ALBERT

Description: Background L-asparaginase is important in successfully treating childhood acute lymphoblastic leukemia (ALL). However, some patients experience hypersensitivity, mechanisms for which have not been fully elucidated. Previous studies, predominately based on populations of European ancestry, have described L-asparaginase hypersensitivity associations with genetic variant tagging the NFATC2, GRIA1, ASNS, and HLA-DRB1genes. The aim of this study was to evaluate the association between L-asparaginase hypersensitivity and genetic variants in Japanese childhood ALL patients. Methods This study included 472 Japanese children with ALL who received E. coli derived L-asparaginase comprising 6000 U/m2 intravenously or 10000 U/m2 subcutaneously according to the Tokyo Children's Cancer Study Group (TCCSG) L84-11, L89-12, L92-13, L95-14, L99-15, L04-16, and more recent protocols. L-asparaginase hypersensitivity was defined as experience of fever, rash, and other allergic reaction after L-asparaginase infusion. Through an ongoing genome-wide association study, patient DNA from saliva were genotyped using the Illumina platform and genome-wide single nucleotide polymorphism (SNP) imputation was performed using the 1000 Genomes Project Phase I Version 3 as the reference population. Tests of association between childhood ALL and SNP genotypes across the candidate loci, NFATC2, GRIA1, and ASNS, were performed using logistic regression and assuming a log-additive model of inheritance. Results We observed 47 (10%) hypersensitivity patients, of which 32 patients could not be infused again. These L-asparaginase hypersensitivities were not associated with patients' gender and age in this population. SNPs previously reported in NFATC2 rs6021191, GRIA1 rs4958351, and ANSN rs3832526 were not significantly associated with L-asparaginase hypersensitivity (P = 0.76, 0.99, and 0.53, respectively). For confirmation, the NFATC2 rs6021191 SNP was directly genotyped in a large subset of the ALL patients, but no association was observed (P = 0.44; 377 patients). Evaluation of other variants within those genes and flanking regions showed evidence of association with rs11482430 (NFATC2, P = 0.01), rs558550699 (GRIA1, P = 0.02), and rs7803055 (ANSN, P= 0.04), although not significant after correction for multiple testing. Conclusion This lack of association with variants reported in populations of European ancestry may be influenced by ethnic specific differences in genetic structure surrounding these variants as exemplified by differences in observed minor allele frequency of the reported NFATC2 rs6021191 and GRIA1 rs4958351 SNPs in Japanese (0.11 and 0.01) and Europeans (〈 0.01 and 0.36). Moreover, the type of L-asparaginase, and/or differences in therapeutic protocol may also contribute to inconsistencies with the previous reports. There is indication of an involvement of genetic variation of NFATC2 in L-asparaginase hypersensitivity, but whether the signal is representing the same regions as the previous reports needs to be further examined. Disclosures No relevant conflicts of interest to declare.

Description: Introduction 6-mercaptopurine (6-MP) is a main component of childhood acute lymphoblastic leukemia (ALL) therapy. The sensitivity of 6-MP is associated with genetic variant of 6-MP metabolism. Recently, the NUDT15 genetic variant has been identified as a risk factor of 6-MP intolerability, and its association with 6-MP-induced toxicities and 6-MP dose in ALL patients have been reported. The frequency of NUDT15 hypomorphic variant is higher in Asian populations than in European and African populations. However, the 6-MP tolerable dose and efficacy for NUDT15-deficient patients remains clear. Our study aimed to evaluate 6-MP tolerable dose, the frequencies of 6-MP induced toxicities, and outcome in 17 ALL patients with NUDT15-deficient genotype. Methods We genotyped NUDT15 genetic variants and evaluated the patients with NUDT15 homozygous variant in Japanese childhood ALL. The NUDT15 variants V18_V19insGV, V18I, R139C, and R139H were genotyped by Sanger sequencing, and the diplotype was precisely determined. The standard initiation dose of maintenance therapy was 6-MP 40 to 50 mg/m2/day and methotrexate 25 mg/m2/week. The 6-MP-induced toxicities were graded by CTCAE version 4.0. The survival rate was estimated by the log-rank test. Results A total of 17 patients with NUDT15 diplotype of *2/*2, *2/*3, *2/*5, *3/*3, *3/*5, and *5/*5 were genotyped as NUDT15 deficient. Fifteen patients were B cell-precursor (BCP) ALL and 2 patients