and the University of Utah Huntsman Cancer Institute (Salt Lake City, UT). Physicians who received the survey were identified through their participation as a clinical collaborator with the California Childhood Leukemia Study or their affiliations with one of the above institutions.27 A single physician at each site was asked to distribute the online survey to all attending physicians, fellows, and nurse practitioners who were members of hematology/oncology or stem cell transplant services. All responses were collected anonymously with no respondent identifying information. Responses were collected using SurveyMonkey Inc. of Palo Alto, CA.43 A reminder email was sent to participants who had not completed the survey after approximately 4 weeks.

mbers of hematology/oncology or stem cell transplant services. All responses were collected anonymously with no respondent identifying information. Responses were collected using SurveyMonkey Inc. of Palo Alto, CA.43 A reminder email was sent to participants who had not completed the survey after approximately 4 weeks. The survey consisted of 11 questions pertaining to demographic information, perceptions regarding the causes of childhood cancer, history-taking behaviors and training, patient experiences, and home practices; with multiple and open-ended responses allowed (see Supplemental Appendix I, Supplemental Digital Content 1, http://links.lww.com/JPHO/A102 for the full-text survey). A Likert scale of 1 to 5 (“strongly disagree” to “strongly agree”) was used to assess attitudes, and 2 questions allowed for open-ended responses. A pilot survey was conducted at Lucile Packard Children’s Hospital at Stanford University (n=22), and slight modifications were made to the instrument based on these results. However, these pilot responses were not included in the final analyses. Descriptive analyses (frequencies, percentages, SDs) were performed overall and by respondents’ characteristics (type of position and years in practice), using SAS 9.2 (SAS Institute, Cary, NC). The survey was approved by the Institutional Review Boards at Stanford University, Dana-Farber Cancer Institute, and the University of Utah.

lyses (frequencies, percentages, SDs) were performed overall and by respondents’ characteristics (type of position and years in practice), using SAS 9.2 (SAS Institute, Cary, NC). The survey was approved by the Institutional Review Boards at Stanford University, Dana-Farber Cancer Institute, and the University of Utah. RESULTS The survey was distributed to 427 physicians and nurse practitioners, with 191 responding (overall response rate of 45%). The majority of respondents were attending physicians, most of whom had over 10 years of practice in pediatric hematology/oncology (Table 1). TABLE 1 Characteristics of Respondents Participants were asked about their beliefs regarding the likely causes of leukemia in children. Their responses included genetics (92%), health status (eg, stress, prenatal care, and nutrition; 25%), environmental exposures (eg, chemicals, contamination, second-hand smoke, infections, and radiation; 78%), and none of the above (7%) (Table 2). Open-ended responses varied, but commonly included: “all of the above,” previous exposures to chemotherapy agents, infertility treatments, and simply “bad luck.” A majority of respondents (61%) agreed that environmental exposures were important contributors to childhood cancer (mean Likert score, 3.65). TABLE 2 Responses to Selected Survey Questions (n=191)

Participants were asked about their beliefs regarding the likely causes of leukemia in children. Their responses included genetics (92%), health status (eg, stress, prenatal care, and nutrition; 25%), environmental exposures (eg, chemicals, contamination, second-hand smoke, infections, and radiation; 78%), and none of the above (7%) (Table 2). Open-ended responses varied, but commonly included: “all of the above,” previous exposures to chemotherapy agents, infertility treatments, and simply “bad luck.” A majority of respondents (61%) agreed that environmental exposures were important contributors to childhood cancer (mean Likert score, 3.65). TABLE 2 Responses to Selected Survey Questions (n=191) When asked about their routine history-taking practices, the participants most frequently obtained information about parental occupations, household tobacco smoke, and radiation exposures (Fig. 1). Nearly 25% reported not asking about any of the factors mentioned in the survey. Only 7% had reported ever receiving training in taking an environmental history. FIGURE 1 As part of your patient’s history, do you collect information on potential exposures to any of the following external factors? (check all that apply).

When asked about their routine history-taking practices, the participants most frequently obtained information about parental occupations, household tobacco smoke, and radiation exposures (Fig. 1). Nearly 25% reported not asking about any of the factors mentioned in the survey. Only 7% had reported ever receiving training in taking an environmental history. FIGURE 1 As part of your patient’s history, do you collect information on potential exposures to any of the following external factors? (check all that apply). Although half of the respondents reported rarely suspecting that a case was related to some factor in the patient’s environment, a large majority (88%) either “frequently” or “occasionally” received questions from parents or family members about potential workplace or environmental exposures contributing to their child’s disease. Forty-four percent of respondents felt either “somewhat uncomfortable” or “not at all comfortable” discussing the disease implications of environmental exposures with patients and their families (Table 2). An overwhelming majority (92%) stated they would find it helpful to have more information regarding the association between childhood cancers and environmental exposures to answer questions from parents, patients, or family members.

ase implications of environmental exposures with patients and their families (Table 2). An overwhelming majority (92%) stated they would find it helpful to have more information regarding the association between childhood cancers and environmental exposures to answer questions from parents, patients, or family members. Respondents with 0 to 5 years in oncology practice were more likely to focus on genetics as a likely cause of childhood leukemia than practitioners with 5 to 10 and 10+ years of experience (98%, 85%, and 91%, respectively; P=0.04). Clinicians with 10+ years in practice were more likely to ask about exposure to pesticides, solvents, and paternal exposures to specific environmental hazards (P=0.01) (Table 3). Responses also varied across types of position (Table 4). Eighty-five percent of nurse practitioners agreed or strongly agreed that environmental exposures were important contributors to cancer induction, with only 1 nurse practitioner disagreeing with this statement (mean Likert score, 4.0). By contrast, only 58% of fellows and 55% of attending physicians agreed or strongly agreed (P=0.03). However, nurse practitioners were also more likely to be uncomfortable with discussing potential sources of exposure in relation to disease with patients and family, compared with other positions (P<0.001). Attending physicians were significantly more likely to ask about pesticide use, solvents, and parental occupation than other provider types while taking a patient’s history (P=0.01, 0.01, and 0.001, respectively) (Table 4). Close to 25% of all respondents did not ask patients or their families about any of the queried factors associated with childhood cancers.

re likely to ask about pesticide use, solvents, and parental occupation than other provider types while taking a patient’s history (P=0.01, 0.01, and 0.001, respectively) (Table 4). Close to 25% of all respondents did not ask patients or their families about any of the queried factors associated with childhood cancers. TABLE 3 Responses to Selected Survey Questions Stratified by Years in Practice TABLE 4 Responses to Selected Survey Questions Stratified by Position Sixty-seven percent of all respondents engaged in at least 1 practice to protect themselves and their families from potentially hazardous environmental exposures (eg, pesticides, cleaning products, organic foods, plastics, etc.). Fellows were significantly less likely to report participating in behaviors in their own home that might avoid exposures to chemicals associated with health risks than were nurse practitioners or attending physicians (P<0.001) (Table 4). Those who attempted to avoid exposures at home were more likely to agree or strongly agree that environmental exposures are important contributors to the development of childhood leukemia (69%; SD, 8%; P=0.001). Among those providers who attempted to avoid home exposures and also agreed or strongly agreed that environmental factors are important contributors to childhood cancers, only 53% were somewhat or very comfortable discussing these issues with their patients.

the development of childhood leukemia (69%; SD, 8%; P=0.001). Among those providers who attempted to avoid home exposures and also agreed or strongly agreed that environmental factors are important contributors to childhood cancers, only 53% were somewhat or very comfortable discussing these issues with their patients. DISCUSSION This survey found that a majority of clinicians agreed that environmental exposures were important contributors to childhood cancers, but remained inconsistent in their history taking for these events. Although most practitioners routinely received questions about the relationship between environmental exposures and disease, few were entirely comfortable addressing these issues. Over 90% of respondents believed they would benefit from more information on this topic. Physician-Patient Communications Previous surveys of the general public in the United States indicate widespread beliefs that the environment plays an important role in various health problems, and that parents would like more information from their pediatricians regarding environmental health topics.44,45 The findings of our survey support these previous results. Many respondents (48%) reported being frequently asked about environmental exposures to potential carcinogens by patients or their families. Greater familiarity with the emerging research on environmental contributions to childhood cancer would allow clinicians to be more responsive to these questions.

revious results. Many respondents (48%) reported being frequently asked about environmental exposures to potential carcinogens by patients or their families. Greater familiarity with the emerging research on environmental contributions to childhood cancer would allow clinicians to be more responsive to these questions. Providers with 5 or fewer years of experience were more likely to highlight genetics as a cause of childhood leukemia compared with providers with more experience, perhaps reflecting differences in curriculum and training that highlight more recent genetic studies. This group was also less likely to incorporate environmental health questions into their routine patient histories. Generational differences might account for this difference, as the more experienced clinicians were educated and trained during the height of the environmental movement, which could lead to greater awareness of environmental impacts on health.46,47

te environmental health questions into their routine patient histories. Generational differences might account for this difference, as the more experienced clinicians were educated and trained during the height of the environmental movement, which could lead to greater awareness of environmental impacts on health.46,47 Barriers to Integration Into Practice Our survey results indicate that clinical hematologists and oncologists engage in a variety of environmental history-taking practices. Many participants reported frequently asking about parental occupation, but not about any specific environmental hazards associated with that occupation. Anecdotal evidence and survey results suggest that clinicians have reservations about the appropriateness of asking the patients’ families questions related to environmental exposures and other carcinogens. One factor contributing to this perception may be the notion that clinicians do not have a major role in assessing etiology, and that such questions could raise the parents’ anxiety and guilt with little benefit to treatment outcomes. Although anticipation of negative parental reaction has been similarly cited by pediatricians as a common barrier to intervening with parents who smoke, the vast majority of smoking parents show strong support for addressing smoking at office visits.48,49

ts’ anxiety and guilt with little benefit to treatment outcomes. Although anticipation of negative parental reaction has been similarly cited by pediatricians as a common barrier to intervening with parents who smoke, the vast majority of smoking parents show strong support for addressing smoking at office visits.48,49 Twenty-five percent of clinicians did not ask about any of the environmental factors mentioned in our survey, whereas 75% routinely asked at least some questions related to assessing environmental exposures. For both groups, a better grounding in the literature could ensure that responses to questions from patients and families regarding environmental hazards are addressed promptly and accurately. This assumption is supported by our result that providers uniformly believed that more information on environmental health research relevant to childhood cancer would be helpful.

re could ensure that responses to questions from patients and families regarding environmental hazards are addressed promptly and accurately. This assumption is supported by our result that providers uniformly believed that more information on environmental health research relevant to childhood cancer would be helpful. Informing Research Agendas Historically, alert clinicians have recognized environmental exposure trends in their patient populations, and brought them to the attention of public health authorities. This was seen in cases of mesothelioma and lung cancer in asbestos workers, and vaginal adenocarcinoma in women born to diethylstilbestrol-exposed mothers, among others.50–52 Investigations of these cancer clusters led to the identification of previously unrecognized human carcinogens. By bringing an awareness of potential environmental etiologies to their oncology practice, clinicians can play an important role in raising issues in the research community and assisting investigators and public health officials in deciding potential areas of study.

ication of previously unrecognized human carcinogens. By bringing an awareness of potential environmental etiologies to their oncology practice, clinicians can play an important role in raising issues in the research community and assisting investigators and public health officials in deciding potential areas of study. Further illustration of this point can be seen in a recent case report, where the authors identified 4 cases of congenital fibrosarcoma linked to prenatal exposure to petroleum derivatives.52 Through the use of a routine pediatric environmental health history questionnaire, the authors were able to compare case histories with toxicological databases and identify exposures in each case to compounds associated with the development of fibrosarcoma in animals.52 Although this case series does not establish a causal association, it may form the hypothesis for a full-scale study.

questionnaire, the authors were able to compare case histories with toxicological databases and identify exposures in each case to compounds associated with the development of fibrosarcoma in animals.52 Although this case series does not establish a causal association, it may form the hypothesis for a full-scale study. Health professionals use peer-reviewed journals, consultations with peers, and conference attendance as their primary sources of reliable information for clinical decision making.53,54 We reviewed abstracts from the 2011 and 2012 American Society of Pediatric Hematology/Oncology (ASPHO) meetings to select those that examined causation or environmental risk factors (characterized broadly to include factors like diet and infectious agents). Of 569 abstracts, 8% dealt with questions of causation, and only 1% mentioned any environmental risk factors. During the same 2-year period, a PubMed search showed that 48 papers were published specifically on the topic of childhood cancers and environmental risk factors. However, these papers appeared primarily in nonclinical journals, such as Environmental Health Perspectives, American Journal of Epidemiology, and Cancer Causes & Control. It is likely that nonclinical journals do not have a widespread readership among busy clinicians.55

hildhood cancers and environmental risk factors. However, these papers appeared primarily in nonclinical journals, such as Environmental Health Perspectives, American Journal of Epidemiology, and Cancer Causes & Control. It is likely that nonclinical journals do not have a widespread readership among busy clinicians.55 We reviewed federal funding to assess the proportion of resources devoted to studying environmental causes of childhood cancers. During 2010 and 2011, the National Institutes of Health awarded 3% to 7% of its total funding for childhood leukemia research to studies evaluating environmental etiologies. (A broad definition of environment was used to include factors such as diet and infection, together with more traditional factors. Only projects that focused on cancer as the primary endpoint were considered. Projects exclusively focusing on adult populations were excluded, as well as those that investigated mechanisms that might be broadly implicated in cancer development.) The majority of funding for this research comes from the National Institute for Environmental Health Sciences (NIEHS). The National Cancer Institute contributed around 1% of its funding for all childhood cancer toward environmental risk research.56,57 Despite an evolving understanding of environmental exposures associated with leukemia, funding for research that might inform activities aimed at prevention of childhood cancer remains limited.

tional Cancer Institute contributed around 1% of its funding for all childhood cancer toward environmental risk research.56,57 Despite an evolving understanding of environmental exposures associated with leukemia, funding for research that might inform activities aimed at prevention of childhood cancer remains limited. Limitations Although this is a relatively small survey, it is the first characterization of pediatric hematology-oncology practitioners’ current perception of the importance of environmental exposures to their patient care responsibilities. Surveys, as a research tool, have disadvantages with respect to the data collected and representativeness of the sample. In our survey design, participants were restricted to specific responses, limiting the potential details collected. Two questions allowed for open-ended responses, which increased the depth of our investigation.

a research tool, have disadvantages with respect to the data collected and representativeness of the sample. In our survey design, participants were restricted to specific responses, limiting the potential details collected. Two questions allowed for open-ended responses, which increased the depth of our investigation. Our survey response rate of 45% may introduce bias in our results, as no information about the nonresponders was collected. For example, if those who had a higher concern for environmental associations were more likely to respond to the survey, these perspectives would have been overestimated in our sample. Email surveys of general practitioners have reported response rates similar to ours,58 however a study found response rates among specialty-fellows to be lower than nonspecialists.59 In general, email surveys have significantly lower response rates than mailed questionnaires.60,61 We aimed to increase the response rate and ensure more representativeness of the sample by sending a reminder email to participants. The survey was distributed to clinicians at selected institutions and does not comprise a representative sample of all practitioners in this field. This may affect the generalizability of our results if clinicians at these institutions differ substantially in their attitudes and practices from their counterparts across the country.

