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Haemophilia B patients with factor IX inhibitors have particularly unmet needs for conventional therapy.Phase II/III clinical trial, multicentre, open-label, prospective, self-controlled study was conducted to assess MC710 prophylaxis in haemophilia B patients with inhibitors.We enrolled haemophilia patients who had received episodic or prophylactic treatment with bypassing agents up to that time. The participants continued their conventional therapy for 24 weeks and then MC710 was prophylactically infused intravenously every 2 or 3 days at 60 to 120 μg as FVIIa per kilogram of body weight for 24 weeks. The primary endpoint was the annual bleeding rate (ABR) requiring bypassing agents, which was compared intraindividually between the conventional therapy period and the MC710 prophylaxis period.A total of 11 male haemophilia B patients were enrolled. The median ABR ratio for each participant (the prophylaxis period ABR divided by the conventional therapy period ABR) was .33 (2.1/6.5), range from .00 to 3.77. ABR ratios for 9 of the 11 patients ranged from .00 to .60, and 3 of the 9 patients had zero bleeding events during the prophylaxis period. Meanwhile, ABR ratios for the remaining two patients were 2.53 and 3.77, respectively. Although a fibrinogen decrease recovered by the dose reduction was reported for only one participant as the sole adverse drug reaction in this study, no thrombotic events or other safety concerns were reported.MC710 prophylaxis is considered to be decrease the bleeding rate in haemophilia B patients with inhibitors without safety concerns.

Background: The present study aimed to examine the effect of magnesium (Mg) supplementation on cisplatin-induced nephrotoxicity (CIN) in pediatric cancer patients. Methods: The present phase II, open-label, multicenter, randomized controlled trial enrolled patients aged less than 20 years who were scheduled to receive cisplatin-containing chemotherapy and randomly allocated them at a ratio of 1:1 to a Mg supplementation arm with even-numbered chemotherapy courses (arm AB) or another arm with odd-numbered courses (arm BA). Analysis objects were reconstructed into two groups depending on whether the chemotherapy course had Mg supplementation (group B) or not (group A). The primary outcome was the proportion of chemotherapy courses resulting in elevating serum creatinine per chemotherapy course. The secondary outcomes included efficacies evaluated using other biomarkers and the safety of the Mg supplementation. Results: Twenty-eight patients were randomly allocated to either group (16 to arm AB and 12 to arm BA). The baseline characteristics of the groups were similar. There was no significant difference in elevated serum creatinine between the groups (group A: 10% vs. group B: 6%; P=0.465), nor was any significant difference observed in other biomarkers during any chemotherapy course. The Mg value during chemotherapy was significantly higher in group B than in group A. No adverse events related to magnesium administration were observed. Conclusions: The study design, which treated a single chemotherapy course as a study object, failed to detect a statistically significant benefit of Mg supplementation for preventing CIN in pediatric cancer patients.

SMARCB1 is mutated in most rhabdoid tumors (RTs) developing in the kidney (RTK) and various other organs. Focal deletions found in patients with 22q11.2 deletion syndrome show breakpoints within clusters of segmental duplications (SDs), and those in some RTs show breakpoints in the 22q11-q12 region. SDs are known to cause focal deletion mediated by non-allelic homologous recombination. The present study identified SMARCB1 alterations in all 30 RTKs, using SNP array CGH, MLPA, and sequence analyses. Twenty-eight tumors had a total of 51 breakpoints forming focal 22q deletion and/or uniparental disomy (22qUPD), and the other two had compound mutation with no breakpoints in 22q. Twenty-four (47.1%) of the 51 breakpoints were within SDs, and occurred in 16 (53.3%) of the 30 tumors. The association of breakpoints with SDs was found not only in focal deletion, but also in 22qUPD, indicating that SDs mediate the first and second hits (focal deletion) and the second hit (22qUPD) of SMARCB1 alteration. Of the 51 breakpoints, 14 were recurrent, and 10 of the 14 were within SDs, suggesting the presence of hotspots in the 22q11.2 region. One recurrent breakpoint outside SDs resided in SMARCB1, suggesting inactivation of the gene by out-of-frame fusion. The association between SDs and focal deletion has been reported in two other types of cancer. RTKs may be the third example of SD-associated tumors. Thus, the present study indicated that RTKs exploit genomic instability in the 22q11.1-11.2 SDs region, and 22qUPD caused by mitotic recombination may also be mediated by SDs. This article is protected by copyright. All rights reserved.

[Background] The prognosis of patients with metastatic Ewing sarcoma family of tumors (ESFT) remains poor. [Procedure] We retrospectively analyzed 57 patients diagnosed with metastatic ESFT between 2000 and 2018 to identify prognostic and therapeutic factors affecting the clinical outcome. [Results] The 3-year overall survival (OS) rate of the entire cohort was 46.8% (95% confidence interval [CI], 33.0-59.4%). Treatment-related death was not observed. Multivariate analysis identified stem cell transplantation (SCT), response to first-line chemotherapy, and bone metastasis as independent risk factors for OS. Objective response rate to first-line chemotherapy was 65.1% in the 43 evaluable patients. There was no significant difference in the response to different types of first-line chemotherapy. Among patients with lung metastasis alone, the 3-year OS rate was higher in 13 patients who received local treatment than in four who did not, although the difference was not significant. [Conclusions] One possible reason for the high OS rates was the absence of treatment-related mortality even in patients receiving SCT, which could be attributed to advances in the management of post-SCT complications. Novel first-line chemotherapy strategies need to be established to improve the disease status prior to SCT in a higher proportion of patients.

BACKGROUND The survival rate in patients with Ewing sarcoma family of tumors (ESFT) in Japan was reported to be

t(17;19)(q22;p13) translocation, which generates an E2A-HLF chimeric transcription product, is rarely found in childhood precursor B-cell acute lymphoblastic leukemia (ALL). This type of ALL is cha...

