Children's Cancer Study Group Research Articles

The correlation of asparaginase enzyme activity levels after PEG-asparaginase administration with clinical characteristics and adverse effects in Chinese paediatric patients with acute lymphoblastic leukaemia.

Studies on asparaginase enzyme activity (AEA) monitoring in Chinese patients receiving PEG-asparaginase remain limited. We monitored AEA in paediatric patients diagnosed with acute lymphoblastic leukaemia (ALL) and treated according to the Chinese Children's Cancer Group study protocols, CCCG-ALL-2015/CCCG-ALL-2020 protocols. We measured the AEA at days 7 ± 1 and 14 ± 1 and analysed their association with patient characteristics and PEG-asparaginase-related adverse effects (AEs). We measured 2147 samples from 329 patients. Mean AEA levels (interquartile range) were 931 iu/L (654-1174 iu/L) at day 7 ± 1 and 664 iu/L (463-860 iu/L) at day 14 ± 1. The AEA levels were higher in younger children and increased with the cumulative dose numbers. PEG-asparaginase inactivation rate was 19.1%, and the silent inactivation (SI) rate was 12.5%. Nine patients were identified with allergic-like reactions. Hypofibrinogenaemia, hypertriglyceridaemia, pancreatitis and thrombosis were associated with older age, whereas hypoglycaemia was associated with younger age. The risk of hypertriglyceridaemia and hypoglycaemia increased with cumulative dose numbers of PEG-asparaginase. Except for hypofibrinogenaemia, elevated AEA levels did not increase the risk of PEG-asparaginase-related AEs. Drug monitoring can be utilized as guidance for treatment decision-making. Individualizing asparaginase doses do not reduce toxicities. The treatment target of PEG-asparaginase remains to achieve sustained and adequate activity.

Causes of death and treatment-related mortality in newly diagnosed childhood acute lymphoblastic leukemia treatment with Chinese Children's Cancer Group study ALL-2015.

To investigate the possible risk factors for death at post-treatment in children with acute lymphoblastic leukemia (ALL). A multivariate competing risk analysis was performed to retrospectively analyze the data of children with ALL who died after treatment with CCCG-ALL-2015 in China and to determine the possible risk factors for death at post-treatment in children with ALL. Age at the first diagnosis of ≥10 years; final risk level of high-risk; D19 minimal residual disease (MRD) (≥0.01%) and D46 MRD (≥0.01%); genetic abnormalities, such as KMT2A-rearrangement, c-Myc rearrangement, and PDGFRB rearrangement; and the presence of CNS3 (all P values, <0.05) were identified as independent risk factors, whereas the risk level at the first diagnosis of low-risk (LR) and ETV6::RUNX1 positivity was considered as independent protective factors of death in children with ALL. Among the 471 cases of death, 45 cases were treated with CCCG-ALL-2015 only, and 163 (34.61%) were treatment-related, with 62.42% due to severe infections. 55.83% of treatment-related mortality (TRM) occurred in the early phase of treatment (induction phase). TRM has a significant impact on the overall survival of pediatric patients with ALL. Moreover, the CCCG-ALL-2015 regimen has a better safety profile for treating children with ALL, with rates close to those in developed countries (registration number: ChiCTR-IPR-14005706; date of registration: June 4, 2014).

Report of Chinese Children's Cancer Group acute lymphoblastic leukemia 2015 multicenter study