s distributed to clinicians at selected institutions and does not comprise a representative sample of all practitioners in this field. This may affect the generalizability of our results if clinicians at these institutions differ substantially in their attitudes and practices from their counterparts across the country. CONCLUSIONS This survey identifies an opportunity for improved training in and awareness of environmental health research among attending physicians, fellows, and nurse practitioners working in the field of pediatric hematology/oncology. To help bridge this gap, environmental health researchers and epidemiologists should publish their relevant findings in journals that are widely read by pediatric hematologists/oncologists and nurse practitioners.

ending physicians, fellows, and nurse practitioners working in the field of pediatric hematology/oncology. To help bridge this gap, environmental health researchers and epidemiologists should publish their relevant findings in journals that are widely read by pediatric hematologists/oncologists and nurse practitioners. Educational opportunities should also be made available at national meetings, as our survey revealed significant interest in increasing baseline knowledge of environmental factors contributing to cancer. Training in environmental history taking, introduction of basic environmental history questions into the electronic medical record, and implementation of self-administered patient questionnaires during medical visits that address environmental exposures may aid in simplifying the collection of relevant information in the medical records of children with cancer. Moreover, the self-administered questionnaire may address the hesitancy of some clinicians to ask these questions during history taking. Ultimately, having gained a better understanding of current research findings, practitioners will utilize this information in ways appropriate to their particular practice setting.

r. Moreover, the self-administered questionnaire may address the hesitancy of some clinicians to ask these questions during history taking. Ultimately, having gained a better understanding of current research findings, practitioners will utilize this information in ways appropriate to their particular practice setting. Our results highlight the need for better integration of environmental health awareness in pediatric oncology practice and training. The translation of rigorous environmental health research findings to clinicians would potentially improve provider-patient communications, enhance data collection, and promote the role of alert clinicians in identifying sentinel events. With the convergence of research from CLIC and other groups, the body of evidence supporting environmental associations with childhood cancers is growing. Hazards, such as those mentioned in the Introduction section, have already gained a significant body of supporting information. As the environment continues to change and more hazards are identified, there is an ever-present need for pediatric oncologists to keep pace with this emerging research. Supplementary Material SUPPLEMENTARY MATERIAL ACKNOWLEDGMENTS In memory of Pat Buffler who guided this work and made epidemiology important in our lifetime. She strongly motivated others to get involved and never saw a reason to fail. The authors would also like to acknowledge Yang Wang, Pagan Morris, and Sandra Luna-Fineman for their contributions.

NTARY MATERIAL ACKNOWLEDGMENTS In memory of Pat Buffler who guided this work and made epidemiology important in our lifetime. She strongly motivated others to get involved and never saw a reason to fail. The authors would also like to acknowledge Yang Wang, Pagan Morris, and Sandra Luna-Fineman for their contributions. Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.jpho-online.com. Supported by the National Institutes of Health (NIEHS)/EPA award numbers 1P01ES018172-01 (NIEHS)/RD83451101 (USEPA). M. Miller and C. Zachek were also supported for this publication by the cooperative agreement award number 1 U61TS000238-01 from the Agency for Toxic Substances and Disease Registry (ATSDR). Its contents are the responsibility of the authors and do not necessarily represent the official views of the Agency for Toxic Substances and Disease Registry (ATSDR). Neither EPA nor ATSDR endorse the purchase of any commercial products or services mentioned in PEHSU publications. The authors declare no conflict of interest.

Adequate iron availability is essential to human development and overall health. Iron is indispensable for numerous biological functions such as oxygen transport, cellular respiration, and immune response. Conversely, a high iron concentration is harmful to both cells and tissues.1,2 Hereditary hemochromatosis is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. The syndrome is a result of genetically determined failure to stop iron entering the circulatory pool when it is not needed. It is associated with pathogenic mutations of at least 5 hemochromatosis genes: HFE (OMIM gene number 613609, phenotype MIM number 235200), TfR2 (OMIM gene number 604720, phenotype MIM number 604250), HJV (OMIM gene number 60837, phenotype MIM number 602390), HAMP (OMIM gene number 606464, phenotype MIM number 613313), and SLC40A1 (OMIM gene number 604653, phenotype MIM number 606069). In addition, it is likely due to a regulatory defect in iron homeostasis.3,4 HFE-related hemochromatosis (OMIM gene number 613609, phenotype MIM number 235200) is the most frequent form of the disease and the most common autosomal recessive disorder in Northern European populations, with a prevalence of 1:200 to 1:250 for homozygosity and a carrier rate of 1:8 to 1:12.5,6 This autosomal recessive disorder is characterized by enhanced intestinal absorption of iron leading to multiple organ damage such as cirrhosis, hepatoma, diabetes mellitus, arthritis, and cardiomyopathy.

rn European populations, with a prevalence of 1:200 to 1:250 for homozygosity and a carrier rate of 1:8 to 1:12.5,6 This autosomal recessive disorder is characterized by enhanced intestinal absorption of iron leading to multiple organ damage such as cirrhosis, hepatoma, diabetes mellitus, arthritis, and cardiomyopathy. The symptomatic phenotype preceded by fatigue, arthropathy and impotence, occurs in homozygotic males predominantly between the fourth and sixth decade of life, depending on gene penetrance and multiple concomitant factors.7 Taking into consideration the widespread prevalence of HFE heterozygosity in populations of Northern European descent, H63D heterozygosity is more prevalent (23.6% to 31.1%) than C282Y heterozygosity (8.6% to 11.9%).8 Interestingly, adult heterozygotic HFE genotypes are associated with the progression of some diseases and may present increased serum iron and transferrin saturation.9–11 However, the intensity of iron storage in HFE carriers in the developmental age is still unknown. The objective of this study was to consider iron metabolism in relation to HFE status in 100 Polish children, living in Poland. Despite the numerous adult studies that have been conducted, this population has not been studied before.12

The symptomatic phenotype preceded by fatigue, arthropathy and impotence, occurs in homozygotic males predominantly between the fourth and sixth decade of life, depending on gene penetrance and multiple concomitant factors.7 Taking into consideration the widespread prevalence of HFE heterozygosity in populations of Northern European descent, H63D heterozygosity is more prevalent (23.6% to 31.1%) than C282Y heterozygosity (8.6% to 11.9%).8 Interestingly, adult heterozygotic HFE genotypes are associated with the progression of some diseases and may present increased serum iron and transferrin saturation.9–11 However, the intensity of iron storage in HFE carriers in the developmental age is still unknown. The objective of this study was to consider iron metabolism in relation to HFE status in 100 Polish children, living in Poland. Despite the numerous adult studies that have been conducted, this population has not been studied before.12 MATERIALS AND METHODS One hundred unrelated, that is, nonkindred, children of Polish origin aged from 4 to 18 years (58 boys and 42 girls, average age 11.4±4.19 y), were recruited for the study. There were no chronic disorders, that is, hemochromatosis or hemoglobinopathies in parental families. Parents denied following a vegetarian diet, iron pills administration, and meat consumption was adequate for the patients age. To exclude acute and chronic illnesses, all patients underwent physical examination, laboratory assays which included a full blood count with reticulocytosis and microscopic evaluation, protein C concentration, aspartate and alanine transaminases activity, bilirubin, creatinine levels, hepatitis B virus surface antigen and antibody against hepatitis C virus antibodies. Full blood count with reticulocytosis was performed using SYSMEX XE 2100, whereas, aspartate transaminase, alanine transaminase, bilirubin, creatinine levels, hepatitis B virus surface antigen, antibody against hepatitis C virus antibodies, ferritin, transferrin, and protein C concentration were performed using ARCHITECT CI 8200 (ABBOTT). All reagents used were supplied by the manufacturer.

whereas, aspartate transaminase, alanine transaminase, bilirubin, creatinine levels, hepatitis B virus surface antigen, antibody against hepatitis C virus antibodies, ferritin, transferrin, and protein C concentration were performed using ARCHITECT CI 8200 (ABBOTT). All reagents used were supplied by the manufacturer. Iron metabolism was also assessed by measuring iron concentration, ferritin, and transferrin saturation (SYSMEX XE 2100, Architect ci 8200, and Test 1 SDL). Abdominal ultrasonography was also performed and no abnormalities were revealed. Patients underwent genetic testing for HFE mutations using real-time polymerase chain reaction and melting curve analysis methods with the HFE H63D S65C C282Y (TibMolbiol) LightMix in-vitro diagnostics kit and LightCycler 2.0 Instrument (ROCHE). Two fragments of the HFE gene were amplified simultaneously: 163 bp fragment containing 2 polymorphisms: c.187C>G (63 H/D), c.193A>T (65 S/C), and 284 bp fragment containing the c.845G>A (282 C/Y) polymorphism. The genotype results were detected in 2 different optical canals based on melting temperatures. The mutant type 63D allele displayed higher temperature (65°C) than wild-type H63 and S65 allele (57°C) and the mutant 65C allele (52°C). The mutant 282Y allele displayed higher temperature (62°C) than the wild-type C282 allele (56°C). The patients’ genotype profiles were compared with the standards supplied by the manufacturer. The study was officially approved by the Independent Bioethics Commission for Research in Gdansk NKBBN/409/2013 in accord with the Helsinki Declaration.

Iron metabolism was also assessed by measuring iron concentration, ferritin, and transferrin saturation (SYSMEX XE 2100, Architect ci 8200, and Test 1 SDL). Abdominal ultrasonography was also performed and no abnormalities were revealed. Patients underwent genetic testing for HFE mutations using real-time polymerase chain reaction and melting curve analysis methods with the HFE H63D S65C C282Y (TibMolbiol) LightMix in-vitro diagnostics kit and LightCycler 2.0 Instrument (ROCHE). Two fragments of the HFE gene were amplified simultaneously: 163 bp fragment containing 2 polymorphisms: c.187C>G (63 H/D), c.193A>T (65 S/C), and 284 bp fragment containing the c.845G>A (282 C/Y) polymorphism. The genotype results were detected in 2 different optical canals based on melting temperatures. The mutant type 63D allele displayed higher temperature (65°C) than wild-type H63 and S65 allele (57°C) and the mutant 65C allele (52°C). The mutant 282Y allele displayed higher temperature (62°C) than the wild-type C282 allele (56°C). The patients’ genotype profiles were compared with the standards supplied by the manufacturer. The study was officially approved by the Independent Bioethics Commission for Research in Gdansk NKBBN/409/2013 in accord with the Helsinki Declaration. Statistical Analyses Statistical analyses were performed using Statistica 10.0 for Windows (StatSoft 2011). Data are presented as mean±SE and were assessed using Mann-Whitney or nonparametric Kruskal-Wallis unpaired test where appropriate. For the analyses variables were assessed for normality with natural log transformations performed on the serum Fe, ferritin, and Ts variables. Significance for the Mann-Whitney and post hoc tests was set at 0.05.

an±SE and were assessed using Mann-Whitney or nonparametric Kruskal-Wallis unpaired test where appropriate. For the analyses variables were assessed for normality with natural log transformations performed on the serum Fe, ferritin, and Ts variables. Significance for the Mann-Whitney and post hoc tests was set at 0.05. RESULTS Molecular testing of 100 autochthonous, healthy Polish children, aged 11.4±0.42 years (58 boys and 42 girls) revealed a predominance of the wild-type HFE gene as 60 children (60%), presented with wild-type HFE. 25 children (25%), presented with heterozygotic H63D mutation, 6 children, presented with heterozygotic C282Y mutation and 6 children, presented with heterozygotic S65S mutation. Two boys were compound heterozygotes (C282Y/S65C, C282Y/H63D), whereas only 1 girl was a H63D homozygote (data presented in Table 1). In the entire group of tested children, laboratory assays performed excluded chronic diseases, abdominal ultrasound imaging visualized normal internal organs, and the mean concentration of iron, ferritin, and transferrin saturation were within normal range (mean values: iron 98.3±3.23 μg/dL, ferritin 33.2±1.98 ng/mL, transferrin saturation Ts, 27.5%±1.00%). The wild-type HFE gene group presented with a mean iron concentration of 83.4±2.75 μg/dL, ferritin 28.5±1.73 ng/mL, and transferrin saturation of 22.2%±0.68%. The groups which had other types of mutations (carriers of C282Y, H63D, S65C, compound heterozygotes, and H63 homozygote) had statistically higher mean iron concentration (120.8±5.24 μg/dL), mean ferritin levels (40.3±4.00 ng/mL), and mean transferrin saturation (35.5%±1.58%) than the wild-type group, data presented in Table 1. The group carrying the H63D mutation presented with a mean iron concentration of 128.8±6.88 μg/dL, ferritin levels of 44.8±5.87 ng/mL, and transferrin saturation of 38.1%±1.97%. Transferrin saturation was statistically higher in H63D carriers when compared with those of the wild-type group. All the values were statistically higher in the H63D mutation carriers when compared with those of the wild-type group. Moreover, mean transferrin saturation was statistically nonsignificant higher in the C282Y mutation carriers (Ts, 32.8%±3.20%) compared with that of the wild-type group (Ts, 22.2%±0.85%).