To evaluate haematopoietic stem cell transplantation (HSCT) in children and adolescents, we reviewed the records of 47 patients who were ≤18 years, had relapsed or refractory anaplastic large cell lymphoma, and received HSCT between 1990 and 2010. At HSCT, complete remission (CR) was less common in allogeneic HSCT recipients (n = 24) than in autologous HSCT recipients (n = 23) (P = 0·01). The autologous and allogeneic HSCT groups differed in terms of 5-year event-free survival (EFS) (38% vs. 50%, P = 0·63), cumulative incidence of progress or relapse (49% vs. 28%, P = 0·25), and treatment-related mortality (12% vs. 25%, P = 0·40). However, these differences were not significant. Patients with non-CR at autologous HSCT had a significantly lower EFS rate (14% vs. 48%, P = 0·03). Conversely, although those with non-CR at allogeneic HSCT had a lower EFS rate, this was not significant (44% vs. 63%, P = 0·26). Reduced-intensity conditioning regimens were used for three of the 16 allogeneic HSCTs received by patients with non-CR. These three patients achieved CR, surviving 32-65 months after HSCT. These results demonstrated that allogeneic HSCT might be a treatment option for patients who do not achieve CR through conventional chemotherapy.

Background Cyclosporine A (CsA) is used widely for graft-versus-host disease (GVHD) prophylaxis in hematopoietic stem cell transplantation (HSCT); however, the optimal schedule of its administration has not been established. Although comparative studies of adult patients undergoing HSCT have demonstrated enhanced efficacy and safety of twice-daily infusion (TD) compared with continuous infusion (CIF) of CsA, to our knowledge, similar studies have not yet been performed in pediatric groups. Procedure A self-administered questionnaire was used to retrospectively compare the clinical outcome and incidence of CsA-associated adverse events of 70 pediatric acute myelogenous leukemia patients who were receiving CsA by TD (n = 36) or CIF (n = 34) as GVHD prophylaxis for their first allogeneic HSCT. Results The cumulative incidences of grade II–IV acute GVHD and chronic GVHD, as well as the overall survival and event-free survival rates, did not differ significantly between the TD and CIF groups; however, the incidence of severe hypertension was significantly higher in the CIF group than the TD group. Conclusions The analysis presented here indicates that TD and CIF administration of CsA have similar prophylactic effect on pediatric GVHD and suggest that TD is associated with a lower rate of toxicity than CIF in pediatric patients undergoing HSCT. Pediatr Blood Cancer 2015;62:291–298. © 2014 Wiley Periodicals, Inc.

Total body irradiation is performed as a preconditioning regimen to inhibit graft-versus-host disease after bone marrow transplantation and to eradicate remaining tumor cells. However, these regimens result in delayed secondary sex characteristics and failure of ovarian function recovery, leading to amenorrhea and infertility. Herein, we report a case of an 11-year-old girl diagnosed with acute lymphocytic leukemia who received induction chemotherapy and prophylactic cranial irradiation. For bone marrow transplantation, she received total body irradiation of 12 Gy with uterine and ovarian shielding at 13 years of age. The patient remained in remission and menarche began at 14 years of age. At 23, she became pregnant and delivered a baby naturally with no abnormalities.

Introduction Recombinant thrombomodulin (rTM), which degrades factors Va and VIIIa by activating protein C, has been developed as a new drug for treating disseminated intravascular coagulation (DIC). Materials and methods Since July 2009, we have treated 25 children with DIC using rTM (380 U/kg/day, or 130 U/kg/day for newborns) as a first-line therapy. Median duration of rTM administration was 5 consecutive days (range, 2–13 days). We employed DIC criteria of the Japan Welfare and Health Ministry. The first day on which rTM treatment was given was defined as day 1. Results Median patients age was 3 years. Underlying diseases were hematological disorders (n = 13) and severe infection (n = 12). Overall, 20 of the 25 patients had recovered from DIC by day 7 and 22 of the 25 patients remained alive at day 28. Median Pediatric Logistic Organ Dysfunction score improved from 11 on day 1 to 2 on day 7 (p = 0.009). Laboratory data (median) on day 7 (prothrombin time (PT) ratio, 1.15; fibrin and fibrinogen degradation products (FDP), 9.6 mg/l; D-dimer, 1.6 mg/l FEU; antithrombin, 112%; protein C, 105%) were significantly improved compared to results on day 1 (PT ratio, 1.39; FDP, 21.6 mg/l; D-dimer, 6.4 mg/l FEU; antithrombin, 86%; protein C, 54%). Whereas, 5 patients failed to respond and serious bleeding events were observed in 2 newborns. Conclusion The efficacy of rTM cannot be assessed from the present dataset, due to several limitations such as the small heterogenous patient cohort, and the lack of age- and disease-matched controls. Nevertheless, this case-series remains important in terms of enabling further prospective control studies to evaluate the efficacy of rTM in children.

Purpose Perifosine is an alkylphospholipid analog that inhibits or modulates signaling through signal transduction pathways such as Akt, which is enhanced in neuroblastoma (NB) by activation of tyrosine kinase receptors. We conducted a phase I study of perifosine in Japanese patients with recurrent or refractory NB. Experimental Design All patients enrolled were over 2 years of age; all had refractory or relapsed NB and a performance status of greater than 50%. Perifosine was orally administered at a loading dose (100–300 mg) on day 1 and at a maintenance dose (50–150 mg) from day 2 onward. Dose-limiting toxicity (DLT) and pharmacokinetics were assessed in Step 1 and safety and efficacy in Step 2. Results Nineteen patients were recruited. No DLT was observed. Adverse reactions occurring in more than 30% of the patients were vomiting (63%), nausea (53%), and diarrhea (37%). The mean plasma concentration of perifosine was 27.5 ± 9.8 μM on day 15 and 27.3 ± 11.5 μM on day 29. The response rate (RR) in 18 patients evaluable according to modified International Neuroblastoma Response Criteria was 0%; the disease control rate (DCR) was 56%. Median progression-free survival (PFS) was 122 days. In 11 patients evaluable according to the Response Evaluation Criteria in Solid Tumors, the RR and DCR were 9% and 55%, respectively. The median PFS was not reached. Conclusions Perifosine monotherapy was well tolerated in Japanese patients with recurrent/refractory NB. Further investigations in combination with other anticancer or molecular targeted agents are warranted.