were T-ALL. Of the 15 BCP ALL patients, 13 were standard risk and 2 were high risk patients according to National Cancer Institute/Rome criteria. Grade 3 leukopenia and grade 4 neutropenia were observed in all 17 patients, and the median observation time were 33 (range 3-95) days and 35 (20-137) days after initiating maintenance therapy, respectively. Grade 3 ALT elevation was observed in 6 patients (35%), and median observation time was 47 (range 19-427) days after initiating maintenance therapy. Moreover, during the early consolidation phase with 6-MP, severe myelosuppression was observed in 11 of these patients. The average 6-MP dose during maintenance therapy was 7.0 (range 2.7-18.3) mg/m2/day. Moreover, 16 of these 17 patients (94%) with NUDT15 deficiency required median 66 (range 5-376) days of therapy interruption. Notably, the average 6-MP dose was 18.3 mg/m2/day, and no therapy interruption occurred during maintenance therapy in patients with NUDT15 *5/*5 diplotype. Therefore, the degree of NUDT15 deficiency influenced 6-MP tolerable dose. The effect of NUDT15 deficiency on treatment outcome was evaluated in 14 patients, who completed treatment. Three patients relapsed at 124-388 days, and two of these three patients died at 877 and 959 days after the end of maintenance therapy, respectively. The overall and event-free survival rate at 4 years were 0.75 and 0.63, respectively. Neither the average 6-MP dose nor the interruption duration was associated with these events. Conclusions NUDT15-deficient genotypes strongly influence intolerability. Patients with NUDT15 deficiency did not tolerate standard 6-MP dose, and physicians should consider reducing 6-MP dose to 7 mg/m2 to avoid therapy interruption. Conversely, NUDT15 *5/*5 genotype displayed only mild NUDT15 deficiency, and the patients with this genotype tolerated 40% of the standard 6-MP dose. Further large-scale studies should be conducted to assess the NUDT15 variant's effect on survival. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: [Introduction] Acute megakaryocytic leukemia of Down syndrome (DS-AMKL) is characterized by excellent outcome with chemotherapy in contrast to non-Down syndrome-related AMKL (non-DS-AMKL). DS-AMKL and non-DS-AMKL have distinct genetic features which may underlie their different clinical characteristics. DS-AMKL is initiated by a GATA1 mutation in the transient abnormal myelopoiesis (TAM) phase and developed with further mutations of other regulators, while non-DS-AMKL is a heterogeneous group which occasionally carry chimeric oncogenes. CBFA2T3-GLIS2 fusion gene is identified in about 30% of children with non-DS-AMKL, and reported as a strong poor prognostic factor in pediatric AMKL. However, CBFA2T3-GLIS2 has never been reported in DS-AMKL and adult AMKL patients. We performed genomic analysis of DS-AMKL including atypical case with difficult clinical course. This is the first report of DS-AMKL harboring the CBFA2T3-GLIS2 fusion gene. [Case] The patient is a 1-year-old female of DS-AMKL with no prior episode of TAM. G-banding analysis revealed the karyotype both of the leukemic cells and normal tissue sample; 47, XX, +21. Chimeric genes of AML1-MTG8, CBFB-MYH, DEK-CAN, MLL-LTG4, MLL-LTG9, MLL-ENL and abnormalities of KIT and FLT3 were not detected. The chemotherapy according to the Japanese Pediatric Leukemia / Lymphoma Study Group AML-D05 protocol, gemtuzumab ozogamicin, IDA-FLAG regimen (idarubicin, fludarabine, cytarabine, filgrastim) and clofarabine-based regimen were tried, but all of them failed to achieve complete remission (CR). She underwent umbilical cord blood transplantation and relapsed on day 35 after transplantation. Once she showed a response to azacitidine, but finally she died on day 293 after transplantation. [Materials and Methods] We performed whole transcriptome sequencing (RNAseq), SNP array analysis, mutational analysis of GATA1 in 6 DS-AMKL samples, which included this refractory sample and five DS-AMKL samples with GATA1 mutations. To analyze gene expression profiling, we applied the hierarchical clustering method and principal component analysis. [Results] RNA sequencing analysis identified a fusion gene involving exon 10 of CBFA2T3 and exon 2 of GLIS2 gene in this refractory sample. This fusion gene was a result of a cryptic inversion on chromosome 16 and the in-frame fusion of both genes. The fusion transcript was validated by reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing. Though SNP array analysis confirmed 21 trisomy, it did not identify other copy number aberrations. PCR