-type group. All the values were statistically higher in the H63D mutation carriers when compared with those of the wild-type group. Moreover, mean transferrin saturation was statistically nonsignificant higher in the C282Y mutation carriers (Ts, 32.8%±3.20%) compared with that of the wild-type group (Ts, 22.2%±0.85%). Mean transferrin saturation of H63D mutation carriers (Ts, 38.1%±1.97%) was higher than that of the S65C mutation carriers (Ts, 26.3%±2.16%), whereas mean transferrin saturation of S65C mutation carriers (Ts, 26.3%±2.16%) was higher than that of the wild-type (Ts, 22.2%±1.16%). The mean transferrin saturation of the H63D and C282Y mutation carriers was higher than the normal range in children. The entire group of children (100 subjects) was also divided into four sex groups: wild-type males (39 subjects), wild-type females (21 subjects), male carriers of HFE mutation (19 subjects), and female carriers of HFE mutation (21 subjects) to evaluate presumptive iron metabolism variations (data presented in Table 1). There were no statistical differences in age, weight, height, and body mass index between sex groups. There were 11 girls who reported regular menstruation in the wild-type female group and 12 in the HFE group. Mean iron concentration, ferritin concentration, and transferrin saturation were statistically higher in the group of male carriers of HFE mutation (iron, 134.8±7.96 μg/dL; ferritin, 49.2±6.96 ng/mL; Ts, 40.7%±2.26%) than in the group of female carriers (iron, 108.2±5.81 μg/dL; ferritin, 32.3±3.65 ng/mL; Ts, 30.9%±1.69%), wild-type male group (iron, 83.4±3.61 μg/dL; ferritin, 30.8±2.37 ng/mL; Ts, 22.0%±0.85%), and wild-type female group (iron, 83.2±4.24 μg/dL; ferritin, 24.2±1.96 ng/mL; Ts, 22.5%±1.16%). Mean iron concentration and transferrin saturation of females carrying the HFE mutation were statistically higher than in wild-type male and wild-type female groups. The level of ferritin was statistically higher in male carriers of HFE mutation (49.2±6.95 ng/mL) than in female carriers (32.2±3.65 ng/mL), wild-type male group (30.8±2.37 ng/mL), and wild-type female group (24.2±1.96 ng/mL). The entire group of children (100 subjects) was also divided into sex subgroups according to different HFE status.

was statistically higher in male carriers of HFE mutation (49.2±6.95 ng/mL) than in female carriers (32.2±3.65 ng/mL), wild-type male group (30.8±2.37 ng/mL), and wild-type female group (24.2±1.96 ng/mL). The entire group of children (100 subjects) was also divided into sex subgroups according to different HFE status. There were 11 groups: wild-type males (39 subjects), wild-type females (21 subjects), male carriers of H63D mutation (12 subjects), female carriers of H63D mutation (13 subjects), male carriers of C282Y mutation (2 subjects), female carriers of C282Y mutation (4 subjects), male carriers of S65C mutation (3 subjects), female carriers of S65C mutation (3 subjects), a single male in the compound heterozygote H63D/C282Y group and S65C/282Y group, and a single female in the H63D homozygote group. Males carrying H63D mutation had statistically higher iron concentration (144.8±10.7 μg/dL) than both wild-type males (83.4±3.62 μg/dL) and females (83.2±4.24 μg/dL). H63D carrier males (55.4±10.3 ng/mL) had statistically nonsignificant higher ferritin concentration than wild-type females (24.2±1.96 ng/mL). Transferrin saturation was statistically higher in both sex groups of H63D carriers (males 44.3%±2.60%; females, 32.5%±1.92%) than in wild-type probands (males, 22.0%±0.85%; females, 22.5%±1.16%), all data are presented in Table 1. TABLE 1 Iron Status Parameters in Control and Various HFE Mutation Group

There were 11 groups: wild-type males (39 subjects), wild-type females (21 subjects), male carriers of H63D mutation (12 subjects), female carriers of H63D mutation (13 subjects), male carriers of C282Y mutation (2 subjects), female carriers of C282Y mutation (4 subjects), male carriers of S65C mutation (3 subjects), female carriers of S65C mutation (3 subjects), a single male in the compound heterozygote H63D/C282Y group and S65C/282Y group, and a single female in the H63D homozygote group. Males carrying H63D mutation had statistically higher iron concentration (144.8±10.7 μg/dL) than both wild-type males (83.4±3.62 μg/dL) and females (83.2±4.24 μg/dL). H63D carrier males (55.4±10.3 ng/mL) had statistically nonsignificant higher ferritin concentration than wild-type females (24.2±1.96 ng/mL). Transferrin saturation was statistically higher in both sex groups of H63D carriers (males 44.3%±2.60%; females, 32.5%±1.92%) than in wild-type probands (males, 22.0%±0.85%; females, 22.5%±1.16%), all data are presented in Table 1. TABLE 1 Iron Status Parameters in Control and Various HFE Mutation Group DISCUSSION In the presented study we attempt to analyze iron metabolism in 100 unrelated, healthy children of Polish origin in relation to their HFE status. The frequency of H63D carriage in our study population was found to be 25%, whereas heterozygosity of C282Y and S65C gene was each 6%. A single patient was found to be a H63D homozygote, whereas 2 boys presented with compound heterozygosity. The prevalence of HFE-hemochromatosis alleles was compatible with other European population reports.5,6,8 Despite the fact that iron status is commonly evaluated in pediatric primary medical care, only deficiency usually elicits diagnostic procedures. Pathologic iron loading in primary hemochromatosis is a prolonged and unpredictable process and without proper treatment may result in multiorgan dysfunction.1–4 Thus early diagnosis, monitoring, and treatment are essential.

uated in pediatric primary medical care, only deficiency usually elicits diagnostic procedures. Pathologic iron loading in primary hemochromatosis is a prolonged and unpredictable process and without proper treatment may result in multiorgan dysfunction.1–4 Thus early diagnosis, monitoring, and treatment are essential. The statistical analysis revealed that mean concentration of iron, ferritin levels, and transferrin saturation were statistically higher in the group of various HFE-associated mutations compared with the wild-type group. A statistical difference in iron concentration, level of ferritin, and transferrin saturation was also noted between H63D carrier groups and wild-type groups. Furthermore, mean transferrin saturation of C282Y and S65C carrier groups was higher than in the wild-type group. It is important to note that in all the groups’ mean iron concentration and ferritin levels were within the normal range, whereas transferrin saturation in the various HFE mutation groups, (H63D and C282Y carriers) was elevated. Our data confirms the high sensitivity and diagnostic value of transferrin in detecting iron storage diseases even in childhood.7 Remarkably, the study showed statistically higher iron concentration and transferrin saturation in boys and girls who are carriers of HFE mutation than in wild-type subjects. In addition, boys carrying the HFE mutation presented with higher iron concentration, transferrin saturation, and ferritin level than girls carrying the same mutation. Notably, the presented data emphasizes the varying influence of HFE mutation with regard to sex even in the developmental age.

an in wild-type subjects. In addition, boys carrying the HFE mutation presented with higher iron concentration, transferrin saturation, and ferritin level than girls carrying the same mutation. Notably, the presented data emphasizes the varying influence of HFE mutation with regard to sex even in the developmental age. The impact of the HFE gene mutation on biochemical features of homozygotic and heterozygotic adults seems to be understandable,2,9–11,13 while its impact in childhood still needs to be accurately observed. In contrast, the clinical course of hemochromatosis is unpredictable since reduced gene penetration, possible concomitant environmental and epigenetic factors also play a role in disease development.14,15 All the more, knowing the HFE status of a child seems to be important as it will allow for close observation of affected children. Notable, there are reports which demonstrate that higher blood ferritin but still within the reference range, is associated with increased risk of some morbidities in adults. Zacharski et al16 revealed that 75% of newly diagnosed cancer cases have been diagnosed among patients who presented with mean ferritin levels of >57 ng/mL during follow-up. Similarly, Mainous et al17 reported that higher transferrin saturation correlated with an increased risk of overall all-cause mortality. In addition, a study performed on a young male demonstrated that blood ferritin higher than 150 ng/mL correlates negatively with cardiovascular fitness.18 Altogether, these data indicate that monitoring body iron stores can be recommended as part of a prevention strategy against diseases especially in the HFE mutation group.

ition, a study performed on a young male demonstrated that blood ferritin higher than 150 ng/mL correlates negatively with cardiovascular fitness.18 Altogether, these data indicate that monitoring body iron stores can be recommended as part of a prevention strategy against diseases especially in the HFE mutation group. Reports considering HFE hemochromatosis in children are uncommon. Researchers are particularly concerned about the validity of screening children or genotyping siblings and spouses. To date, multiple screening programmes: neonatal, descendant, and population have been suggested,11,19–23 but rare phenotypical manifestation, patient stigmatization and economical aspects have derailed progress. On the basis of the data that iron storage is a slow and prolonged process2 and children carrying HFE mutation do not present increased ferritin levels,24 Delatycki et al25 stated that C282Y homozygotes present with iron overload very rarely during the first 2 decades, and the only abnormality they present with is biochemical. However, the risk of iron accumulation cannot be excluded in instances of coexisting circumstances in childhood. Researchers6,26,27 described an association between HFE mutation variants, iron storage and concomitant disease progression. There are also preliminary reports considering the impact of HFE variants and susceptibility to acute lymphoblastic leukemia in childhood.6,28,29 Taking into consideration presented data and cited authors the impact of HFE mutation in childhood still needs further investigation.

age and concomitant disease progression. There are also preliminary reports considering the impact of HFE variants and susceptibility to acute lymphoblastic leukemia in childhood.6,28,29 Taking into consideration presented data and cited authors the impact of HFE mutation in childhood still needs further investigation. CONCLUSIONS In our study, we demonstrated variations in iron metabolism in relation to various HFE-associated mutations in children of Polish origin. The individual course of hemochromatosis seems to be unique which makes diagnostic procedures, monitoring and proper treatment unquestionable. This preliminary investigation demonstrates allelic impact on potential disease progression from childhood. Supported by grant DS RIK/2016 funded by Gdansk University of Physical Education and Sport, Faculty of Rehabilitation and Kinesiology. B.K.-H. designed the study and wrote the paper. M.L. performed the research. J.A. designed and performed the research. W.Z., M.M., E.M., and J.J.K. performed the research. E.A.-D. contributed essentials reagents or tools. The authors declare no conflict of interest.

Congenital dyserythropoietic anemia (CDA) type IV has been linked to a heterozygous, gain-of-function mutation of the erythroid transcription factor KLF1; E325K.1–5 This report describes a child with CDA type IV due to KLF1 E325K who presented with fetal anemia and nonimmune hydrops fetalis, postnatal transfusion dependence, and only partial response to splenectomy. She also exhibited complete 46 XY sex reversal. Detailed laboratory and genetic analyses provided insights into our patient’s severe hemolytic anemia phenotype.

IV due to KLF1 E325K who presented with fetal anemia and nonimmune hydrops fetalis, postnatal transfusion dependence, and only partial response to splenectomy. She also exhibited complete 46 XY sex reversal. Detailed laboratory and genetic analyses provided insights into our patient’s severe hemolytic anemia phenotype. CASE REPORT The patient, now 10 years of age, was the second child born to a nonconsanguineous Ashkenazi Jewish couple. Genetic amniocentesis revealed a fetal karyotype of 46 XY. Pregnancy surveillance identified hydrops fetalis and fetal anemia, hemoglobin 4 g/dL, at 25-week gestation. Hemolytic anemia from blood group incompatibility was excluded. The fetus received 2 in utero transfusions before an elective Cesarean section at 33-week gestation. Birth weight was 1360 g. The infant was hydropic with splenomegaly; there were no skeletal deformities. Despite the karyotype results, the neonate was phenotypically female with a normal vagina, uterus, and what appeared to be ovaries were seen on ultrasound. There was severe anemia and pRBC transfusions were administered. She required transfusion support approximately once every 4 to 6 weeks for the first several years of life. No genetic cause for the hemolytic anemia nor for the sex reversal was identified despite extensive laboratory testing. Complete androgen insensitivity syndrome was excluded by clinical testing. Splenectomy was performed at 4 years of age because of the concerns for iron accumulation and only a modest response to deferasiriox. Transfusion requirement abated, although moderate to severe anemia requiring occasional transfusion persists.

. Complete androgen insensitivity syndrome was excluded by clinical testing. Splenectomy was performed at 4 years of age because of the concerns for iron accumulation and only a modest response to deferasiriox. Transfusion requirement abated, although moderate to severe anemia requiring occasional transfusion persists. Presplenectomy, postsplenectomy, and recent blood counts are shown in Table 1. The peripheral smear presplenectomy showed spherocytes, spiculated erythrocytes, and nucleated red blood cells, with some clover leaf nuclei. After splenectomy, there was marked normoblastosis, up to 10-fold in excess of the total leukocyte count, with prominent large, round macrocytes (Fig. 1A). The fetal hemoglobin level was 34.6%. Bone marrow morphology showed a predominantly erythroid marrow (83% erythroid precursors, 3% lymphocytes, 15% myeloid cells). Of the erythroid precursors, the majority were orthochromatic with 3% binucleate forms and 3% cells with clover leaf nuclei. There were some tight clusters of orthochromatic erythroblasts, similar to that described in early fetal erythropoiesis (Fig. 1B). Delayed enucleation of orthochromatic normoblasts was suggested by the asynchrony of the nuclear versus cytoplasmic maturation, with centrally spaced nuclei. Peripheral blood smears from both parents were normal. TABLE 1 Hematologic Findings Before and After Splenectomy

Presplenectomy, postsplenectomy, and recent blood counts are shown in Table 1. The peripheral smear presplenectomy showed spherocytes, spiculated erythrocytes, and nucleated red blood cells, with some clover leaf nuclei. After splenectomy, there was marked normoblastosis, up to 10-fold in excess of the total leukocyte count, with prominent large, round macrocytes (Fig. 1A). The fetal hemoglobin level was 34.6%. Bone marrow morphology showed a predominantly erythroid marrow (83% erythroid precursors, 3% lymphocytes, 15% myeloid cells). Of the erythroid precursors, the majority were orthochromatic with 3% binucleate forms and 3% cells with clover leaf nuclei. There were some tight clusters of orthochromatic erythroblasts, similar to that described in early fetal erythropoiesis (Fig. 1B). Delayed enucleation of orthochromatic normoblasts was suggested by the asynchrony of the nuclear versus cytoplasmic maturation, with centrally spaced nuclei. Peripheral blood smears from both parents were normal. TABLE 1 Hematologic Findings Before and After Splenectomy FIGURE 1 Laboratory studies. A, Peripheral blood smears. Peripheral blood smears from the proband after splenectomy shows spherocytes, unusually large round macrocytes, spherocytes, spiculated red blood cell and nucleated red blood cells; magnification ×1000. B, Bone marrow smear. Tight clusters of orthochromic erythroblasts are seen in bone marrow smears from the proband; a clover leaf form noted at the upper edge and binucleate form middle right. C, Osmotic gradient ektacytometry. Red cell deformability studies by osmotic gradient ektacytometry shows reduced deformability in the proband (low DI max); the rightward shift suggests presence of large erythrocytes with low MCHC. Ektacytometry of erythrocytes from the father and mother shows a pattern consistent with mild spherocytosis. D, Eosin-5′-maleimide-binding (EMA) binding. EMA studies of erythrocytes from the proband and both parents demonstrated decreased fluorescence consistent with a defect in the erythrocyte membrane. Patterns in dominant spherocytosis (HS) are shown for comparison. E, Isoelectric focusing of hemoglobin. Isoelectric focusing shows the presence of embryonic hemoglobin in erythrocytes from the proband. F, Sanger sequencing. Sanger sequencing confirmed heterozygosity for the G to A substitution in the KLF1 gene and wild-type status for the mother and father (not shown).

r comparison. E, Isoelectric focusing of hemoglobin. Isoelectric focusing shows the presence of embryonic hemoglobin in erythrocytes from the proband. F, Sanger sequencing. Sanger sequencing confirmed heterozygosity for the G to A substitution in the KLF1 gene and wild-type status for the mother and father (not shown). Osmotic gradient ektacytometry performed postsplenectomy demonstrated a unique pattern indicating presence of erythrocytes with low mean corpuscular hemoglobin concentration in the proband; erythrocytes from the parents revealed a pattern consistent with mild hereditary spherocytosis (Fig. 1C).6 Eosin-5′-maleimide-binding (EMA) studies of erythrocytes from the proband and both parents demonstrated decreased fluorescence consistent with a defect in the erythrocyte membrane (Fig. 1D). Erythrocyte enzyme analyses, including erythrocyte pyruvate kinase activity, were normal.