In 1996, the Japan Wilms Tumor Study (JWiTS) group was founded to elucidate the efficacy and safety of the regimen established by the National Wilms Tumor Study (NWTS) group in the USA, and a multicenter cooperative study (JWiTS-1) was started in Japan. This report reviews the results of JWiTS-1.A total of 307 patients with malignant renal tumor were enrolled in the JWiTS-1 study between 1996 and 2005. Central pathological diagnosis and follow-up data were available in 210 cases. The protocol regimens were similar to the NWTS-5 regimens. Clinical stage was classified according to the Japanese Staging System.Five-year overall survival (OS) rate was 91.1% for nephroblastoma, 72.9% for clear cell sarcoma of the kidney (CCSK), and 22.2% for rhabdoid tumor of the kidney (RTK). In the nephroblastoma patients, 5-year OS was 90.5% for stage I disease, 92.2% for stage II, 90.9% for stage III, 86.7% for stage IV, and 78.7% for stage V.The OS of patients in the JWiTS-1 study were comparable with the results of other multicenter studies in the USA and Europe. The outcome for patients with nephroblastoma and CCSK was fair. In contrast, the cure rate for those with RTK was not satisfactory. New treatment strategies are needed for patients with RTK.

PURPOSE Three familial cases of each of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for neutrophil elastase (Ela2) gene mutation. The contents of the neutrophil-specific granule proteins cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were also analyzed in SCN. METHODS Genomic DNA was extracted from the patients' peripheral blood or bone marrow, and the coding sequence of the Ela2 gene was amplified by polymerase chain reaction and subjected to direct sequencing. The contents of antimicrobial peptides were analyzed by flow cytometry. RESULTS Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN (W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case was a novel mutation of Ela2. Prophylactic treatment for growth factors or antibiotic prophylaxis against bacterial infection was useful for lowering the frequency of infectious episodes. Adult patients tended to have less frequent infections compared with minors in the same family. The contents of both cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were significantly reduced in SCN compared with healthy controls. CONCLUSIONS Prophylaxis by growth factor or antibiotics is useful for decreasing risks of bacterial infections in SCN and CN. Adults were likely to have less frequent infections than children in familial cases of SCN and CN with the same mutation of Ela2.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood. There are well-recognized prognostic factors in NB such as age at diagnosis, organ of origin, stages, MYCN gene amplification, and expression of H-ras, trkA and survivin. Moreover, we investigated the expression of vascular endothelial growth factor (VEGF), tyrosine hydroxylase (TH), p53, stem cell factor (SCF) and c-kit of its receptor with quantitative real-time polymerase chain reaction (PCR) in 22 NBs and 4 other tumors (one malignant lymphoma, one malignant teratoma, and 2 rhabdomyosarcomas) samples. The correlation between patients' prognoses and the expression of TH or c-kit was newly recognized, particularly the good prognosis in patients in whom c-kit highly expressed and the poor prognosis contrarily associated with low or no expression, although the SCF of its ligand had no relationship with patient prognosis. It is possible that tumors without c-kit expression can not react with SCF (via the autocrine or paracrine system) and remain immature. It may be that this is a new critical clinical event in NB patients.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (20 x 10(9)/l), 15 showed hemoglobinor =8 g/dl and 14 showed platelet countor =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Malignant rhabdoid tumor of the kidney (MRTK) is a rare but highly aggressive tumor in children, and knowledge about the molecular signature of this tumor is limited. We report the molecular genetic alterations and gene expression profile of an MRTK tumor that arose in a 4-month-old Japanese girl. Fluorescence in situ hybridization and Southern blot analyses revealed a homozygous deletion of an approximately 0.29-Mb genomic region bordered by the Rgr and DDT genes in these tumor cells. This deleted region encodes SMARCB1, a candidate tumor suppressor gene for MRTK. Using a high-density oligonucleotide DNA array, we found increased expression of 25 genes, including genes involved in the cell cycle (10 genes), DNA replication (3 genes), cell growth (5 genes), and cell proliferation (5 genes), in this MRTK tumor sample, compared with a noncancerous kidney (NK) sample. On the other hand, 64 genes, including 4 genes regulating apoptosis, were found to show decreased expression in this MRTK tumor sample, compared with the NK sample. Among these alterations, we found alterations of expression of some genes, such as IGF2, MDK, TP53, and TNFSF10, in this MRTK tumor, as described previously. The molecular genetic alterations and altered pattern of gene expression found in this case may have contributed to the biological characteristics of the MRTK tumor that arose in our patient.