analysis did not detect GATA1 mutation in this refractory sample, which can be identified in other DS-AMKL samples. Expression analysis elucidated DS-AMKL with CBFA2T3-GLIS2 fusion had distinct expression profile from DS-AMKL with GATA1 mutations. [Discussion] CBFA2T3-GLIS2 fusion is the most common chimeric oncogene identified in non-DS-AMKL children, but has never been detected in DS-AMKL patients. Patients with non-DS-AMKL, especially holding CBFA2T3-GLIS2 fusion gene, have poorer outcomes than DS-AMKL. DS-AMKL patients generally have GATA1 mutations, show high sensitivity to chemotherapy, and can be treated with less intensive chemotherapy. However, our case had no GATA1 mutation and could not achieve CR despite intensive chemotherapy and transplantation. Thus, it is suggested this fusion gene caused the resistance to chemotherapies including hematopoietic stem cell transplantation in our case. Therefore, our case suggests patients with DS-AMKL should be surveyed genomic investigations including RNAseq and mutational analysis of GATA1 to identify their molecular biological subtypes before treatments are initiated. In case that fusion genes are detected in DS-AMKL patients, they must undergo highly intense chemotherapies, looking ahead to transplantation from the beginning of the treatment. Moreover, in case of harboring CBFA2T3-GLIS2 fusion gene, some potential therapies have been proposed, so that efficacy of such new therapies should be validated in a cell line-derived xenograft or patient-derived xenograft model. [Conclusion] DS-AMKL is generally known to show superior outcome, but DS-AMKL without GATA1 mutation and with CBFA2T3-GLIS2 fusion gene shows resistance to chemotherapies. For DS-AMKL patients, it is desirable to perform genomic analysis including RNAseq before chemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Background Response to induction chemotherapy is one of the most important predictors of outcome in acute myeloid leukemia (AML) as well as cytogenetics and molecular genetics. Measurement of minimal residual disease (MRD) by flow cytometry is an informative method for assessment of initial treatment response, but the heterogeneity of leukemia-associated antigens and antigen shifts during treatment limit its sensitivity and specificity. We prospectively evaluated the prognostic prevalence of MRD monitoring using multi-color flow cytometry in children with AML treated on the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Patients and methods From January 2007 to October 2010, 34 children with newly diagnosed de novo AML were enrolled on the AML-MRD study conducted by the Tokyo Children's Cancer Study Group. The median age at the diagnosis was 8 years (1 month- 15 years), and 17 patients were boys and 17 were girls. They were all treated with the JPLSG AML-05 trial. In AML-05, enrolled patients received two induction courses and those achieving a complete remission (CR) were treated according to risk classification at which all the core binding factor-AML cases and good initial response based on morphology after induction 1 were assigned to low risk (LR) group, cases presented with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), unfavorable cytogenetics including monosomy7, 5q-,t(16;21), and with poor initial response were assigned to high risk (HR) group, the others were assigned to intermediate risk (IR) group. Allogeneic hematopoietic stem cell transplantation (HSCT) was indicated for the HR patients after the third or later treatment courses. Among the 34 patients enrolled on the AML-MRD study, 8 presented with LR, 22 with IR, and 4 with HR regarding the cytogenetics and FLT3-ITD status. MRD of 63 bone marrow samples were analyzed after induction 1 (BMA-2) and induction 2 (BMA-3) by four-color flow cytometry using 9 AML-associated antigens. A threshold level for MRD-positivity was set at the point of 0.1%. Results Sixty-two (98.4%) of 63 bone marrow samples were evaluable for MRD. Thirteen (39.4%) of 33 samples and 8 (27.6%) of 29 showed MRD-positivity at BMA-2 and BMA-3 respectively. Among the patients with MRD-positivity, 12 at BMA-2 and 7 at BMA-3 were diagnosed as achieving CR by morphology. MRD was associated neither specific FAB subtype nor white blood cell count at diagnosis, but all 3 patients with FLT3-ITD showed the MRD-positivity at BMA-2. Although 3-year probability of event free survival (3-yr EFS) at BMA-2 or BMA-3 was similar between patients with and without MRD; 53.8% (n= 13) vs 70.0% (n= 20) (p=0.30) and 50.0% (n= 8)vs 62.0%(n= 21) (p=0.36), respectively, 3-yr EFS of those with MRD at BMA-2 or BMA-3 was significantly worse compared to those without MRD; 33.3% (n= 9) vs 83.3% (n= 12) (p=0.02) and 20.0%(n= 5) vs 76.9% (n= 13) (p=0.04), respectively, in the group of the IR cytogenetics and negative FLT3-ITD. Multivariate analysis indicated that the MRD detected by multi-color flow cytometry was solely associated with worse outcome in this group. Conclusion Highly sensitive detection of MRD by multi-color flow cytometry was possible in children with AML. The present study suggests that MRD monitoring may have a prognostic relevance in childhood AML with the IR cytogenetics and negative FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