ld hereditary spherocytosis (Fig. 1C).6 Eosin-5′-maleimide-binding (EMA) studies of erythrocytes from the proband and both parents demonstrated decreased fluorescence consistent with a defect in the erythrocyte membrane (Fig. 1D). Erythrocyte enzyme analyses, including erythrocyte pyruvate kinase activity, were normal. The severity of anemia, the elevated nucleated red blood cell (nRBC) count, high fetal hemoglobin (HbF) and elevated mean corpuscular volume suggested that the high HbF is not likely due to the known disorders of familial hereditary persistence of high fetal hemoglobin. Rather the findings were similar to the observations described in the first 2 cases of CDA type IV due to KLF1 E325K.1,2 CD44 expression on red cells was used as a screening tool and additional confirmatory tests were performed. CD44 expression (CD71− population) was absent on mature erythrocytes from the proband (Fig. 2B) compared with its expression on mature erythrocytes from the parents and a control. CD44 was also reduced or absent in CD71+ cells from the proband (reticulocytes and nucleated red blood cells [CD45−, DRAQ5+, CD71+ cells) in contrast to its presence on reticulocytes and nRBC from a thalassemia major patient with elevated numbers of circulating nRBC and on cells in a normal control bone marrow (Fig. 2C). CD44 expression was present in patient’s lymphocytes but was decreased compared with control values (not shown). Sanger sequencing of the KLF1 gene, performed as described,2 revealed the proband was heterozygous for the E325K KLF1 variant associated with CDA type IV (NM_006563:c.G973A:p.E325K) (Fig. 1E). Neither parent carried the E325K variant (Fig. 1F; data on father not shown), suggesting this occurred de novo or 1 parent is mosaic.

sequencing of the KLF1 gene, performed as described,2 revealed the proband was heterozygous for the E325K KLF1 variant associated with CDA type IV (NM_006563:c.G973A:p.E325K) (Fig. 1E). Neither parent carried the E325K variant (Fig. 1F; data on father not shown), suggesting this occurred de novo or 1 parent is mosaic. FIGURE 2 A, Fetal hemoglobin staining by flow cytometry—2 distinct population of cells can be seen suggesting a heterocellular pattern of staining for fetal hemoglobin (shown in red). B, CD44 staining of peripheral blood red blood cell (RBC). CD71 was used to mark reticulocytes and immature red cells. Red cells from the proband are deficient in CD44 expression and as well the immature RBC containing reticulocytes and nucleated red blood cell (nRBC). Normal staining in the parents and in a sickle cell anemia patient with high reticulocytes and nRBC are shown for comparison. C, CD44 staining in nRBC was further assessed by using the nuclear stain DRAQ5. CD45 was used to mark white blood cell/myeloid fraction (column A). In a normal control marrow, erythroid precursors (CD45−, DRAQ5+, CD71+—column D) showed the expected positive staining for CD44 (top row) and in a child with β-thalassemia major with high nRBC (bottom row). In contrast the nRBC in the proband (middle row) show markedly decreased staining with CD44.

fraction (column A). In a normal control marrow, erythroid precursors (CD45−, DRAQ5+, CD71+—column D) showed the expected positive staining for CD44 (top row) and in a child with β-thalassemia major with high nRBC (bottom row). In contrast the nRBC in the proband (middle row) show markedly decreased staining with CD44. Identification of the KLF1 E325K mutation prompted detailed hemoglobin analyses based on previous cases. Isoelectric focusing of erythrocyte hemoglobin yielded a pattern identical to that published by Arnaud and colleagues, indicating the presence of embryonic hemoglobins (Fig. 1F). Presence of ζ-globin was confirmed by proteomic analysis of low–molecular-weight bands on the sodium dodecyl sulfate polyacrylamide gel electrophoresis of red cell membranes (not shown). By flow cytometry the distribution of HbF was heterocellular (Fig. 2A).

, indicating the presence of embryonic hemoglobins (Fig. 1F). Presence of ζ-globin was confirmed by proteomic analysis of low–molecular-weight bands on the sodium dodecyl sulfate polyacrylamide gel electrophoresis of red cell membranes (not shown). By flow cytometry the distribution of HbF was heterocellular (Fig. 2A). Additional genetic variants contributing to the patient’s clinical course were sought. With parental consent; patient and parental samples were subjected to whole exome sequencing (WES) to identify other coinherited mutations in red cell genes. Data were processed using the SeattleSeq annotation platform (http://snp.gs.washington.edu/SeattleSeqAnnotation137) to identify the pathologic variants. Erythrocyte membrane protein genes were initially analyzed due to the abnormal erythrocyte EMA binding and ektacytometry results in the proband and her parents. Numerous nonsynonymous coding sequence variants in the α-spectrin gene (SPTA1) were found in the proband (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A231).7

were initially analyzed due to the abnormal erythrocyte EMA binding and ektacytometry results in the proband and her parents. Numerous nonsynonymous coding sequence variants in the α-spectrin gene (SPTA1) were found in the proband (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A231).7 The proband inherited several variant SPTA1 alleles from the father and mother (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/JPHO/A231) including the αLELY allele (NM_003126:c.5572C>G:p.L1858V), which may result in reduced α-spectrin membrane content.8,9 Another variant allele was the αBughill allele (NM_003126:c.2909C>A:p.A970D), inherited from the mother. This allele is associated with recessive hereditary spherocytosis, is associated with a mild hereditary spherocytosis “carrier state,”10 and is frequently inherited with the αLEPRA mutation. The αLEPRA allele was not detected in either parent or the proband. No functional studies of the αBughill allele have been performed to inform its contribution to spectrin function, but αBughill homozygous patients without the αLEPRA mutation exhibit mild spectrin deficiency and the hereditary spherocytosis carrier state.10 No predicted pathogenic mutations were found in the coding regions or intron/exon junctions of the β-spectrin, ankyrin-1, band 3, protein 4.2 or other erythrocyte membrane genes. These studies do not exclude deep intronic or regulatory element mutations or small genic deletions.

ereditary spherocytosis carrier state.10 No predicted pathogenic mutations were found in the coding regions or intron/exon junctions of the β-spectrin, ankyrin-1, band 3, protein 4.2 or other erythrocyte membrane genes. These studies do not exclude deep intronic or regulatory element mutations or small genic deletions. Other red cell disease genes were examined for potential variants contributing to the proband’s phenotype. Hypomorphic variants with significantly elevated CADD scores were noted in SEC23B (NM_001172745:c.1467C>G:p.H489Q), the CDA type II gene, and in YARS2 (NM_001040436:c.572G>T:p.G191V0), a pathogenic variant in a gene linked to a type of sideroblastic anemia. DISCUSSION Phenotypic features and the clinical course of 5 cases of KLF1 E325K-associated CDA type IV have been reported (Table 2). Review reveals 2 distinctive clinical courses, one characterized by a mild course, with childhood anemia or transfusion dependence in infancy that resolves spontaneously, and the other characterized by a more severe course. Severely affected patients present with fetal anemia and hydrops fetalis followed by postnatal transfusion dependence; splenectomy is palliative but not curative. To gain insight into our patient’s severe hematologic phenotype and the sex reversal phenotype, detailed laboratory and genetic analyses were performed. TABLE 2 Clinical and Laboratory Findings in Cases of KLF1 E325K-associated Congenital Dyserythropoietic Anemia Type IV

DISCUSSION Phenotypic features and the clinical course of 5 cases of KLF1 E325K-associated CDA type IV have been reported (Table 2). Review reveals 2 distinctive clinical courses, one characterized by a mild course, with childhood anemia or transfusion dependence in infancy that resolves spontaneously, and the other characterized by a more severe course. Severely affected patients present with fetal anemia and hydrops fetalis followed by postnatal transfusion dependence; splenectomy is palliative but not curative. To gain insight into our patient’s severe hematologic phenotype and the sex reversal phenotype, detailed laboratory and genetic analyses were performed. TABLE 2 Clinical and Laboratory Findings in Cases of KLF1 E325K-associated Congenital Dyserythropoietic Anemia Type IV Abnormal EMA binding and ektacytometry of erythrocytes from the patient and parents identified functional abnormalities of the erythrocyte membrane. Analyses of WES data, while finding numerous membrane protein gene variants, did not provide a specific genetic diagnosis for the mild HS/HS carrier phenotype in either parent. The mother’s mild clinical picture could be attributed to the αBugHill allele as previously described,10,15 or other as yet unidentified variants. It is possible that the numerous membrane protein gene variants, for example in SPTA1, in cis or trans may lead to a cumulative effect on the protein of interest, with variants interacting to produce a clinical phenotype as described in other proteins.16

previously described,10,15 or other as yet unidentified variants. It is possible that the numerous membrane protein gene variants, for example in SPTA1, in cis or trans may lead to a cumulative effect on the protein of interest, with variants interacting to produce a clinical phenotype as described in other proteins.16 Other variant alleles in 2 other red blood cell disease-associated genes, SEC23B and YARS2, that may have contributed to the severity of our patient’s clinical course were also identified. Hypomorphic mutations in SEC23B have been associated with mild phenotypes in CDA type II.17 Similarly, a wide spectrum of phenotypic and genotypic variability have been described in the YARS2 mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome, including isolated anemia presenting in adulthood.18 Taken together, we hypothesize that coinheritance of variants in relevant erythrocyte genes contribute to the clinical course in our patient and other E325K-linked CDA IV patients with severe clinical phenotypes.

y, lactic acidosis, and sideroblastic anemia syndrome, including isolated anemia presenting in adulthood.18 Taken together, we hypothesize that coinheritance of variants in relevant erythrocyte genes contribute to the clinical course in our patient and other E325K-linked CDA IV patients with severe clinical phenotypes. In 2 of the cases with severe phenotypic features, the patients were phenotypic males with urogenital anomalies.2 One patient had micropenis with hypospadias and had growth retardation unresponsive to human growth hormone while our case had sex reversal. In both cases, the etiology of these findings remains unexplained. A child with a KLF1 null phenotype due to 2 different loss of function mutations who presented with anemia and hydrops fetalis and severe postnatal growth retardation was recently described.19 This patient did not exhibit urogenital anomalies.

reversal. In both cases, the etiology of these findings remains unexplained. A child with a KLF1 null phenotype due to 2 different loss of function mutations who presented with anemia and hydrops fetalis and severe postnatal growth retardation was recently described.19 This patient did not exhibit urogenital anomalies. The KLF1 E325K mutation has a strong dominant negative effect, like the mutation in the homologous amino acid, E339D, in the neonatal anemia nan mouse. This mutation not only alters affinity of KLF1 for its cognate binding sites across the genome, it also binds to degenerate KLF1 motifs not normally occupied by KLF1 in a promiscuous manner. Together, these alterations lead to numerous changes in erythroid gene expression, altering expression of direct KLF1 target genes and inducing ectopic expression of genes not typically expressed.20,21 KLF1 mutant erythrocytes may have defects in proteins involved in growth and differentiation, maintenance of membrane integrity, hemoglobins and heme biosynthesis, iron homoeostasis, and regulation of metabolism.22

ession of direct KLF1 target genes and inducing ectopic expression of genes not typically expressed.20,21 KLF1 mutant erythrocytes may have defects in proteins involved in growth and differentiation, maintenance of membrane integrity, hemoglobins and heme biosynthesis, iron homoeostasis, and regulation of metabolism.22 The role of KLF1 in erythropoiesis has recently been further refined.7 In normal erythropoiesis, there is an orderly, progressive reduction in CD44 expression from proerythroblast to the orthochromatic erythroblast along with changes in the distribution of CD44 within the cell.23,24 Similar to reported cases, loss of CD44 appears near total in mature erythrocytes (CD71− fraction, Fig. 2B). Circulating orthochromatic erythroblasts (normoblasts; CD45−, DRAQ5+, CD71+ fraction, Fig. 2C) are predominantly negative for CD44, a finding not noted in the previously published reports. The loss of CD44 in early erythroid precursors could play an important role in KLF1 E325K-mediated disruption of the transition from fetal to adult erythropoiesis in the KLF1 mutant cases.25 In addition, emerging data show that CD44, along with several other genes, for example SDF1/CXCLR4, VCAM/VLA-4, are involved in the hematopoietic stem cell and bone marrow niche interaction.25,26 The presence of trace amounts of embryonic hemoglobin, the unusually large round macrocytes, the enucleation defect (Fig. 1) and the heterocellular pattern of fetal hemoglobin (Fig. 2) taken together suggest the persistence of a clone of fetal erythroid cells.

topoietic stem cell and bone marrow niche interaction.25,26 The presence of trace amounts of embryonic hemoglobin, the unusually large round macrocytes, the enucleation defect (Fig. 1) and the heterocellular pattern of fetal hemoglobin (Fig. 2) taken together suggest the persistence of a clone of fetal erythroid cells. The exact cause of the 46 XY sex reversal in our patient is not yet determined. Our patient did not have any other congenital anomalies, associated with syndromic gonadal dysgenesis cases.27 In the WES studies, an evaluation of exons and exon-intron boundary regions of the following genes linked to male sex reversal syndromes failed to identify a pathogenic mutation—SRY, AMH. AMHR, ATRX1, DAX1, DHH, DMRT1, FOG2, GATA4, HOXB2, HOXB3, MAP3K, NR5A1, SOX9, and WT1 (genes are from OMIM). Clinical management of KLF1 E325K-assoiated CDA IV remains empiric. Severe anemia is treated with transfusion support. An attempt to treat anemia in one case with erythropoietin failed.2 We did not prescribe erythropoietin, but in vitro erythroid cultures from our patient failed to grow, while there was normal growth of granulocyte, macrophage colonies (not shown, studies kindly done by James Palis, Rochester). It is important to monitor for iron overload and treat when indicated.

ythropoietin failed.2 We did not prescribe erythropoietin, but in vitro erythroid cultures from our patient failed to grow, while there was normal growth of granulocyte, macrophage colonies (not shown, studies kindly done by James Palis, Rochester). It is important to monitor for iron overload and treat when indicated. Poor somatic growth was described in 1 patient that was unresponsive to growth hormone and thyroid supplementation.2 This is less likely attributable to end organ effects from iron accumulation, as transfusion dependency abated after splenectomy at age 4 and one would have expected ferritin levels to have normalized soon thereafter. In our case, ferritin values normalized 9 months after splenectomy without chelation and remain normal to date. In cases with growth and endocrine changes, imaging studies with magnetic resonance imaging (T2* or superconducting quantum interference device) may be necessary to exclude tissue iron accumulation. Although our patient can maintain a hemoglobin of 9 to 10 g/dL and shows no evidence of growth retardation, there is a mild high receding forehead and mid face dysplasia reminiscent of Cooley anemia with skull radiographs and computerized tomography showing marked expansion of the diploic space. Thus of the 5 cases included in this review, all 3 karyotypic males with CDA IV seem to have a more severe, often transfusion-dependent course compared with the milder course in the 2 females with the same mutation. Because of ongoing health concerns in our patient, hematopoietic stem cell transplantation, an option for severely affected KLF1 E325K CDA IV patients, was considered but deferred for now as the only sibling was not a full match on HLA typing.

rse compared with the milder course in the 2 females with the same mutation. Because of ongoing health concerns in our patient, hematopoietic stem cell transplantation, an option for severely affected KLF1 E325K CDA IV patients, was considered but deferred for now as the only sibling was not a full match on HLA typing. Summary A comparison of the first 5 cases with KLF1 E325K mutation suggests that the spectrum of clinical effects of this mutation can vary from moderate to severe, transfusion-dependent anemia. The presence of modifier alleles in non-KLF1 genes associated with congenital anemia may lead to the wide variability in clinical phenotypes observed. The concurrent presence of urogenital anomalies in 2 of the karyotypic males remains unexplained. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.jpho-online.com.