Two cases of hepatoblastoma with unique karyotypic changes are described. One case was that of a 2-year-old boy with an unbalanced chromosomal translocation involving 4q35 as the sole chromosomal abnormality. The clonal karyotype of this tumor was 46,XY,add(4)(q35)[3]/46,XY[9]. In the other case, that of a 2-year-old boy, karyotypic analyses revealed the clonal karyotype as 57,XY,+del(1)(p22),+2,+5,+6,+7,+8,+del(12)(p12),+18,+19,+20,+22[4]/46,XY[12]. Review of these two cases, together with previous reports, underscored the significance of numerical and/or structural chromosomal abnormalities of 1q, 4q, 2, 8, and 20 in the development of hepatoblastoma. The present results show that imbalance of the terminal region of 4q could be the sole chromosomal abnormality in a hepatoblastoma. We also found that imbalance of chromosomal regions on chromosomes 1 and 12 may contribute to the development of hepatoblastoma.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood, and among all childhood malignancies is second in prevalence only to leukemia. In NB we need to both make an accurate diagnosis and rapidly analyze the expression of genetic prognostic factors such as MYCN, H-ras, and trkA. Moreover, it has recently become important to analyze the expression of survivin mRNA, a member of the inhibitor of apoptosis protein family. Expression of the survivin gene is related to tumorigenesis and inhibition of apoptosis in some malignant tumors. We investigated its expression by reverse transcription-polymerase chain reaction (RT-PCR) in NB cell lines (SK-N-SH, NB-39, and IMR-32), two normal blood cell samples, and 13 clinical NB tumor samples. All three NB cell lines had high levels of mRNA expression for this gene, but normal blood cells had no expression. We detected expression of survivin mRNA in 7 of the 13 NB tumor samples (54%). Two NB patients were in stage I disease, 6 in stage II, and 5 in stage IV(A). Quantitative analysis by RT-PCR revealed that the ratio between survivin mRNA and human glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH) mRNA was very low in stages I and II (0-0.017). In contrast, in advanced NBs (stage IV(A)) the ratio was much higher (0-0.050). The prognoses of the three patients in the advanced stage who had high ratios of expression were poor. A high level of expression of survivin mRNA indicates a high grade of malignancy, high likelihood of recurrence, and poor prognosis.

The authors describe a 15-year-old boy with hepatosplenic γδ T-cell lymphoma associated with hemophagocytic syndrome (HPS) along with isochromosome 7q and trisomy 8. He presented with prolonged fever, mild anemia, thrombocytopenia, and hepatosplenomegaly. Physical examination, radiography

Ewing's sarcoma (ES) is one of the most malignant bone and soft tissue tumors in childhood. Morphologically, ES belongs to the small round cell tumors (SRCT). ES, peripheral primitive neuroectodermal tumor (PNET), and Askin's tumor are classified as ES family tumors (ESFT) because they share a common chromosomal translocation. The EWS-FLI1 chimeric gene is generated by t (11; 22). Other reciprocal translocations resulting in formation of chimeric genes between EWS and ETS family genes (ERG, ETV1, E1AF, and FEV) are t (21; 22), t (7; 22), t (17; 22), and t (2; 22), respectively. Although it is generally difficult to distinguish ES from SRCT, we could easily and quickly distinguish ES from other SRCT by using reverse transcription polymerase chain reaction (RT-PCR). We looked for specific chimeric genes in 23 tumor samples, including three ES clinical samples. We detected five chimeric genes in the three ES samples. Three chimeric genes, all EWS-FLI1, were detected in one ES sample. Different chimeric genes, EWS-ERG and EWS-ETV1, were detected in the other two ES samples. Moreover, because we could not detect specific chimeric genes in samples from non-ESFT, it may be possible to use this technique to diagnose ESFT and to detect tumor cell contamination before hematopoietic stem cell transplantation.

The biological behavior of neuroblastomas detected through mass screening (MS,/=1 year of age) and that of mass screening-negative later-presenting (MSN,1 year of age) neuroblastomas have been reported to differ in many studies. To investigate the biological differences between these two groups, we analyzed the differences in mRNA profiles.We analyzed the mRNA profiles of MS and MSN neuroblastomas using differential display, and cloned and sequenced the bands differentially expressed between these two groups. Using the RNA analysis by polymerase chain reaction (RNA-PCR) method, the relative amount of mRNA in tumor tissue in each sample was measured. Associations between relative amount of mRNA and clinical and genetic variables related to patient prognosis and the effect of the level of mRNA expression on survival probability were investigated using statistical methods.Using differential display and RNA-PCR, we found that the mRNA for the human homologue of the yeast cdc10 gene (hCDC10) identified in Saccharomyces cerevisiae was expressed at a higher level in the MS group of patients than in the MSN group of patients (0.554 +/- 0.197 for MS neuroblastoma, n = 24 and 0.244 +/- 0.179 for MSN neuroblastoma, n = 10, P0.01), and this difference was suggested to be independent of the histologic subtype of tumor. A high level of hCDC10 mRNA expression in neuroblastomas (relative amount of hCDC10 mRNA0.35) was also suggested to be associated with younger age at diagnosis (/=1 year of age, P0.01), favorable clinical stage (I, II, and IVs, P0. 01), and favorable histology in the Shimada classification (P0. 01), whereas a low level of hCDC10 mRNA expression (relative amount of hCDC10 mRNA/=0.35) was suggested to be associated with the progression of clinical stage (P0.01) and N-myc gene amplification (1 copy, P0.05). Patients with neuroblastomas with a high level of hCDC10 mRNA expression were suggested to have a better prognosis than those with a low level of hCDC10 mRNA expression (P0.01).A high level of hCDC10 mRNA expression in neuroblastomas may be associated with favorable clinical and biological characteristics, and the expression of hCDC10 mRNA in neuroblastomas may affect the clinical and biological characteristics of this type of tumor.