Description: Development of prophylactic cranial irradiation (CRT) has greatly contributed to the improvement of outcome of childhood ALL. However, late complications, including secondary malignant neoplasms, arising from CRT are becoming big problems. Furthermore, the prognosis of the patients with relapse in central nervous system (CNS) after CRT is extremely poor. To eliminate CRT in the treatment for majority of the patients with B-cell precursor (BCP) ALL, we had attempted to reduce the indication of CRT by conducting randomizations in 3 studies. We present here the long-term results of 4 consecutive clinical trials, to assess the effect of reducing the proportion of patients who received CRT on the treatment outcome. Of the 2,116 children with newly diagnosed ALL with 1–18 years of age who were enrolled to the 4 consecutive TCCSG ALL studies between 1989 and 2003, 1845 BCP ALL patients were evaluable: 354 Pts in L89-12 protocol (1989–1992), 291 Pts in L-92-13 (1992–1995), 536 Pts in L95-14 (1995–1999) and 664 Pts in L99-15 (1999–2003). The probability of event-free survival (EFS) and overall survival (OS) were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. The initial status of CNS disease was examined after 7 or 10 days of pre-phase treatment consisted of steroid and/or other drugs. The definition of the CNS status was as follows: CNS positive = CNS-1 with symptomatic CNS disease, CNS-2, CNS -3 and traumatic lumbar puncture (TLP) with leukemic blasts; CNS negative = CNS-1 and TLP without leukemic blasts. In L89-12 study, all but a half of the standard risk group (SR) patients had received CRT. After this study, no CRT was given for the patients in SR. Patients in the intermediate risk group (IR) were randomly assigned into either high-dose methotrexate (HD-MTX) arm or CRT arm in L92-13 study. In L95-14 study, patients with initial leukocyte count exceeding 50,000/ul received CRT, whereas the others were treated with HD-MTX in IR. A part of patients in the high risk group (HR) in L95-14 study proceeded to stem cell transplantation during the first remission. In L99-15 study, only patients with high WBC, more than 100,000/ul, at diagnosis received CRT. Overall, no increase in CNS relapse was observed. The EFS rate and the OS rate at 8 years in L99-15 study were significantly improved compared to those in L89-12 study. Details of the results are shown in the table. By intensifying systemic chemotherapy and intrathecal injections, the proportion of the patients who received CRT in L99-15, which was the last study, was reduced to as small as 4% without increase in CNS relapse. These remaining 4% of the patients who had received CRT included those with CNS-3 disease (= 0.3 %), and they are the ultimate target who needs innovative treatment. In conclusion, we have succeeded to reduce the proportion of the patients with BCP ALL receiving CRT from 79% to 4% in the past 20 years, without compromising the treatment outcome. Table. Treatment and outcome in each study L89-12 L92-13 L95-14 L99-15 LP: lumbar puncture, IT: intrathecal injection, *randomization, **Pts with WBC higher than 50,000/ul at diagnosis received CRT. ***Pts with WBC higher than 100,000/ul at diagnosis received CRT. Rate of CNS positive 2.0% 3.4% 1.3% 2.1% Date of 1st LP/IT Day8 Day8 Day11 Day8 Rate of Pts receiving CRT 79% 36% 23% 4% Dose of CRT 18Gy 1y:12Gy, 2-18y: 18Gy 1y:12Gy, 2–18y: 18Gy 1–6y:12Gy, 7–18y: 18Gy CNS directed therapy SR*: A; HD-MTX 9g B; CRT IR: CRT HR: CRT SR: HD-MTX 6g IR*: A; HDMTX 6g B; CRT HR: CRT SR: HD-MTX 9g IR**:A;MTX9g B;CRT HR: CRT SR: HD-MTX 9g IR***: HD-MTX 9g (*HD-or LD-Ara-C) HR***: HD-MTX 6g Rate of Pts receiving SCT 0% 0% 15% 8% 8y-EFS 65+/−3% 55+/−3% 76+/−2% 74+/−2% 8y-OS 76+/−2% 79+/−2% 84+/−2% 86+/−2% Rate of BM relapse 25.6% 31.1% 17.6% 18.7% Rate of Isolated CNS relapse 2.6% 0.72% 1.2% 1.5% Rate of all CNS relapse 3.2% 1.4% 1.7% 3.2%