Summary A comparison of the first 5 cases with KLF1 E325K mutation suggests that the spectrum of clinical effects of this mutation can vary from moderate to severe, transfusion-dependent anemia. The presence of modifier alleles in non-KLF1 genes associated with congenital anemia may lead to the wide variability in clinical phenotypes observed. The concurrent presence of urogenital anomalies in 2 of the karyotypic males remains unexplained. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.jpho-online.com. ACKNOWLEDGMENTS Dr Senthil Sundaram provided invaluable help to the primary author in getting started with exome sequencing studies and the use of various annotation Websites. A special thanks to Dr Laurence Boxer (University of Michigan) for his encouragement and sharing in the care of this child. The authors acknowledge the contributions of James Hoyer (Mayo Laboratories for Isoelectric focusing studies of hemoglobin), James Palis (Rochester, NY for in vitro erythroid colony studies), Douglas Whitten (Michigan State University, Lansing Michigan for proteomic studies), and Adele Kruger for assistance with WES analyses. The authors also acknowledge the contribution of the child and her parents to these investigative studies.

moglobin), James Palis (Rochester, NY for in vitro erythroid colony studies), Douglas Whitten (Michigan State University, Lansing Michigan for proteomic studies), and Adele Kruger for assistance with WES analyses. The authors also acknowledge the contribution of the child and her parents to these investigative studies. Y.R.: designed diagnostic strategies and wrote the manuscript; G.G., M.G., and S.B.: executed and interpreted laboratory studies; P.G.G.: performed genetic analyses and assisted in writing the manuscript; R.M.J.: supervised laboratory testing, assisted in genetic analyses, and assisted in writing the manuscript. Presented in part in abstract form at the American Society of Hematology annual meeting [ASH Annual Meeting Abstracts. 2011;118(21):2101]. Supported by the Georgie Ginopolis Chair Award (Y.R.); the Melissa Ann Krinsky Research Fund, the David Carr Memorial Fund and NIDDK RO1DK104046. The authors declare no conflict of interest.

Protein C, the key component of the PC anticoagulant system, is an important vitamin K-dependent serine protease zymogen; it is synthesized mostly in the liver and secreted to the plasma as an inactive zymogene and regulates the physiologic coagulation cascade by inactivating factors Va and VIIIa upon activation by thrombin.1–3 PC is encoded by the protein C gene (PROC) on chromosome 2q13-q14, which is composed of 9 exons and spans about 11.2 kb.4 Heterozygous individuals have an ~7-fold increased risk of venous thrombosis compared with normal individuals. The homozygous (or compound heterozygous) state of protein C deficiency (PCD) is much rarer.5,6 The prevalence of PCD in the healthy Chinese population is about 0.29%.7 Although the prevalence of PCD in VTE patients rises to about 14% to 19%, the rate of PCD caused by a missense mutation of PROC is about 55% in China.8,9 Severe congenital PCD is an uncommon yet life-threatening coagulopathy that usually presents with symptoms of purpura fulminans (PF) and severe disseminated intravascular coagulation as early as in the neonatal period.

to about 14% to 19%, the rate of PCD caused by a missense mutation of PROC is about 55% in China.8,9 Severe congenital PCD is an uncommon yet life-threatening coagulopathy that usually presents with symptoms of purpura fulminans (PF) and severe disseminated intravascular coagulation as early as in the neonatal period. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most frequent hereditary cerebral small vessel disease. CADASIL is caused by mutations in the neurogenic locus notch homolog protein 3 (NOTCH3) gene on chromosome 19q12,10 which is involved in cell signaling and differentiation.11 There is currently no treatment for this disorder.12 Magnetic resonance imaging (MRI) studies have established microbleeds and subcortical lacunar lesions as additional radiologic features of CADASIL. At this point, there has been no case of suspected CADASIL with infancy onset that has the characteristics of cerebral white matter lesion and NOTCH3 mutation. Here, we studied a rare patient who was diagnosed with congenital PCD and CADASIL. This was a sporadic case diagnosed as both congenital PCD and CADASIL, and the first report of infancy onset of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most frequent hereditary cerebral small vessel disease. CADASIL is caused by mutations in the neurogenic locus notch homolog protein 3 (NOTCH3) gene on chromosome 19q12,10 which is involved in cell signaling and differentiation.11 There is currently no treatment for this disorder.12 Magnetic resonance imaging (MRI) studies have established microbleeds and subcortical lacunar lesions as additional radiologic features of CADASIL. At this point, there has been no case of suspected CADASIL with infancy onset that has the characteristics of cerebral white matter lesion and NOTCH3 mutation. Here, we studied a rare patient who was diagnosed with congenital PCD and CADASIL. This was a sporadic case diagnosed as both congenital PCD and CADASIL, and the first report of infancy onset of CADASIL. MATERIALS AND METHODS A 5-month-old boy was referred to Shenzhen Children’s Hospital (China) for evaluation after frequent episodes of refractory PF had developed in his right calf over a month. The patient was a test-tube baby (G4P1), and his mother had a history of abortion and 2 miscarriages. We performed the PC activity testing of this patient and his immediate family, including his parents and all of his grandparents, to investigate the coagulation status; in addition, the testing of PROC gene sequencing was performed among this family members.

and his mother had a history of abortion and 2 miscarriages. We performed the PC activity testing of this patient and his immediate family, including his parents and all of his grandparents, to investigate the coagulation status; in addition, the testing of PROC gene sequencing was performed among this family members. At the age of 11 months, our patient could not stay sitting up and had not shown any verbal skills. His physical and cognitive development was evaluated with Bayley Scales of Infant Development, with scores of 58 for mental status and 29 for psychomotor status, supporting that the patient had demonstrated developmental delays and cognitive impairment at that point of time. Thereafter, the patient was subjected to cranial MRI and NOTCH3 gene sequencing. The changes of NOTCH3 protein with skin tissue was performed using an anti-NOTCH3 antibody (ab23426; Abcam, UK) by immunohistochemistry staining (Supplemental Digital Content 1, http://links.lww.com/JPHO/A291). RESULTS Results of Protein C Activity and Coagulation Function of Family Members The results of protein C activity and coagulation function of these family members had shower in Table 1. All of the tested indicators were abnormal for the patient; protein C activity level of the proband for him, his mother, and his father were reduced to 3%, 32%, and 53%, respectively. His prothrombin time, thrombin time, and activated partial thromboplastin time were increased to 18.6, 36.9, and 51.7 seconds. Factor V activity had increased to 138%, and the remaining indicators were also reduced for the patient.

band for him, his mother, and his father were reduced to 3%, 32%, and 53%, respectively. His prothrombin time, thrombin time, and activated partial thromboplastin time were increased to 18.6, 36.9, and 51.7 seconds. Factor V activity had increased to 138%, and the remaining indicators were also reduced for the patient. TABLE 1 The Results of Protein C Activity and Coagulation Function of Family Members Analysis of PROC Genotype and Plasma Protein C Phenotype Nucleotide sequence analysis demonstrated that the proband was confirmed as a compound homozygous for (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene (Fig. 1). His parents were confirmed as heterozygous. His grandfather, maternal grandfather, mother, and father all had low PC activity but were asymptomatic. FIGURE 1 A, The pedigree of the family with protein C deficiency. The index patient was indicated with an arrow. B, Results of PROC gene sequencing for this family. *indicates the mutation location in this sequencing report.

Analysis of PROC Genotype and Plasma Protein C Phenotype Nucleotide sequence analysis demonstrated that the proband was confirmed as a compound homozygous for (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene (Fig. 1). His parents were confirmed as heterozygous. His grandfather, maternal grandfather, mother, and father all had low PC activity but were asymptomatic. FIGURE 1 A, The pedigree of the family with protein C deficiency. The index patient was indicated with an arrow. B, Results of PROC gene sequencing for this family. *indicates the mutation location in this sequencing report. Results of MRI NOTCH3 Gene Sequencing and Immunohistochemistry The patient’s cranial MRI showed hyperintense lesions in the cerebral white matter on T2-weighted images; cranial MRI of the patient and his mother are shown in Figure 2. On the basis of the above clinical manifestation and imaging findings, hereditary cerebral microangiopathy was suspected for this patient. Molecular analysis of the NOTCH3 gene was performed, with automatic sequencing of exon 24 showing that the patient had a heterozygous mutations in exon 24(c.4348G>A), leading to a pathogenic amino acid substitution of p.A1450T (showed in Fig. 3). His maternal grandmother and his mother were asymptomatic but had the same mutation of NOTCH3 gene. Results of immunohistochemistry showed that NOTCH3 protein was positive in the skin’s vascular smooth muscle of the patient (showed in Fig. 4) and indirectly proved that heterozygous mutation of the NOTCH3 gene caused CADASIL.

ternal grandmother and his mother were asymptomatic but had the same mutation of NOTCH3 gene. Results of immunohistochemistry showed that NOTCH3 protein was positive in the skin’s vascular smooth muscle of the patient (showed in Fig. 4) and indirectly proved that heterozygous mutation of the NOTCH3 gene caused CADASIL. FIGURE 2 A, Magnetic resonance imaging scan of the brain showing multiple hyperintense centrum ovale white matter lesions on T2-weigthed images. B, Magnetic resonance imaging scan of the brain showing multiple hyperintense periventricular white matter lesions on T2-weighted images. C, Mother's magnetic resonance imaging scan of the brain showing multiple hyperintense centrum ovale white matter lesions on T2-weigthed images. D, Mother's magnetic resonance imaging scan of the brain showing multiple hyperintense periventricular white matter lesions on T2-weighted images. FIGURE 3 A, The pedigree of the family with NOTCH3 protien. The index patient was indicated with an arrow. B, Results of NOTCH3 gene sequencing for this family. FIGURE 4 Immunohistochemistry of NOTCH3 protein with skin tissue. Immunohistochemistry data showed the NOTCH3 protein was positive in the patient’s skin vascular smooth muscle (scar, ×200).

FIGURE 3 A, The pedigree of the family with NOTCH3 protien. The index patient was indicated with an arrow. B, Results of NOTCH3 gene sequencing for this family. FIGURE 4 Immunohistochemistry of NOTCH3 protein with skin tissue. Immunohistochemistry data showed the NOTCH3 protein was positive in the patient’s skin vascular smooth muscle (scar, ×200). DISCUSSION Hereditary PCD was first reported in 1981. The coagulopathy in PCD is caused by impaired inactivation of factors Va and VIIIa by activated PC after the propagation phase of coagulation activation. There have been >160 PC gene mutations reported. The incidence of asymptomatic PCD has been reported to be between 1/200 and 1/500 in the population, whereas the incidence of clinically significant PCD has been estimated to be 1 in 20,000 in 1995.13 The majority of symptomatic heterozygous PC-deficient patients may develop venous thrombosis and/or pulmonary embolism, which usually begins when the patient is between the ages of 15 and 30 years. Homozygous or complex heterozygous PROC mutations usually lead to neonatal fulminant purpura, and the patient may soon develop a broad blood clot in the microcirculatory system shortly during the neonatal period.14 Our patient was confirmed as homozygous for the (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene with neonatal fulminant purpura symptom. The implication of this mutation is to be further studied.

broad blood clot in the microcirculatory system shortly during the neonatal period.14 Our patient was confirmed as homozygous for the (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene with neonatal fulminant purpura symptom. The implication of this mutation is to be further studied. Most cases of severe PCD have been managed with combination therapy of PC replacement and warfarin as the preferred anticoagulant.15,16 As PC concentrate is not available in China, to relieve the patient’s thrombosis, we gave the child initially one dose (15 mL/kg) of fresh-frozen plasma (FFP) every day for 40 days until his skin lesions were completely resolved. In response, there was obvious improvement of his coagulation function. However, PC activity of the patient dropped consistently below 13% with a recurrence of PF in his calf, because of which we stopped FFP for 3 days. Consequently, the patient received 1 dose of FFP every day for 5 days and his skin lesions resolved again, but he still presented with thrombosis, because of which combination therapy was stopped; his PC activity was consistently below 10%, which made us speculate that the PC activity did not normalize even with FFP therapy.

uently, the patient received 1 dose of FFP every day for 5 days and his skin lesions resolved again, but he still presented with thrombosis, because of which combination therapy was stopped; his PC activity was consistently below 10%, which made us speculate that the PC activity did not normalize even with FFP therapy. CADASIL was initially thought to be a rare disorder, but increasing numbers of families have been identified worldwide.17–21 The disease is clinically characterized by transient ischemic attacks, strokes, progressive subcortical dementia, migraine with aura, and mood disturbances. For a typical CADASIL patient, widespread areas of increased signal in the white matter are associated with focal hyperintensities in the basal ganglia, thalamus, and brain stem in MRI; the extent of white matter signal abnormalities is highly variable.22 MRI signal abnormalities can also be detected during a presymptomatic period of variable duration.23 CADASIL was an autosomal dominant inherited disease, characterized by midadult onset of cerebrovascular disease and dementia, but the majority of mutations are found in exon 4. Most studies indicate that pathogenic mutations have been found throughout exons 2 to 24, which are the exons that encode the EGF reporter. However, the mutation hotspots of CADASIL are located in exon 4 of the NOTCH3 gene;24 the prevalence of mutations in other exons varies between countries; in the French, German, and English CADASIL population, exon 3 is the second most frequently mutated exon, whereas, in Dutch CADASIL patients, exon 11 is the second most frequently mutated exon.25 These identified mutations were clustered in exons 3, 4, 5, and 11 in mainland Chinese patients,26,27 but the exon 11 was a mutational hotspot in Taiwanese patients.28 In this study, we had reported a heterozygous mutation in exon 24 (c.4348G>A), which leads to a pathogenic amino acid substitution of p.A1450T. Immunohistochemistry results revealed that the NOTCH3 protein was positive in the skin tissue of our patient, which showed that these mutations had caused CADASIL. Nevertheless, his maternal grandmother and his mother also had the same mutation of the NOTCH3 gene, but no clinical features of CADASIL such as cognitive impairment or migraine with aura were shown, which suggests that heterozygous mutations of c.4348G>A(p.A1450T) in exon 24 were either sex-dependent pathogenic mutations or not pathogenic mutations.

mother and his mother also had the same mutation of the NOTCH3 gene, but no clinical features of CADASIL such as cognitive impairment or migraine with aura were shown, which suggests that heterozygous mutations of c.4348G>A(p.A1450T) in exon 24 were either sex-dependent pathogenic mutations or not pathogenic mutations. Chen et al27 found that most patients of Chinese origin were carrying p.Arg607Cys and p.Arg544Cys mutations, not p. Ala 1450Thr mutations. This was consistent with Rutten et al’s25 study, which demonstrated that missense mutations in NOTCH3 that are not altering a cysteine residue are unlikely to be pathogenic. However, according to the research of Li et al,29 a novel pathogenic variant of the NOTCH3 gene, which was a heterozygous mutation of c.128G>C in exon 2, caused a cysteine to serine substitution at codon 43 in 2 Chinese CADASIL patients.29 Moreover, it explains the phenomenon why his maternal grandmother and mother were not CADASIL. We suspected PCD to cause or aggravate CADASIL. Further research and analysis may help unravel the reasons of early onset in this patient.