Two cases of fetal hepatoblastoma with unique karyotypic changes are described. One was a 17-month-old boy with multiple unbalanced chromosomal translocations, resulting in four types of derivative chromosomes involving chromosomal loci at 1q21, 1q32, 2q23, 6q27, 7p22, and 21p12, partial tetrasomy of 1q, partial trisomy of 2q, and partial monosomy of 21p. The clonal karyotype of this tumor was 46,XY,der(2)t(1;2)(q32;q37), der(6)t(1;6)(q12;q27), der(7)t(2;7)(q23;p22), der(21)t(2;21)(q23;p12). In the other case, a 4-year-old girl, karyotypic analyses revealed trisomy 2 and 8, and the clonal karyotype of this case was 48,XX, + 2, + 8. Review of these cases together with previous reports suggested the significance of chromosomal changes including numerical abnormalities of 1q, 2 (or 2q), 20, and 8 (or 8q), and breakage of 1q and 2q in the development of hepatoblastoma. The results presented herein underscore the significance of numerical abnormalities of chromosomal regions 1q and 2q and of chromosome 8 in the development of hepatoblastoma, in addition to abnormalities of 6q27, 7p22, and 21p12 ∼ 13 as other chromosomal loci that may be responsible for the pathogenesis of this embryonal type of tumor.

The main purpose of the present study was to determine the response rate to immunosuppressive therapy combined with recombinant human granulocyte-colony stimulating factor (rhG-CSF) and its efficacy for preventing infections in patients with severe aplastic anemia. The treatments included one course of antithymocyte globulin, cyclosporin A, methylprednisolone, danazole and rhG-CSF. Three patients had very severe aplastic anemia and two had moderate aplastic anemia. One patient relapsed 13 months following the first course of therapy and received a second course. Five patients received six courses of treatment and the response rate at 6 months was 83.3%. All patients achieved an absolute neutrophil count of greater than 1.0 x 10(9)/L within 40 days. All patients with a complete response are transfusion-free and doing well. All five patients are currently alive and have not had any episode of infection for 17-53 months. The results of the study indicate that this therapy may improve the poor prognosis of young patients with severe aplastic anemia. It has a good response rate and induces a rather rapid increase in the neutrophil count, which protects against life-threatening bacterial and fungal infections.

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49-84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.

BACKGROUND: Minimal residual disease (MRD) level after induction (Time Point1:TP1) and before consolidation therapy (Time Point2:TP2) has a strong impact in prediction of outcome for childhood acute lymphoblastic leukemia and it has clinical utility of a prognostic factor to stratify the risk groups in many current studies. We measured MRD levels on various time points to evaluate their prognostic significance in MRD-based augmented therapy. PATIENTS & METHODS: From June 2004 to September 2009, we prospectively assigned 333 consecutive children with ALL, 1〜19 years of age, to receive one of three treatment protocols on the stratification based on National Cancer Institute (NCI) risk criteria. NCI standard risk:SR, NCI high risk:HR and those with a white blood cell count≧1000x109 per L was defined as high-high risk:HHR. Patients were stratified again at TP2, patients with MRD level over 10-3 were assigned to salvage arm, treated in augmented therapy. Among 333 patients, 326 were eligible and 245 were MRD quantifiable. Then, we re-evaluated those samples by RQ-PCR according to the guideline of Euro MRD. Finally 167 cases were analyzed at 7 time points of MRD quantification in the CCLSG ALL2004 study. RESULTS: The overall 5-year event free survival (5-EFS) rate for ALL2004 was 82.5±2.1% (n=326), and 5-EFS of SR group (n=267), HR group (n=86) and HHR group (n=33) were 84.8±2.5%, 80.1±4.3% and 74.7±7.8%, respectively. In the SR group, 5-EFS of standard therapy group (n=124) with TP2 MRD TP1, at week 7, at the end of induction, 5-year relapse free survival (5-RFS) was 87.4.% in MRD negative cases (n=115) and 58.3% in MRD positive cases (n=36), and the differences were significant. TP2,week13, during consolidation, and TP3, at week 19 or 22, and at TP4, at week 26 to 28, had significant difference on 5-RFS with cut off of both 10-3 and 10-4. TP7, at the end of therapy, 2 patients were MRD positive and were thought molecular relapse. Additionally, we investigated changes in MRD levels of relapsed cases (n=35). MRD remained cases (n=4), MRD late disappeared cases (n=4), and MRD converted to positivity cases (n=4) were observed. CONCLUSIONS: The impact of predictive power of PCR MRD at TP1 and TP2 was validated, still more TP3 and TP4 were also statistically significant in CCLSG ALL2004 study. Even the patients whose final MRD level was negative could relapse, it is needed to investigate other prognostic factor except MRD. Disclosures Imai: Juno Therapeutics: Patents & Royalties.

Background The majority of minimal residual disease (MRD)-positive patients with acute lymphoblastic leukemia (ALL) have poor outcomes. The ALL2000 study was performed to evaluate the efficacy of augmented chemotherapy based on MRD-restratification in childhood ALL. Procedure Between 2000 and 2004, 305 eligible patients with precursor B or T-cell ALL were enrolled in the ALL2000 study. The ALL941-based therapy protocol utilized PCR MRD assays using Immunoglobulin and T-cell receptor gene rearrangements. They were initially stratified into three risk-groups according to leukocyte count and age, and MRD levels were measured at weeks 5 (TP1) and 12 (TP2) for a second stratification. From week 14, patients with MRD levels received an increase in therapy (one risk group higher), while the remainder continued to receive the initial risk-adapted therapy. Results The overall 5-year event-free survival (EFS) rate for ALL2000 was 79.7 ± 2.4%. MRD stratification was feasible for 234 of 301 patients (77%) who achieved complete remission. The EFS rate of the MRD stratifiable (MRD) group was 82.5 ± 2.6%, considerably superior to the 74.7 ± 5.7% of MRD non-stratifiable (Non-MRD) group (P = 0.084) and the 74.4 ± 2.1% for ALL 941 (P = 0.012). MRD-positive patients at TP2 showed inferior outcomes as compared with MRD-negative cases, but the difference did not reach a statistically significant level in any risk groups or immunophenotypes. Conclusions These results suggest that augmented therapy for MRD-positive patients at TP2 contributed to better outcomes of the ALL2000 study. Pediatr Blood Cancer. 2010;55:1287–1295. © 2010 Wiley-Liss, Inc.