Objective: To evaluate the outcomes and identify the prognostic factors of childhood acute lymphoblastic leukemia (ALL) treated with Chinese Children's Cancer Group study ALL-2015 (CCCG-ALL-2015) protocol. Methods: There were two randomization studies in CCCG-ALL-2015 study. A total of 7 640 newly diagnosed ALL patients in 20 hospitals of multi-institutional study group between January 2015 and December 2019 were treated with unified protocol. CCCG-ALL-2015 protocol featured risk-directed therapy based on morphology, immunophenotype, and genetic features, and adjusted according to minimal residual disease (MRD) assay on day 19 and day 46 of induction, which totally omitted prophylactic cranial irradiation. Two randomized controlled trails were designed. Children with Philadelphia chromosome-positive ALL (Ph+ALL) were randomly treated with dasatinib (Ph-D group) or imatinib (Ph-I group). During the latter half of continuation therapy, children were randomly treated with seven pulses of vincristine plus dexamethasone (group A) or not (group B). The survival rates of different groups were compared. Event-free survival (EFS) and overall survival (OS) curves were estimated according to the Kaplan-Meier method and compared by Log-rank test. Cox proportional hazards model was used for multivariate analysis. Results: Of the 7 640 enrolled patients, there were 4 521 males and 3 119 females, 7 508 (98.3%) entered complete remission. The 5-year EFS was 60.1% (95%CI 49.8%-72.5%) in the Ph-D group and 39.4% (95%CI 26.9%-57.7%) in the Ph-I group (χ2=5.00, P=0.020). Between patients with lower risk (LR) and intermediate and higher risk (I/HR) treated with (group A) or without (group B) additional pulse of vincristine plus dexamethasone, there were no difference in 5-year EFS and OS. The one-sided 95% upper confidence bound for the difference in 5-year EFS and OS were 0.02 and 0.01 for LR, 0.05 and 0.01 for I/HR, establishing non-inferiority, lower than 0.05. The follow-up time was 3.5 (2.4,4.8) years. The 5-year OS was 90.9% (95%CI 90.2%-91.7%), and EFS was 80.1% (95%CI 79.0%-81.2%). The 5-year cumulative risk of any relapse was 15.3% (95%CI 14.3%-16.3%).The cumulative risk of isolated central nervous system (CNS) relapse was 1.9% (95%CI 1.6%-2.2%), and any CNS relapse 2.7% (95%CI 2.3%-3.1%). The 5-year cumulative risk of death during remission was 1.3% (95%CI 1.0%-1.6%). In the multivariate analysis, the independent factors associated with inferior EFS of patients with B-ALL were age≥10 years, male sex, white blood cell count ≥50×109/L, CNS2 or CNS3 status, BCR-ABL fusion, KMT2A rearrangements, without ETV6-RUNX1 fusion and the presence of MRD>0.01% at day 19 or 46 (all P<0.05). Among patients with T-ALL, the significant predictors for lower EFS were the BCR-ABL1 fusion and MRD≥0.10% at day 46 (both P<0.05). Conclusions: Patients treated with CCCG-ALL-2015 protocol without the use of prophylactic cranial irradiation have favorable outcomes, and the cumulative risk of isolated CNS relapse and cumulative risk of death during remission are low. Intensive chemotherapy including dasatinib yield superior results compared to imatinib in the treatment of Ph+ALL. Vincristine plus dexamethasone pulses can be omitted for ALL children without decrease in treatment ontcome during the latter half of continuation therapy.

Prominence of NUDT15 genetic variation associated with 6-mercaptopurine tolerance in a genome-wide association study of Japanese children with acute lymphoblastic leukaemia.

Inherited genetic variation is associated with 6-mercaptopurine (6-MP) dose reduction and frequent toxicities induced by 6-MP. However, the tolerable dose for 6-MP is not fully predicted by the known variation in NUDT15 and TPMT among Asian children with acute lymphoblastic leukaemia (ALL). We performed a genome-wide association study (GWAS) related to 6-MP dose among Japanese children with ALL. This GWAS comprised 224 patients previously enrolled in Tokyo Children's Cancer Study Group clinical studies with replication attempted in 55 patients. Genome-wide single nucleotide polymorphism (SNP) genotypes were evaluated for association with average 6-MP dose during the initial 168 days of maintenance therapy. Possible associations were observed across five gene-coding regions, among which only variants at 13q14.2 were significant and replicated genome-wide (rs116855232, NUDT15, β=-10.99, p=3.7 × 10-13 ). Notable findings were observed for variants in AFF3 (rs75364948, p=2.05 × 10-6 ) and CHST11 (rs1148407, p=2.09 × 10-6 ), but were not replicated possibly due to small numbers. A previously reported candidate SNP in MTHFR was associated with higher average 6-MP dose (rs1801133, p=0.045), and FOLH1 (rs12574928) was associated in an evaluation of candidate regions (padjust =0.013). This study provides strong evidence that rs116855232 in NUDT15 is the genetic factor predominantly associated with 6-MP tolerable dose in children in Japan.

Malignant extracranial germ cell tumours: A first national report by the South African Children's Cancer Study Group.