caused a cysteine to serine substitution at codon 43 in 2 Chinese CADASIL patients.29 Moreover, it explains the phenomenon why his maternal grandmother and mother were not CADASIL. We suspected PCD to cause or aggravate CADASIL. Further research and analysis may help unravel the reasons of early onset in this patient. The mean age of CADASIL patients who have the clinical symptoms is 45 years, and the duration of the disease varies between 10 and 40 years, and can also be observed as early as 20 years of age.23 Mosca et al30 had reported 140 CADASIL patients from Italy and China who were in the age group of 21 to 73 years. Abramycheva et al31 had reported 30 white CADASIL patients who were in the age group of 22 to 73 years. Cognitive decline initially manifests with a decrease in executive function, followed by a slowly progressive or stepwise deterioration in cognitive function, which becomes apparent while performing daily activities around the age of 50.32 In this study, we reported the case of an 11-month-old boy with CADASIL who had also been diagnosed with PCD earlier. The infant maybe the youngest among CADASIL patients reported in the world. The case was noted if cognitive decline occurred in an infant or baby; the diagnosis about CADASIL maybe necessary.

e of 50.32 In this study, we reported the case of an 11-month-old boy with CADASIL who had also been diagnosed with PCD earlier. The infant maybe the youngest among CADASIL patients reported in the world. The case was noted if cognitive decline occurred in an infant or baby; the diagnosis about CADASIL maybe necessary. CONCLUSIONS In conclusion, we studied a rare case in which both congenital PCD and CADASIL were diagnosed in an infant boy; congenital PCD was attributable to a compound that was homozygous for (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene, and CADASIL was caused by missense mutation in exon 24 of NOTCH3 and PCD. He was a sporadic patient with congenital PCD and CADASIL in the world; it maybe that the deficiency of protein C led to early onset of CADASIL. Moreover, it supports that CADASIL patients can be encountered even at the infant stage. In contrast, it shows that the gene sequencing of PROC gene and NOTCH3 gene may have important value for genetic guidance and prenatal diagnosis. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.jpho-online.com.

CONCLUSIONS In conclusion, we studied a rare case in which both congenital PCD and CADASIL were diagnosed in an infant boy; congenital PCD was attributable to a compound that was homozygous for (A-G)-12 at the transcription initiation site in the promoter region of the PROC gene, and CADASIL was caused by missense mutation in exon 24 of NOTCH3 and PCD. He was a sporadic patient with congenital PCD and CADASIL in the world; it maybe that the deficiency of protein C led to early onset of CADASIL. Moreover, it supports that CADASIL patients can be encountered even at the infant stage. In contrast, it shows that the gene sequencing of PROC gene and NOTCH3 gene may have important value for genetic guidance and prenatal diagnosis. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.jpho-online.com. ACKNOWLEDGMENTS The authors thank the patient and his family for allowing them to use the medical documentation and information leading to the present article; the authors also thank Gang Xu (MD, Clinical Laboratory Department of Shenzhen Children’s Hospital) for technical and material support, and Dr Yungen Gan (MD, Radiologist, Radiology Department of Shenzhen Children’s Hospital) for imaging technical assistance. This study was supported by Sanming Project of Medicine in Shenzhen (No. SZSM201512033). The authors declare no conflict of interest.

During the past decade, immunotherapy has been a scientific breakthrough for the treatment of cancer. Adoptive cell therapy (ACT) using genetically modified T cells expressing chimeric antigen receptors (CAR) has demonstrated remarkable success in the treatment of pediatric B-cell leukemia with response rates as high as 70% to 90%.1–3 However, there are subtypes of pediatric leukemia with unknown tumor antigens, or the targeted tumor antigens are not expressed and CAR cell therapy is not an option. Therefore, there remains a need to identify leukemia-specific T cells that can be used for ACT. For patients who are not candidates for CAR T-cell therapy, ACT using leukemia antigen-experienced polyclonal T cells (in combination with chemotherapy or other targeted therapies) may be an option.

therapy is not an option. Therefore, there remains a need to identify leukemia-specific T cells that can be used for ACT. For patients who are not candidates for CAR T-cell therapy, ACT using leukemia antigen-experienced polyclonal T cells (in combination with chemotherapy or other targeted therapies) may be an option. The T-cell immune inhibitory receptor, PD1, is upregulated following T-cell receptor activation and remains elevated with chronic antigen stimulation.4 Recently, melanoma-reactive CD8+ T cells were found to reside in the subset of PD1+CD8+ melanoma-infiltrating lymphocytes.5,6 Therefore, enrichment of PD1+ T cells is a reasonable strategy to optimize the anticancer efficacy of ACT. Since bone marrow is a reservoir for leukemic blasts, we hypothesized that bone marrow-derived T cells would express PD1 and may serve a source of leukemia-reactive cells. The objective of this study was to phenotype bone marrow-derived T cells from pediatric leukemia patients to determine whether these T cells would be amenable for ACT. Since many leukemia patients are heavily pretreated with chemotherapy before immunotherapy, T cells were analyzed following induction chemotherapy. Using matched pairs of T cells obtained from bone marrow and peripheral blood, our results show the percentage of CD8+ T cells expressing activation markers PD1, CD69, and CD45RO is significantly elevated in bone marrow as compared with peripheral blood. Importantly, bone marrow-derived T cells expanded ex vivo when obtained following induction chemotherapy.

from bone marrow and peripheral blood, our results show the percentage of CD8+ T cells expressing activation markers PD1, CD69, and CD45RO is significantly elevated in bone marrow as compared with peripheral blood. Importantly, bone marrow-derived T cells expanded ex vivo when obtained following induction chemotherapy. MATERIALS AND METHODS Patient Samples Bone marrow and peripheral blood samples were obtained in accordance with guidelines set forth in the approved Children’s Hospital of Wisconsin Institutional Review Board protocol, IBC20150066. Informed consent was obtained. T-Cell Purification and Ex Vivo Expansion Mononuclear cells were purified from bone marrow and peripheral blood using SepMate tubes (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s guidelines. Cells were activated once with anti-CD3 and anti-CD28-conjugated magnetic Dynabeads (Thermo Fisher Scientific, Carlsbad, CA) at a 1:1 ratio of cells to beads. Cells were cultured in RPMI 1640 media (Gibco) with 20 U/mL interleukin 2 (IL2), 5 ng/mL IL7, and 5 ng/mL IL15 for 9 to 12 days. Media and cytokines were replaced every 2 to 3 days during culture.

-CD3 and anti-CD28-conjugated magnetic Dynabeads (Thermo Fisher Scientific, Carlsbad, CA) at a 1:1 ratio of cells to beads. Cells were cultured in RPMI 1640 media (Gibco) with 20 U/mL interleukin 2 (IL2), 5 ng/mL IL7, and 5 ng/mL IL15 for 9 to 12 days. Media and cytokines were replaced every 2 to 3 days during culture. Flow Cytometry Cells were stained with antibodies to T-cell markers: CD3 (UCHT1), CD4 (OKT4), CD8a (SK1); inhibitory receptors: CD279 (PD1, clone J105) and CD223 (LAG3, clone 17B4); activation markers: CD69 (FN50), CD45RO (UCHL1), and memory markers: CD197 (CCR7, clone 3D12), CD122 (mikβ3), and CD127 (ebioRDR5). Cells were stained with 7-AAD viability dye and gated on live cells. Flow cytometric analysis was performed on a BD Biosciences LSRII (Franklin Lakes, NJ) flow cytometer, and resulting data analyzed using FlowJo software (Tree Star Inc., Ashland, OR). Apoptosis The Caspase-Glo 3/7 assay (Promega, Madison, WI) was used according to the manufacturer’s protocol. Cells were assayed between 9 and 12 days of ex vivo expansion. Thereafter, 4 μM camptothecin was added to cells for 2.5 hours as a positive control.

Flow Cytometry Cells were stained with antibodies to T-cell markers: CD3 (UCHT1), CD4 (OKT4), CD8a (SK1); inhibitory receptors: CD279 (PD1, clone J105) and CD223 (LAG3, clone 17B4); activation markers: CD69 (FN50), CD45RO (UCHL1), and memory markers: CD197 (CCR7, clone 3D12), CD122 (mikβ3), and CD127 (ebioRDR5). Cells were stained with 7-AAD viability dye and gated on live cells. Flow cytometric analysis was performed on a BD Biosciences LSRII (Franklin Lakes, NJ) flow cytometer, and resulting data analyzed using FlowJo software (Tree Star Inc., Ashland, OR). Apoptosis The Caspase-Glo 3/7 assay (Promega, Madison, WI) was used according to the manufacturer’s protocol. Cells were assayed between 9 and 12 days of ex vivo expansion. Thereafter, 4 μM camptothecin was added to cells for 2.5 hours as a positive control. RESULTS Following Induction Chemotherapy, Bone Marrow-derived CD8+ T Cells Have an Activated Phenotype Bone marrow and peripheral blood samples were obtained from pediatric leukemia patients following one month of induction chemotherapy. Induction chemotherapy included vincristine, pegaspargase, dexamethasone, and daunorubicin with a subset of patients also receiving bortezomib. Table 1 shows patient clinical descriptors. Since bone marrow is a preferred site for homeostatic proliferation of cytotoxic memory CD8+ T cells,7,8 it was of interest to determine if activated bone marrow-derived T cells would be present and able to expand ex vivo following induction chemotherapy. This was particularly relevant since there is an anticipated need to obtain T cells for ACT in patients following chemotherapy. Patient matched bone marrow and peripheral blood buffy coats were stained with antibodies and analyzed for T-cell subsets by flow cytometry. For patients 006, 008, and 010, the bone marrow sample was not adequate to be used for flow cytometric analysis. There was a significant (P<0.05) difference in the percentage of bone marrow-derived CD8+ T cells expressing activation markers PD1, CD45RO, and CD69 as compared with peripheral blood CD8+ T cells (Fig. 1A). This difference was not observed for CD4+ T cells (Fig. 1B). Figure 1C shows expression of the activation markers relative to each other on bone marrow-derived CD8+ T cells. There was no difference in the expression of memory markers, CD197 (CCR7), CD122, or CD127 on CD4+ and CD8+ T cells obtained from the bone marrow and peripheral blood when analyzed before activation and expansion (data not shown).

ion of the activation markers relative to each other on bone marrow-derived CD8+ T cells. There was no difference in the expression of memory markers, CD197 (CCR7), CD122, or CD127 on CD4+ and CD8+ T cells obtained from the bone marrow and peripheral blood when analyzed before activation and expansion (data not shown). TABLE 1 Clinical Characteristics and Disease Burden of Pediatric Leukemia Patients FIGURE 1 T-cell subsets in bone marrow and peripheral blood of pediatric patients following postinduction chemotherapy. Percentage of PD1+, CD69+, and CD45RO+ CD8+ T cells (A) and CD4+ T cells (B) in BM and PB. C, The expression of PD1, CD69, and CD45RO in individual patients. *P<0.05 as calculated by Wilcoxon matched pairs signed rank test. BM indicates bone marrow; PB, peripheral blood.

c patients following postinduction chemotherapy. Percentage of PD1+, CD69+, and CD45RO+ CD8+ T cells (A) and CD4+ T cells (B) in BM and PB. C, The expression of PD1, CD69, and CD45RO in individual patients. *P<0.05 as calculated by Wilcoxon matched pairs signed rank test. BM indicates bone marrow; PB, peripheral blood. Following Ex Vivo Expansion, PD1 and LAG3 are Upregulated on CD4+ and CD8+ T Cells Obtained From Bone Marrow and Peripheral Blood To demonstrate the ability to expand T cells ex vivo, cells were activated once with anti-CD3/CD28-conjugated beads and cultured in the presence of low dose IL2 (20 U/mL), IL7 (5 ng/mL), and IL15 (5 ng/mL) for 9 to 12 days. Although there were individual differences in the ability of bone marrow-derived T cells to expand, there was >10-fold expansion in 8 of 10 bone marrow samples tested (Fig. 2A). There was significantly (P<0.05) greater fold expansion of peripheral blood-derived T cells as compared with bone marrow-derived T cells. When tested for apoptosis postexpansion, there was no difference between bone marrow and peripheral blood-derived T cells (Fig. 2B). Importantly, Figure 2C shows that despite a low postinduction chemotherapy white blood count in patients 003, 004, and 010, their bone marrow-derived T cells expanded >25-fold. These data show that in the majority of patients tested, bone marrow-derived T cells expanded ex vivo and had an apoptotic profile similar to peripheral blood-derived T cells.

t despite a low postinduction chemotherapy white blood count in patients 003, 004, and 010, their bone marrow-derived T cells expanded >25-fold. These data show that in the majority of patients tested, bone marrow-derived T cells expanded ex vivo and had an apoptotic profile similar to peripheral blood-derived T cells. FIGURE 2 Fold expansion, apoptosis, and checkpoint receptor expression on ex vivo expanded T cells. A, Comparison of fold expansion between BM-derived and PB-derived T cells. B, Caspase 3/7 apoptosis of BM-derived and PB-derived T cells. C, Correlation of postinduction chemotherapy complete white blood cell count and fold expansion of BM-derived T cells. D, Percentage of CD4+ and CD8+ T cells present in the PB and BM pre-activation and expansion (pre-expansion) and post-activation and expansion (post-expansion). E, PD1 and LAG3 checkpoint receptor expression pre-expansion and post-expansion. *P<0.05 as calculated by Wilcoxon matched pairs signed rank test. BM indicates bone marrow; PB, peripheral blood.