Neuroblastoma (NB), which is a malignant tumor of young children derived from neural crest cells that occurs in children, exhibits a wide range of clinical behaviors, from spontaneous regression to rapid progression. Advanced NB patients have a poor prognosis, and recently, autologous bone marrow transplantation (BMT) and autologous peripheral blood stem cell transplantation (PBSCT) have been attempted to improve the prognosis of these patients. In this study, we attempted to detect the expression of tyrosine hydroxylase (TH), neuroendocrine protein gene product (PGP) 9.5, ELAVL-4 and GD2 synthetase (GALGT), all of which are highly expressed in NBs, by the reverse transcription-polymerase chain reaction (RT-PCR) technique in order to detect minimal residual disease (MRD) in the bone marrow (BM) and peripheral blood (PB). Analysis of various tumor cell lines (Ewing's sarcoma, hepatoma, leukemias, and breast cancer cell lines in addition to NBs), and human normal samples (BM and PB cells) revealed that TH was the most specific marker for the detection of NB. On the other hand, PGP9.5 was the most sensitive marker, and was detected even when there was only one positive cell per 10(7) negative cells. We concluded that TH is a better marker before the diagnosis of NB while PGP9.5 is a better marker to detect MRD after the diagnosis. Here, we describe our results on useful markers to detect MRD in patients with NB.

Ewing's sarcoma (ES) is one of the most malignant tumors of bone and soft tissue in children and young adults. ES belongs to a group of small round cell tumors (SRCTs) that also includes neuroblastoma, rhabdomyosarcoma, and malignant lymphoma. However, ES exhibits several specific chimeric genes (EWS-FLI1, EWS-ERG, EWS-ETV1, EWS-E1AF, and EWS-FEV) caused by chromosomal translocations that are not shared by other SRCTs. These chimeric genes regulate the expression of various other genes; that is, they activate inhibitors of DNA binding 2 (Id2) gene expression or they suppress transforming growth factor beta II (TbetaRII) receptor gene expression. The regulation of these chimeric genes may affect critical cell signal transductions, such as signals involved in cell cycle and apoptosis in ES tumor cells. Using an antisense oligodeoxynucleotide against a sequence containing the ATG initiation codon of the EWS-FLI1 chimeric gene that specifically reacts with the EWS-FLI1 and EWS-ERG chimeric genes, we were able to regulate the cell cycle through the down-regulation of Id2. Here, we report that treatment with an antisense oligodeoxynucleotide against this chimeric gene was very useful for inducing the regression of ES tumor growth; thus, this chimeric gene may be an important target for the treatment of ES patients.

The purpose of this study is to evaluate the efficacy of intraoperative radiation therapy (IORT) and the problem of securing the IORT field in advanced pediatric neuroblastoma. Between 1996 and 2005, 12 children received IORT for advanced pediatric neuroblastoma patients. Electron beam energies ranged from 10 to 12 MeV and median dose was 10 Gy (8-12 Gy). All of them had surgery with IORT against the primary tumor site and the abdominal aorta surroundings. A gross total resection (GTR) was achieved in 10 patients and subtotal resection (STR) was two patients. All of 12 patients were classified as high risk. Nine patients were alive 17-120 (mean 48 months) after diagnosis. Local tumor control was achieved in 100% of patients, of whom one experienced local recurrence outside the IORT field. At the operation, it was difficult to secure the IORT field because of the angle of the radiation cylinder in three patients. One of the three of these patients experienced local recurrence outside of the IORT field in the upper side of superior mesenteric artery and two of three patients had an external beam radiation after surgery, and there was no local recurrence. One patient had a postoperative ileus, and one patient had transient diarrhea and hydronephrosis. For advanced neuroblastoma patients, IORT produced excellent local control after surgery. However, there is a problem of securing the IORT field. For local control, it is necessary to add an external beam radiation after IORT when it is difficult to secure the IORT field.

The outcome of patients with Ewing Sarcoma Family of Tumors (ESFT) in our country is poor in comparison with American and European. We performed a review of the relevant data regarding the current status in the treatment of ESFT. The results of the major American and European cooperative groups were discussed, including the treatment strategies used and the prognostic factors identified for both localized and metastatic ESFT. We also discussed a new trial for the establishment of the standard treatment of ESFT in our country.

Hematopoietic stem cell transplant (HSCT) has been performed on patients with Hunter's syndrome. If applied, evaluation of recovery in various organs is needed for long-term follow-up. However, it remains unclear whether HSCT is effective against the neurological involvement in Hunter's syndrome, and morphological evaluation of recovery is inconsistent. We observed the degree of cutaneous nerve involvement in patients with Hunter's syndrome ultrastructurally before and after HSCT. Electron microscopic studies revealed that membrane-bound clear vacuoles were still observed in the cytoplasm of endoneurial fibroblasts and Schwann cells 2 months and 2 years, respectively, after HSCT. On the other hand, only a few vacuoles were present in dermal fibroblasts at 2 months after HSCT, and these disappeared within 2 years. These results suggest that the persistence of clear vacuoles in endoneurial fibroblasts and Schwann cells indicates a disturbed internal condition in the endoneurium 2 years after HSCT. Skin biopsies can be used in patients with Hunter's syndrome to study peripheral nerves for long-term follow-up to evaluate morphological efficacy.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood and, among all childhood malignancies, is second only to leukemia. NB originates before birth in the neural crest, which develops into the adrenal medullae and sympathetic ganglia. In the adrenal medulla, tyrosine hydroxylase (TH) is the first enzyme in the pathway of catecholamine synthesis. We used reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of TH mRNA in NB and Ewing's sarcoma cell lines, small round cell tumors (SRCTs) containing NB, and other clinical tumor samples (osteosarcoma, osteochondroma, and Wilms' tumor). In total, we analyzed 33 clinical tumor samples. TH mRNA was expressed in all three NB cell lines examined, but not in two ES cell lines or in a breast cancer cell line. We detected TH mRNA in 23 of 25 NB tumor samples (92%), but in none of the SRCTs or other clinical tumor samples. This RT-PCR technique showed a sensitivity for TH mRNA of one NB cell per 10(5) negative cells. Based on these results, the detection of TH mRNA is very useful both as a tumor marker for NB and for detecting minimal residual disease. Therefore, we can use this method to detect tumor cell contamination before hematopoietic stem cell transplantation.