To determine the overall survival (OS) and prognostic factors influencing outcomes in children and adolescents with malignant extracranial germ cell tumours (MEGCTs) in preparation for the development of a harmonised national treatment protocol. A retrospective folder review was undertaken at nine South African paediatric oncology units to document patient profiles, tumour and treatment-related data and outcomes for all children with biopsy-proven MEGCTs from birth up to and including 16years of age. Between 1 January 2000 and 31 December 2015, 218 patients were diagnosed with MEGCTs. Female sex (hazard ratio [HR] 0.284, p=.037) and higher socio-economic status (SES) (HR 0.071, p=.039) were associated with a significantly lower risk of death. Advanced clinical stage at diagnosis significantly affected 5-year OS: stage I: 96%; stage II: 94.3%; stage III: 75.5% (p=.017) and stage IV (60.1%; p<.001). There was a significant association between earlier stage at presentation and higher SES (p=.03). Patients with a serum alpha-fetoprotein (AFP) level of more than 33,000ng/ml at diagnosis had significantly poorer outcomes (p=.002). The use of chemotherapy significantly improved survival, irrespective of the regimen used (p<.001). The cohort demonstrated a 5-year OS of 80.3% with an event-free survival (EFS) of 75.3%. Stage, the use of chemotherapy and an elevated serum AFP level of more than 33,000ng/ml were independently predictive of outcome. The relationship between SES and outcome is important as the implementation of the new national protocol hopes to standardise care across the socio-economic divide.

The Epigenetic and Genetic Landscapes of Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionAlthough genetic basis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has been well characterized and we reported recurrent SPI1 fusion cases exhibiting uniformly dismal outcome, no comprehensive studies have yet explored the epigenetic profiles and their potential contribution to clinicopathological features of T-ALL.MethodsA total of 79 cases with pediatric T-ALL samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of childhood Leukemia Study (JACLS) including 7 cases of SPI1 fusions were analyzed in this study. To describe epigenetic landscape of T-ALL, we performed methylation array analysis using Illumina HumanMethylation EPIC array, and conducted combined analysis of methylation data with our previous data of whole transcriptome sequencing (WTS) and targeted-capture sequencing for 158 ALL-related genes.ResultsAfter normalization, 939 probes were selected to identify the most variable methylated probes, when a standard deviation of the beta-values across the samples was above 0.38. Unsupervised consensus clustering clearly identified 4 distinct sample clusters. Combined analyses with WTS data revealed that these 4 clusters were characterized by TAL1 fusions/high expression with intermediate methylation (M1 cluster; n=39), high TLX/HOX expression with intermediate methylation (M2 cluster; n=20), high TLX//HOX expression with high methylation (M3 cluster; n=11), and SPI1 fusions/high expression with low methylation (M4 cluster; n=9), respectively. Interestingly, these methylation clusters were well correlated with genetic features, expression status, and clinical outcomes. In M1 cluster, PTEN abnormalities and UPS7 mutations were especially enriched, and these aberrations were observed mutually exclusive. Furthermore, promoter regions in TAL1 and its target ALDH1A2, important for retinoic acid synthesis, were significantly hypomethylated and highly expressed in this cluster. In M2 cluster, genes related to chromatin remodeling and JAK-STAT were frequently mutated. PRR5L and DEPTOR, both related to AKT1 activation, were differentially methylated and expressed, suggesting an important role of PI3K-AKT1 pathway. M3 cluster was characterized by frequent mutations of epigenetic regulators related genes, such as PHF6, EZH2, and DNM2 . In contrast, mutations in JAK-STAT and PI3K-AKT1 pathways were less frequent. Since the methylation profile of M3 cluster was similar to normal T cells, abnormal DNA methylation status may be not crucial for the leukemogenesis in this group. Intriguingly, all SPI1 fusion cases were classified into M4 cluster, and remaining 2 cases without SPI1 fusions in this cluster showed very high SPI1 expression. Importantly, M4 cluster was characterized by high expression of RASGRP4, a family of Ras guanine nucleotide exchange factors, and low methylation of RASGRP4 promoter. In addition, FGFR1 expression was uniformly repressed and abnormalities of CDKN2A (89%) and KRAS/NRAS (44%) were frequently observed in this group. Furthermore, it should be noted that patients in M4 cluster showed significantly poor prognosis compared to other clusters; eight out of nine cases were died (Log-rank p =4.4×10-7). SPI1 fusion cases exhibited not only unique cytological and gene expression profiles but also distinctive methylation status.ConclusionsBased on DNA methylation profiles, pediatric T-ALL is clustered into 4 distinct subtypes, which exhibited remarkable correlation with genetic signatures, expression features, and clinical outcomes. PI3K-AKT pathway may be essential in M1/M2 cluster, whereas genetic abnormalities of epigenetic regulators were characteristics in M2/M3 clusters. In addition, the involvement of JAK-STAT pathway would be unique in M2 cluster. Given that cases with M4 cluster showed a catastrophic prognosis and very high expression of RASGRP4 with high frequency of RAS mutation, our findings implicate that targeting therapy of RAS may improve the prognosis of the patients in this group. Although our cohort is limited, our results suggested that the biological phenotype of T-ALL is mediated by both genetic and epigenetic regulations, and explorations for aberrant DNA methylation along with genetic alterations might be helpful for a new therapeutic strategy for T-ALL. DisclosuresNo relevant conflicts of interest to declare.