lls present in the PB and BM pre-activation and expansion (pre-expansion) and post-activation and expansion (post-expansion). E, PD1 and LAG3 checkpoint receptor expression pre-expansion and post-expansion. *P<0.05 as calculated by Wilcoxon matched pairs signed rank test. BM indicates bone marrow; PB, peripheral blood. Prior to ex vivo expansion, the CD4:CD8 ratio was ~1:1 for both peripheral blood and bone marrow-derived T cells (Fig. 2D). Following ex vivo expansion, the CD4:CD8 T-cell ratio was 1:4 regardless of the T-cell source. These data suggest CD8+ T cells have a greater ex vivo expansion potential than CD4+ T cells. Following ex vivo expansion, T cells were analyzed for expression of checkpoint receptors PD1 and LAG3. Regardless of the T-cell source, there was a significant increase in the percentage of CD4+ and CD8+ T cells expressing PD1 and LAG3 (Fig. 2E). An increase in the percentage of T cells expressing PD1 and LAG3 was not surprising as activated or chronically stimulated T cells upregulate coinhibitory molecules.9

and LAG3. Regardless of the T-cell source, there was a significant increase in the percentage of CD4+ and CD8+ T cells expressing PD1 and LAG3 (Fig. 2E). An increase in the percentage of T cells expressing PD1 and LAG3 was not surprising as activated or chronically stimulated T cells upregulate coinhibitory molecules.9 DISCUSSION Following induction chemotherapy for pediatric acute lymphocytic leukemia, the bone marrow was shown to have a greater percentage of PD1+CD8+ T cells than peripheral blood. There is evidence that CD8+ T cells expressing PD1 contain the subset of T cells that are functional against cancer.5,6 In murine models of multiple myeloma and acute myeloid leukemia, our own data has demonstrated PD1 as a biomarker for functional leukemia-reactive T cells that are effective against leukemia in vivo when administered as ACT.10 Therefore, PD1 expression on T cells obtained from pediatric leukemia patients may be a marker of leukemia reactivity. Despite a greater percentage of PD1+ T cells in the bone marrow, it is important to note that PD1+ T cells were also present in peripheral blood. It is possible that both bone marrow and peripheral blood-derived PD1+CD8+ T cells have anti-leukemia reactivity. Peripheral blood-derived T cells are more easily obtained and have greater expansion potential than bone marrow-derived T cells, thus peripheral blood-derived T cells enriched for PD1 could also be a source of anti-leukemic cells.

bone marrow and peripheral blood-derived PD1+CD8+ T cells have anti-leukemia reactivity. Peripheral blood-derived T cells are more easily obtained and have greater expansion potential than bone marrow-derived T cells, thus peripheral blood-derived T cells enriched for PD1 could also be a source of anti-leukemic cells. Following ex vivo expansion there was a significant increase in the percentage of T cells expressing PD1 and LAG3. In a murine model of ovarian cancer, PD1 and LAG3 were shown to physically associate, traffic to the immunologic synapse and synergize to dampen T-cell signaling.11 Blockade of LAG3 and PD1 pathways in preclinical cancer models has had synergistic effects increasing the antitumor CD8+ T-cell response.12 These data suggest that administration of anti-PD1 and anti-LAG3 blocking antibodies in combination with ACT of T cells expressing PD1 and/or LAG3 is a reasonable approach to facilitate the antitumor efficacy of adoptively transferred T cells.

has had synergistic effects increasing the antitumor CD8+ T-cell response.12 These data suggest that administration of anti-PD1 and anti-LAG3 blocking antibodies in combination with ACT of T cells expressing PD1 and/or LAG3 is a reasonable approach to facilitate the antitumor efficacy of adoptively transferred T cells. There are multiple variables to consider when producing effective antileukemia T cells for ACT. During induction chemotherapy, patients are treated with multiple drugs and the direct effects (eg, induction of apoptosis) or indirect effects (eg, elimination of leukemia blast cells or lymphopenia13) of these therapies on T-cell activation, ex vivo proliferation and effector responses is largely unknown. It has been reported that bortezomib (a proteasome inhibitor) augments CD8+ T-cell effector function.14 However, it has also been reported that bortezomib induces apoptosis of activated T cells.15 It would be of interest to investigate the effects of bortezomib and other chemotherapeutics on ex vivo T-cell expansion and anti-leukemia effector function. How best to ex vivo activate and expand T cells to produce durable and effective anti-leukemia T cells is also under investigation. In this study, low dose IL2 (20 U/mL) with 5 ng/mL IL7 and 5 ng/mL of IL15 was used to support lymphocyte proliferation. The influence of cytokines and culture conditions on differentiation or proliferation of T-cell subsets requires further study.

urable and effective anti-leukemia T cells is also under investigation. In this study, low dose IL2 (20 U/mL) with 5 ng/mL IL7 and 5 ng/mL of IL15 was used to support lymphocyte proliferation. The influence of cytokines and culture conditions on differentiation or proliferation of T-cell subsets requires further study. In summary, we have shown that peripheral blood and bone marrow-derived T cells can be expanded ex vivo when obtained from pediatric leukemia patients following induction chemotherapy. There is a greater percentage of PD1+ T cells in the bone marrow as compared with peripheral blood. The bone marrow is a reservoir for leukemic blasts, thus PD1 expression may be a marker of activated leukemia-experienced T cells. However, a comparison of the anti-leukemia specificity (against patient leukemia blast cells) of bone marrow and peripheral blood-derived CD8+or CD8+PD1+ T cells is required to determine the best personalized source of anti-leukemic T cells to be used for ACT. Midwest Athletes Against Childhood Cancer (MACC) Fund, Children’s Research Institute, Children’s Hospital of Wisconsin. The authors declare no conflict of interest.

Childhood leukemia is the most common cancer among children and adolescents younger than 18 years of age in the United States,1 which accounts for 29% of all childhood cancers,2 and acute lymphoblastic leukemia (ALL) is the most commonly observed subtype which accounts for ∼80% of childhood leukemia.1 Currently, the 5-year survival rate of childhood ALL is promising which is approaching 90%.1 Although the survival rate of childhood ALL looks favorable, the disease remains an intractable public health problem due to its increasing incidence. For example, McNally and Eden3 observed a significant increase in the incidence of childhood ALL at about 1% per year in well-developed countries. Therefore, there is a great need to study the etiology of childhood ALL, which can provide evidence for early intervention and prevention.

m due to its increasing incidence. For example, McNally and Eden3 observed a significant increase in the incidence of childhood ALL at about 1% per year in well-developed countries. Therefore, there is a great need to study the etiology of childhood ALL, which can provide evidence for early intervention and prevention. However, there is no consistent evidence to suggest that a single environmental or nutritional factor causes the disease, and some risk factors remain theoretical.4 In 2004, the International Agency for Research on Cancer (IARC) reported that there was a borderline association between parental smoking and the risk of childhood leukemia.5 In a recent systematic review and meta-analysis, Klimentopoulou et al6 reported that maternal smoking during pregnancy was not associated with the risk of childhood ALL. Momen et al7 also reported that maternal smoking was not associated with childhood ALL. These indicate that the etiologic association between prebirth parental smoking and childhood ALL may be attributed to paternal smoking. However, currently available epidemiologic studies investigating the effect of prebirth paternal smoking to the risk of childhood ALL are inconsistent.8–11

ociated with childhood ALL. These indicate that the etiologic association between prebirth parental smoking and childhood ALL may be attributed to paternal smoking. However, currently available epidemiologic studies investigating the effect of prebirth paternal smoking to the risk of childhood ALL are inconsistent.8–11 Presently, many potential parents choose to smoke. According to the data from the Centers for Disease Control and Prevention (CDC), ∼17.6% of adult males in the United States between the ages of 25 and 44 years choose to smoke12; this age range also corresponds to the period during which most of them choose to give birth or rear offspring. Prebirth paternal smoking is known to be associated with many childhood adverse health events, including but not limited to obesity, impaired lung function, asthma, and headache.13–15 Given these adverse effects, if paternal smoking before conception or during pregnancy increases the risk of childhood ALL, it will be more justifiable for us to boost tobacco control or smoking cessation program by informing and educating potential fathers of the hazards of smoking. Therefore, we performed this systematic review and meta-analysis of currently available epidemiology studies.

ng pregnancy increases the risk of childhood ALL, it will be more justifiable for us to boost tobacco control or smoking cessation program by informing and educating potential fathers of the hazards of smoking. Therefore, we performed this systematic review and meta-analysis of currently available epidemiology studies. METHODS Search Strategy A systematic literature search of 4 electronic databases (PubMed, Embase, Web of Science, and Scopus) was performed on March 1, 2019, using keywords and controlled vocabularies that were related to “smoking,” “child,” and “leukemia” (Supplementary A, Supplemental Digital Content 1, http://links.lww.com/JPHO/A333). We also hand-searched the reference lists of previous systematic reviews of similar topics to obtain more eligible studies.

March 1, 2019, using keywords and controlled vocabularies that were related to “smoking,” “child,” and “leukemia” (Supplementary A, Supplemental Digital Content 1, http://links.lww.com/JPHO/A333). We also hand-searched the reference lists of previous systematic reviews of similar topics to obtain more eligible studies. Study Identification We followed the instructions of the Meta-analysis of Observational Studies in Epidemiology (MOOSE)16 and established restrictive selection criteria before study identification. In title/abstract screening, we considered studies that met the following criteria: (1) observational epidemiology study (a case-control, cohort, cross-sectional, nested case-control, and case-cohort study); (2) the exposure of interest was paternal smoking before conception or during pregnancy; (3) the outcome of interest was the risk of childhood ALL; and (4) written in English. In the full-text review process, we read the articles that were identified in the title/abstract screening process to determine whether they met the additional criteria: (1) full-text written in English; (2) reported the disease subtype (should be childhood ALL); (3) reported the exposure time window (before conception vs. during pregnancy); and (4) reported effect measure for paternal smoking or raw data that could be used to calculate unadjusted effect measure. The title/abstract screening and the full-text review were performed independently by reviewer pairs. Any discrepancy between the reviewers was resolved by discussion. Studies selected from the full-text review were included for systematic review and meta-analysis, and we performed data extraction and quality assessment for them independently.

ing and the full-text review were performed independently by reviewer pairs. Any discrepancy between the reviewers was resolved by discussion. Studies selected from the full-text review were included for systematic review and meta-analysis, and we performed data extraction and quality assessment for them independently. The process of study identification and selection is presented in Figure 1 which includes essential details according to the requirement of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).17 FIGURE 1 Flow chart for study identification and selection. Data Extraction and Quality Assessment After reading the selected articles, reviewers recorded information about the study characteristics, participants, exposure/outcome measurement, and measures of association; particularly, risk ratio (RR) was treated as the proximate measure of odds ratio due to the fact that childhood ALL was rare among the population. Moreover, we referred to The Newcastle-Ottawa Scale for assessing the quality of nonrandomized studies in meta-analyses18 to evaluate the quality of included studies regarding aspects of representativeness, measurement reliability, and statistical adjustment (Supplementary B, Supplemental Digital Content 2, http://links.lww.com/JPHO/A334). This process was also performed in an independent manner by reviewers, and discrepancies were solved by discussion.

quality of included studies regarding aspects of representativeness, measurement reliability, and statistical adjustment (Supplementary B, Supplemental Digital Content 2, http://links.lww.com/JPHO/A334). This process was also performed in an independent manner by reviewers, and discrepancies were solved by discussion. Synthesis Methods For qualitative synthesis, we summarized the study characteristics of each individual study; for studies treating smoking as an ordinal variable, we first summarized the RRs of each exposure level qualitatively (Supplementary C, Supplemental Digital Content 3, http://links.lww.com/JPHO/A335) before the subsequent quantitative synthesis. In the overall meta-analysis, we pooled the measures of the association from studies treating smoking as a dichotomous variable by a random-effects model,19 and we weighted the RRs according to the SEs of ln(RR). A sensitivity meta-analysis was performed by including studies adjusting for certain covariates (child age at diagnosis, sex, race/ethnicity, parental age at birth, socioeconomic status, and maternal alcohol consumption during pregnancy) and studies with good quality in exposure measurement. In the dose-response analysis, we first synthesized studies reporting RRs of ordinal exposure by using RRs of the highest smoking consumption in a random-effects model. Then, we utilized a 2-stage restricted cubic spline model with 3 knots20 to investigate the dose-response association between frequency of paternal smoking and risk of childhood ALL. We used a relatively conservative approach to determine the dose in the restricted cubic spline model; particularly, the dose was determined by using the middle point of a range (eg, if the smoking was 10 to 20 cigarettes/d, the dose=15 cigarettes/d) or the lower bound (eg, for smoking>20 cigarettes/d, the dose=20 cigarettes/d) if the range was not reported.

ch to determine the dose in the restricted cubic spline model; particularly, the dose was determined by using the middle point of a range (eg, if the smoking was 10 to 20 cigarettes/d, the dose=15 cigarettes/d) or the lower bound (eg, for smoking>20 cigarettes/d, the dose=20 cigarettes/d) if the range was not reported. To investigate statistical heterogeneity, we calculated the Cochrane Q statistic and I2. The former was a weighted sum of squares following the χ2 distribution;21 I2 was an indicator for inconsistency across studies, and it was interpreted as the proportion of variation between different studies among the total variation observed.22 Substantial statistical heterogeneity existed if the P-value derived from the Cochrane Q statistic was <0.05,21 and we also used “I2>50%” as the evidence of substantial statistical heterogeneity.21,22 To investigate publication bias, we visually inspected the funnel plots and performed Egger tests for studies included in the overall meta-analysis.23 The Egger test indicated a publication bias if P-value <0.05. Trim and fill were utilized to obtain an adjusted RR if publication bias existed.24 The statistical analyses were all performed with stratification based on time window (smoking before conception vs. during pregnancy), and they were performed in STATA 13.0 (StataCorp, LLP, College Station, TX).

To investigate publication bias, we visually inspected the funnel plots and performed Egger tests for studies included in the overall meta-analysis.23 The Egger test indicated a publication bias if P-value <0.05. Trim and fill were utilized to obtain an adjusted RR if publication bias existed.24 The statistical analyses were all performed with stratification based on time window (smoking before conception vs. during pregnancy), and they were performed in STATA 13.0 (StataCorp, LLP, College Station, TX). RESULTS Included Studies We retrieved 2127 articles from the electronic databases, and we identified 2 additional articles from reference lists. After deduplication, we read the titles and abstracts of 971 articles and excluded 940 of them. In the full-text review, we read the full-text of 31 articles and excluded 14 of them. Finally, 17 studies8–11,25–37 were included in the systematic review and meta-analysis. The reasons for exclusion are shown in Figure 1.