To determine the minimum levels of l-asparaginase (ASNase) activity necessary to maintain l-asparagine (Asn) depletion under ASNase treatment in acute lymphoblastic leukemia (ALL). We measured ASNase activity using an enzyme coupling method with a limit of detection of 2 U/l and examined the relationship between ASNase activity and Asn levels in blood samples from 14 children with ALL. In all but one patient showing high ASNase antibody titers, minimum ASNase activity to maintain Asn depletion levels below the limit of detection (40 ng/ml) ranged from 6 to 180 U/l with a median value of 16 U/l. In 11 patients, the enzyme activity corresponding to minimum detectable Asn levels ranged from 2 to 32 U/l with a median value of 6.5 U/l. Patients with an ASNase activity of 2 U/l or an undetectable activity (

Ewing's sarcoma (ES), most commonly an undifferentiated tumor of bone, belongs to the enigmatic diagnostic category of small round cell tumors (SRCT) of childhood. The consistent presence of the translocation t (11; 22) in the vast majority of tumors provides evidence for a common histogenesis in ES and its family of tumors (ESFT), and also provides a unique diagnostic characteristic to discriminate this tumor family from SRCT. Molecular analysis of this translocation has revealed that it forms a chimeric gene between EWS on chromosome 22 and FLI-1 on chromosome 11. Similarly, the variant t (21; 22), t (7; 22), t (17; 22), and t (2; 22) rearrangements also form chimeric genes between regions of EWS and the ETS gene family (ERG, ETV1, E1AF, and FEV). Detection of these specific chimeric genes would provide a method for diagnosis of ESFT. We have developed a procedure for simultaneous detection of the chimeric genes by reverse transcription polymerase chain reaction (RT-PCR) with a mixture of primers. We conclude that the detecting those chimeric genes by this method can be easy and useful for diagnosis of ESFT. Moreover, by defining the specific chimeric gene it is possible to detect the tumor cell contamination in autologous blood stem cell transplantation.

To the editor: The placental syncytiotrophoblast layer is a barrier that protects the fetus from infection and cancer metastasis. Even if mother-derived cells enter the fetus, they are usually rejected and cannot be engrafted. Materno-fetal transmission of leukemia/lymphoma is therefore extremely

The recurrent translocation t(10;11) is associated with acute myeloid leukemia (AML). The AF10 gene on chromosome 10 at band p12 and MLL at 11q23 fuse in the t(10;11)(p12;q23). Recently, we have identified ABI1 as a new partner gene for MLL in an AML patient with a t(10;11)(p11.2;q23). The ABI1 is a human homologue of the mouse Abl-interactor 1 (Abi1), encoding an Abl-binding protein. The ABI1 protein exhibits sequence similarity to homeotic genes, and contains several polyproline stretches and a src homology 3 (SH3) domain. To clarify the clinical features of t(10;11)-leukemias, we investigated 6 samples from acute leukemia patients with t(10;11) and MLL rearrangement and detected MLL-AF10 chimeric transcripts in 5 samples and MLL-ABI1 in one. The patient with MLL-ABI1 chimeric transcript is the second case described, thus confirming that the fusion of the MLL and ABI1 genes is a recurring abnormality. Both of the patients with MLL-ABI1 chimeric transcript are surviving, suggesting that these patients have a better prognosis than the patients with MLL-AF10. To investigate the roles of AF10 and ABI1 further, we examined the expression of these genes in various cell lines and fresh tumor samples using the reverse transcriptase-polymerase chain reaction method. Although AF10 was expressed in almost all cell lines similarly, the expression patterns of ABI1 were different between leukemia and solid tumor cell lines, suggesting the distinctive role of each isoform of ABI1 in these cell lines. We also determined the complete mouse Abi1 sequence and found that the sequence matched with human ABI1 better than the originally reported Abi1 sequence. Further functional analysis of the MLL-AF10 and MLL-ABI1 fusion proteins will provide new insights into the leukemogenesis of t(10;11)-AML.

Background and Objectives Recombinant thrombomodulin (rTM), a new type of natural anticoagulant, has been available in Japan since 2008 for the treatment of disseminated intravascular coagulation (DIC). Since limited information is available on its efficacy in children, we compared the efficacy of rTM to that of conventional treatment. Patients and Methods Of 41 children with DIC, 21 were treated with rTM as a first-line therapy (rTM group) and 20 weere treated with conventional treatment (control group). The day on which anti-DIC treatment was first given was defined as day 1. We used the DIC criteria of the Japan Welfare and Health Ministry. We evaluated the overall survival at day 28 after anti-DIC treatment, the cumulative incidence of DIC resolution, and the total fresh frozen plasma (FFP) and platelet transfusion volumes during the course of DIC. Results Clinical outcomes are summarized in the Table. Both groups showed comparable overall survival (95.2% in the rTM group and 95.0% in the control group). In addition, the cumulative incidence of DIC resolution in the rTM group (90.5%) was not different from that in the control group (70.0%) (p=0.210) (Figure). However, the median FFP and platelet transfusion volumes in the rTM group (0 U/kg and 0.28 U/kg) were lower those in the control group (0.14 U/kg and 0.76 U/kg) (p=0.041 and 0.063, respectively). Conclusion The use of rTM in children with DIC should reduce the social and ethical problems as well as the biological risks associated with human-derived blood products. Although the limited advantages of rTM were shown, the present study was limited by the small and heterogeneous patient population. Therefore, larger prospective studies are warranted to fully evaluate the efficacy of rTM. Disclosures: No relevant conflicts of interest to declare.