After deduplication, we read the titles and abstracts of 971 articles and excluded 940 of them. In the full-text review, we read the full-text of 31 articles and excluded 14 of them. Finally, 17 studies8–11,25–37 were included in the systematic review and meta-analysis. The reasons for exclusion are shown in Figure 1. Study Characteristics The study characteristics are summarized in Table 1. The included studies encompassed a wide time span (1977-2011), and they were conducted in various geographic locales. They yielded a total of 9127 childhood ALL cases. Among them, 8 studies8,9,11,25,28,30,32,33 reported effect measures for paternal smoking before conception and during pregnancy, 3 studies10,35,37 only reported effect measures for smoking during pregnancy, and 6 studies26,27,29,31,34,36 only reported effect measures for smoking before conception. Seven studies8,25–27,32,34,35 reported RRs of dichotomous exposure (smoking vs. nonsmoking), 5 studies28–31,36 reported RRs of ordinal exposure, and 5 studies9–11,33,37 reported RRs on both scales. The self-report approach was utilized to collect information about paternal smoking, and 7 studies10,27,31–33,35,37 collected paternal smoking data via surrogate measurement based on answers from the mothers. In terms of the study setting, most studies were population-based, and only 2 studies9,36 were clinic-based. TABLE 1 Study Characteristics

Study Characteristics The study characteristics are summarized in Table 1. The included studies encompassed a wide time span (1977-2011), and they were conducted in various geographic locales. They yielded a total of 9127 childhood ALL cases. Among them, 8 studies8,9,11,25,28,30,32,33 reported effect measures for paternal smoking before conception and during pregnancy, 3 studies10,35,37 only reported effect measures for smoking during pregnancy, and 6 studies26,27,29,31,34,36 only reported effect measures for smoking before conception. Seven studies8,25–27,32,34,35 reported RRs of dichotomous exposure (smoking vs. nonsmoking), 5 studies28–31,36 reported RRs of ordinal exposure, and 5 studies9–11,33,37 reported RRs on both scales. The self-report approach was utilized to collect information about paternal smoking, and 7 studies10,27,31–33,35,37 collected paternal smoking data via surrogate measurement based on answers from the mothers. In terms of the study setting, most studies were population-based, and only 2 studies9,36 were clinic-based. TABLE 1 Study Characteristics The included studies had some methodologic strengths. For example, all of the studies ascertained childhood ALL cases by robust approach (eg, medical record, diagnosis report, and cancer registry); in addition, they all used match method when selecting controls, which benefited statistical efficiency. However, they also had some methodologic limitations. First, the studies measured paternal smoking via answers from mothers were less reliable than direct measurement, which might lead to misclassification. Second, 1 study26 only had 85 cases, which could introduce imprecision to effect measure. Third, 2 of the included studies9,36 were clinic-based; of them, the study populations were systematically different from the community populations with respect to exposure status and general health status, which introduced some methodologic heterogeneity. Moreover, these studies were conducted in different time periods and locations, and these temporal and socioeconomic heterogeneities could not be adjusted by the random-effects model.

m the community populations with respect to exposure status and general health status, which introduced some methodologic heterogeneity. Moreover, these studies were conducted in different time periods and locations, and these temporal and socioeconomic heterogeneities could not be adjusted by the random-effects model. Overall Meta-Analysis In overall meta-analysis for RRs of dichotomous exposure, 8 studies8,11,25–27,32–34 were included to investigate the association between paternal smoking before conception and childhood ALL risk, and 9 studies8–11,25,32,33,35,37 were synthesized for smoking during pregnancy. The synthesized RR for smoking before conception (Fig. 2) was 1.15 (95% confidence interval [CI]: 1.04-1.27), and all individual studies showed point estimates >1. For smoking during pregnancy (Fig. 2), all of the included studies had point estimates >1, and a positive and statistically significant synthesized effect measure (RR=1.20, 95% CI: 1.12-1.28) was obtained as well. In addition, we did not observe substantial statistical heterogeneity in either of these subgroups stratified by time window (before conception: I2=16.8%, PCochrane=0.298; during pregnancy: I2=0.0%, PCochrane=0.476). FIGURE 2 Overall meta-analysis stratified by the exposure time window. CI indicates confidence interval; RR, risk ratio.

Overall Meta-Analysis In overall meta-analysis for RRs of dichotomous exposure, 8 studies8,11,25–27,32–34 were included to investigate the association between paternal smoking before conception and childhood ALL risk, and 9 studies8–11,25,32,33,35,37 were synthesized for smoking during pregnancy. The synthesized RR for smoking before conception (Fig. 2) was 1.15 (95% confidence interval [CI]: 1.04-1.27), and all individual studies showed point estimates >1. For smoking during pregnancy (Fig. 2), all of the included studies had point estimates >1, and a positive and statistically significant synthesized effect measure (RR=1.20, 95% CI: 1.12-1.28) was obtained as well. In addition, we did not observe substantial statistical heterogeneity in either of these subgroups stratified by time window (before conception: I2=16.8%, PCochrane=0.298; during pregnancy: I2=0.0%, PCochrane=0.476). FIGURE 2 Overall meta-analysis stratified by the exposure time window. CI indicates confidence interval; RR, risk ratio. Sensitivity Analysis Table 2 presents the results of sensitivity analysis. By synthesizing studies adjusting for age at diagnosis, socioeconomic status, sex, race/ethnicity, and maternal alcohol consumption during pregnancy, we observed that the synthesized RRs were all statistically significant without substantial statistical heterogeneity in both exposure time windows. For studies adjusting for parental age at birth, the synthesized RR was only statistically significant for smoking during pregnancy; however, the CIs of synthesized RRs of the 2 exposure time windows were overlapped to a great extent, which indicated a statistical homogeneity between these pooled RRs. By synthesizing studies with good quality in exposure measurement, we observed that the synthesized RRs of the 2 exposure time windows were statistically homogeneous and marginally significant without substantial statistical heterogeneity.

reat extent, which indicated a statistical homogeneity between these pooled RRs. By synthesizing studies with good quality in exposure measurement, we observed that the synthesized RRs of the 2 exposure time windows were statistically homogeneous and marginally significant without substantial statistical heterogeneity. TABLE 2 Sensitivity Analysis Dose-Response Analysis For smoking before conception (Fig. 3), a total of 8 studies9,11,28–31,33,36 were included in the random-effects model by using RRs of the highest level of exposure; the synthesized RR was marginally significant (RR=1.33, 95% CI: 0.99-1.80) with substantial statistical heterogeneity (I2=58.8%, PCochrane=0.018). The 2-stage nonlinear curve (Fig. 4A) showed that the effect measure increased gradually as daily smoking consumption increased; however, it was not significant until the daily consumption reached 16 cigarettes/d, and the synthesized RR reached 2.0 at about 35 cigarettes/d. For smoking during pregnancy (Fig. 3), 5 studies10,28,30,33,37 were synthesized in the random-effects model by the same approach as mentioned before; the synthesized RR was statistically significant (RR=1.32, 95% CI: 1.09-1.60) with substantial statistical heterogeneity (I2=50.9%, PCochrane=0.086). The 2-stage nonlinear curve (Fig. 4B) showed a similar pattern as compared with Figure 4A; a significant effect measure was observed after 11 cigarettes/d, and it reached 1.4 at 20 cigarettes/d.

tatistically significant (RR=1.32, 95% CI: 1.09-1.60) with substantial statistical heterogeneity (I2=50.9%, PCochrane=0.086). The 2-stage nonlinear curve (Fig. 4B) showed a similar pattern as compared with Figure 4A; a significant effect measure was observed after 11 cigarettes/d, and it reached 1.4 at 20 cigarettes/d. FIGURE 3 Meta-analysis by synthesizing RRs of the highest smoking consumption. CI indicates confidence interval; cig/d, cigarette per day; PY, pack-year; RR, risk ratio. FIGURE 4 A, Dose-response curve by restricted cubic spline model (smoking before conception). The solid line is the fitted line, dash lines are the lines for 95% confidence interval, and dot line is the reference line. B, Dose-response curve by restricted cubic spline model (smoking during pregnancy). The solid line is the fitted line, dash lines are the lines for 95% confidence interval, and dot line is the reference line. cig/d indicates cigarette per day; RR, risk ratio. Publication Bias On the basis of the visual inspection of funnel plots and the results of Egger tests (Fig. 5), we observed evidence for publication bias for both groups. After trim and fill adjustment, the synthesized RR was 1.11 (95% CI: 0.96-1.28) for smoking before conception, and it was 1.19 (95% CI: 1.11-1.27) for smoking during pregnancy. FIGURE 5 Funnel plots, Egger tests, and trim/fill adjusted effect measures stratified by time windows. CI indicates confidence interval; RR, risk ratio.

Publication Bias On the basis of the visual inspection of funnel plots and the results of Egger tests (Fig. 5), we observed evidence for publication bias for both groups. After trim and fill adjustment, the synthesized RR was 1.11 (95% CI: 0.96-1.28) for smoking before conception, and it was 1.19 (95% CI: 1.11-1.27) for smoking during pregnancy. FIGURE 5 Funnel plots, Egger tests, and trim/fill adjusted effect measures stratified by time windows. CI indicates confidence interval; RR, risk ratio. DISCUSSION According to the results, paternal smoking before conception and during pregnancy were both associated with an increased risk of childhood ALL. This association was further examined and confirmed by sensitivity analysis and trim and fill adjustment as the estimate did not change too much after these adjustments. We also observed a monotonic nonlinear dose-response relation between paternal smoking and risk of childhood ALL in both time windows, and the risk was shown to be significant when daily smoking consumption was higher than a certain threshold of usage.

nt as the estimate did not change too much after these adjustments. We also observed a monotonic nonlinear dose-response relation between paternal smoking and risk of childhood ALL in both time windows, and the risk was shown to be significant when daily smoking consumption was higher than a certain threshold of usage. A recent systematic review and meta-analysis of this topic was updated in 2011. Liu et al38 came to the same conclusion as ours, and they reported that paternal smoking was positively associated with the risk of childhood ALL regardless of the exposure time windows. However, Liu et al38 did not utilize the linear or nonlinear model to investigate dose-response relation; instead, they utilized RRs of the highest smoking consumption in the meta-analysis when smoking was reported as an ordinal variable. Moreover, they did not conduct trim and fill to adjust for publication bias in meta-analysis. These issues were carefully considered and addressed in our study, which made our outcome more robust.

they utilized RRs of the highest smoking consumption in the meta-analysis when smoking was reported as an ordinal variable. Moreover, they did not conduct trim and fill to adjust for publication bias in meta-analysis. These issues were carefully considered and addressed in our study, which made our outcome more robust. The pathogenesis mechanism of childhood ALL is not fully understood, but it may be related to sperm DNA damage and oxidative stress.39,40 Jenkins et al39 reported smoking was associated with increased risk of the variance in sperm DNA methylation patterns, and it might explain the relationship between paternal smoking and the risk to child health. One of the major carcinogenic polycyclic aromatic hydrocarbons in tobacco smoke is benzo[a]pyrene, which can form DNA adducts in sperm cells and cause subsequent damage.41 According to the 2-hit hypothesis42 of neoplasm, carcinogenesis is more likely to be initiated during the early life cycle if the zygote with impaired germ-line DNA grows up.43 Thus, the impaired sperm cell DNA, after zygote formation, may increase the risk of childhood ALL among offspring. Benzene is another major carcinogen in tobacco smoke. A previous systematic review reported that exposure to excessive benzene during pregnancy could increase the risk of childhood ALL; Zhou et al44 reported a positive association (odds ratio=1.25, 95% CI: 1.09-1.45) between benzene and risk of childhood ALL by synthesizing data from 28 case-control studies. In addition, smoking can diminish the levels of antioxidants and cause excessive oxidative stress in germ-line cells, which can also increase the risk of gene mutation45–47; for example, Fraga et al46 reported that seminal alpha-tocopherol levels were decreased in smokers by ∼30% as compared with nonsmokers. In addition to smoking, Donkin and Barrès48 demonstrated that environmental factors may alter the phenotype of the next generation through remodeling of the epigenetic blueprint of spermatozoa.

le, Fraga et al46 reported that seminal alpha-tocopherol levels were decreased in smokers by ∼30% as compared with nonsmokers. In addition to smoking, Donkin and Barrès48 demonstrated that environmental factors may alter the phenotype of the next generation through remodeling of the epigenetic blueprint of spermatozoa. The human fetal liver serves as the primary organ for hematopoiesis during part of the embryonic period,49 which suggests that pathogenesis in fetal liver can be associated with subsequent risk of hematopoietic malignancies. Ning et al50 found a positive dose-response relationship between benzene levels in tobacco smoke and the frequency of micronucleus-containing polychromatic erythrocytes in blood from mouse fetal livers, indicating its potential harm to the hematopoietic system; also, DeMarini51 reported that the offspring of environmental tobacco smoke-exposed female mice exhibited increased levels of micronuclei in liver tissues and peripheral blood, and they also found that such exposure could increase the risk of sister chromatid exchanges in mouse fetal livers.

o the hematopoietic system; also, DeMarini51 reported that the offspring of environmental tobacco smoke-exposed female mice exhibited increased levels of micronuclei in liver tissues and peripheral blood, and they also found that such exposure could increase the risk of sister chromatid exchanges in mouse fetal livers. At the genetic level, overexpression or down-regulation of important fetal genes can be associated with the risk of childhood ALL. For example, Amson et al52 reported that human pim-1 proto-oncogene (PIM) was overexpressed in fetal liver and hematopoietic malignancies but not in normal human tissues; this suggested the possibility that carcinogens in tobacco smoke might increase the risk of subsequent hematopoietic malignancies by affecting the structure of PIM or influencing regulatory genes of PIM. By inspecting umbilical cord blood of infants whose mothers were exposed to passive smoking during pregnancy, Votavova et al53 detected the down-regulation of several genes that were associated with cellular defense responses and cellular immunity (GNLY, CD160, CD40, PRDM1, and SOCS3); this indicated that paternal smoking during pregnancy might affect the immune surveillance of the offspring, which could increase the risk of childhood ALL.

l53 detected the down-regulation of several genes that were associated with cellular defense responses and cellular immunity (GNLY, CD160, CD40, PRDM1, and SOCS3); this indicated that paternal smoking during pregnancy might affect the immune surveillance of the offspring, which could increase the risk of childhood ALL. Our study has several strengths. First, we searched articles in 4 electronic databases, which generated a broad scope. Second, we utilized a 2-stage nonlinear dose-response model to investigate dose-response relation, which has never been done in previous systematic review and meta-analysis. However, our study still has some limitations. First, leukemia carcinogenesis can be related to the synergistic effect of both parents smoking. However, none of the included studies adjusted for maternal smoking status in the multivariable model. Second, all of the included studies used case-control design, which could not examine the temporality; in addition, in case-control studies, recall bias was likely to be introduced when researchers used a self-report method to collect smoking data. Given that the cases’ parents were more likely to recall smoking status correctly than the controls’ parents, a differential measurement error was likely to be introduced in the original studies, which could bias the estimate. Third, smoking is a time-dependent variable, but case-control studies cannot measure the smoking prospectively and longitudinally as compared with cohort studies, which makes it difficult for us to consider the influence of smoking variation during pregnancy to the effective measures. More importantly, paternal smoking during pregnancy might not be the direct source of passive smoking or environmental tobacco smoke; for example, fathers and mothers might be at different geographic sites when fathers consumed tobacco, which could influence the actual exposure levels to mothers. Fourth, the approach used to determine the dose in 2-stage nonlinear model was conservative, as we did not know the distribution of each exposure level; thus, choosing the middle point or using the lower bound as a surrogate might lead to bias and imprecision to the pooled estimate. Fifth, only 2 of the included studies were conducted in Asia, and this makes our conclusion less generalizable to Asian population. Last, articles that are not in English were excluded, which may lead to relevant articles were omitted.

ower bound as a surrogate might lead to bias and imprecision to the pooled estimate. Fifth, only 2 of the included studies were conducted in Asia, and this makes our conclusion less generalizable to Asian population. Last, articles that are not in English were excluded, which may lead to relevant articles were omitted. In conclusion, we observe that paternal smoking before conception or during pregnancy is associated with an increased risk of childhood ALL; the dose-response analysis also confirms this relation and suggests the possibility of carcinogenesis threshold for paternal smoking. More large sample size cohort studies are needed in the future in which multiple measurements of smoking should be conducted. Our study has some public health implications; particularly, our results support the etiologic role of paternal smoking to childhood ALL, and this can make it justifiable for health practitioners to initiate health education or smoking cessation programs for potential fathers. Supplementary Material SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.jpho-online.com. The authors declare no conflict of interest.