9574 Background: Survival rate of ESFT in Japan was reportedly less than 50% in 1990s. The JESS started in order to improve this condition by conducting clinical trials for ESFT in Japan. Methods: This is a phase II, non-randomized trial to test safety and efficacy of multidisciplinary treatment for non-metastatic ESFT. Age under 30 is eligible. Chemotherapy regimens are alternating vincristine 1.5 mg/m2 on day 1, doxorubicin 37.5 mg/m2 on days 1 and 2, cyclophosphamide 1200 mg/m2 on day 1 (VDC) and ifosfamide 1800 mg/m2 on days 1 through 5 and etoposide 100 mg/m2 on days 1 through 5 (IE) repeating every 21 days for 52 weeks. Local treatment includes surgical resection and/or radiation therapy, of which dosage varies from 0 to 55.8 Gy based on margin of the resection and pathological response. Primary endpoint is 3 year progression free survival (PFS). Statistical design was made to test whether the lower limit of 90% confidence interval (CI) of PFS will exceed the threshold of 60%. Planned sample size is 53 patients including 10% ineligible patients. Results: Fifty-three patients were registered from December 2004 to May 2008. Among the 53 patients, 7 patients were judged ineligible after the registration (metastatic disease 1, changed diagnoses 6). Forty six patients were considered as per protocol set and used for efficacy analyses. Three year PFS is 71.7% (90% CI: 0.59 – 0.81). Estimated 5 year PFS and OS are both 69.6%. Radiographic response rate (RR) in evaluable 38 patients is 71.1% and pathological RR in 25 is 52.0%. In safety data, hematological toxicities are significant such as grade 4 neutropenia observed in 98%. Although there is no previously unknown adverse event reported, there are three patients who experienced secondary malignancies (ALL 1, MDS 1, osteosarcoma 1). Conclusions: A multi-disciplinary treatment comprising standard multi-agent chemotherapy with vincristine, doxorubicin, cyclophosphamide, ifosfamide and etoposide improved outcome of Ewing sarcoma family of tumor in Japan although statistical confirmation of absolute efficacy was not successful.

Abstract 2554 Aims: Children with Ph+ALL generally have a poor prognosis when treated with chemotherapy alone. The timing and duration of the use of imatinib has not been determined. We investigated a role of imatinib immediately before HSCT. Methods: All the patients with ALL were screened for diagnosis of Ph+ALL using RT-PCR. Children with Ph+ALL were enrolled on JPLSG Ph+ALL04 Study within 1 week of initiation of treatment for ALL. Treatment regimen consisted of 5 therapeutic phases: Induction phase (5-drug induction), Intensification phase (high-dose cytarabine and BFM Ib), Re-induction phase (4-drug re-induction), 2 weeks of Imatinib monotherapy phase (23 weeks after diagnosis), and HSCT phase (Etoposide+CY+TBI conditioning). Before and after each phase, minimal residual disease (MRD), the amount of BCR-ABL transcripts, was measured with the real-time PCR method (cut-off 50 copies/microgram RNA). The study was registered in UMIN-CTR (Medical Information, University hospital Medical Information Network - Clinical Trials Registry): UMIN ID C000000290. Results: During the period 2004–08, 42 patients were registered in the Ph+ALL04 study. Out of 42 patients, 37 patients (88%) achieved CR and 7 of 37 patients also achieved MRD-negative after induction phase. There were 13 patients who had no MRD at the beginning of imatinib monotherapy phase, and 14 patients were MRD-negative after imatinib phase, consequently, 14 patients were MRD-negative at the time of HSCT. Six patients relapsed before HSCT. In total, 31 patients received HSCT in 1st CR. All the patients had engraftment and no patients died because of complications of HSCT. Five patients relapsed after HSCT and 4 of the 5 patients were MRD-negative before HSCT and the other patient had detectable MRD although it was less than 50 copies. Twenty-six patients continue to be in 1st CR and MRD-negative for median of 3 years after diagnosis. The 3-year event-free survival rate and over-all survival rate for all the patients was 57% and 80%, respectively (figure 1). Five patients did not achieve CR after induction phase and they were treated with imatinib-contained chemotherapy. Four of the 5 patients achieved CR. All of the 4 patients received cord blood transplantation and remains in continued CR. Interpretation: The chemotherapy we employed was based on the previous high-risk regimen of TCCSG (Tokyo Children's Cancer Study Group) L-99-15 Study. The chemotherapy was intensive enough to induce MRD-negative in 13 at the time of imatinib phase and 31 of 42 patients were in CR at the time of HSCT (around 25–28 weeks after diagnosis). We planned to assess the efficacy of imatinib immediately before HSCT but it was not possible because of the low amount of MRD in most patients at the beginning of imatinib phase. Conclusion: Although EFS and OS was excellent in this study, 88% of induction rate appeared unsatisfactory and relapse occurred before HSCT in 6 out of 37 patients who achieved CR after induction phase. Earlier and longer use of imatinib may improve EFS in children with Ph+ALL and HSCT may be omitted in a subset of patients who achieve an early and deep remission status. Disclosures: No relevant conflicts of interest to declare.