RNA Sequencing Illustrates the Genetic Backgrounds of Pediatric Acute Myeloid Leukemia

BackgroundThe cytogenetic heterogeneity of acute myeloid leukemia (AML) has been recognized for more than three decades, and some chromosomal aberrations and gene mutations have been adopted for the risk stratification. However, approximately 50% of patients have disease relapse. Thus, novel and accurate prognostic markers are needed to improve the risk stratification. We have performed some targetting sequencing and gene expression array, and identified that PR DM16 and MECOM -overexpressing patients had significantly worse overall survival (OS) and event-free survival (EFS). Thisoverexpression was especially helpful for stratifying patients with FLT3 -ITD positive and/or normal karyotype. To establish a more appropriate risk classification, we investigated the correlations among genetic aberrations, gene fusions, and gene expression, which are significant prognostic markers for pediatric AML, and proposed a new risk stratification. In addition, we performed the whole-transcriptome analysis to reveal the comprehensive understanding of 57 patients whose aberrant genetic backgrounds were unclear.Patients and MethodsThis study enrolled 369 patients with de novo AML who had been registered with the Japan Children's Cancer Group study, JPLSG, AML-05 trial between November 2006 and December 2010. Patients diagnosed with acute promyelocytic leukemia or Down syndrome-associated AML were excluded from this study. We investigated gene mutations in KIT, CEBPA, NPM1, FLT3 -ITD, and MLL- PTD, gene fusions of RUNX1-RUNX1T1, CBFB-MYH11, KMT2A (MLL)- rearrangements, NUP98-NSD1, CBFA2T3-GLIS2, NUP98-KDM5A, FUS-ERG, and DEK-NUP214, and gene expression analyses of PRDM16 and MECOM . Although most of these patients were classified into five risk-dependent subgroups based on their genetic aberrations, the genetic background of 57 patients (15%), particularly in patients with complex karyotypes, was unclear. Thus, we additionally performed the whole-transcriptome analysis for these patients to reveal their genetic backgrounds.ResultsWe could re-stratify 369 patients into five new prognostic subgroups based on the genetic aberrations. Because genetic backgrounds of patients with acute megakaryocytic leukemia (AMKL) were different from non-AMKL patients, we proposed another stratification scheme for 34 patients with AMKL. For 335 patients with AML, as the first step, super low and extreme high groups were created. The super low group (5-year OS, 94%; 5-year EFS, 84%) consisted of CBFB-MYH11, KMT2A-R with low MECOM expression, NPM1 mutation, and CEBPA biallelic mutations. Conversely, the extremely high group (5-year OS, 24%; 5-year EFS, 16%) consisted of FUS-ERG, MLL-AF6, DEK-NUP214, CBFA2T3-GLIS2, MLL -PTD, NUP98-NSD1, MLL -R with MECOM and/or PRDM16 high, and FLT3 -ITD with high PRDM16 gene expression. Then, we chose low and intermediate groups as the second step. The low group (5-year OS, 86%; 5-year EFS, 76%) consisted of RUNX1-RUNX1T1 without KIT, MLL -R with low MECOM and PRDM16, and the intermediate group (5-year OS, 85%; 5-year EFS, 58%) consisted of RUNX1-RUNX1T1 with KIT, FLT3 -ITD with low MECOM, and PRDM16, according to the retrospective EFS and OS. For the remaining 57 patients (15%) whose genetic background was unclear, non-complete remission (CR) patients were assigned to the extremely high group, and patients who achieved CR were assigned to the high risk group (5-year OS, 53%; 5-year EFS, 44%). We performed the whole-transcriptome analysis for these 57 patients and identified gene fusions of PICALM-MLLT10 in 3, RPN1-MECOM in 2, CREBBP-KAT2A in 3, RUNX1 -rearrangements in 2, CBFA2T3 -rearrangement in 2, RARB -rearrangement in 1, and ETV6 -rearrangements in 3. The whole-transcriptome analysis was used to reveal the genetic background of these patients, particularly in patients with a complex karyotype. For the remaining 34 patients with AMKL, patients with CBFA2T3-GLIS2 or NUP98-KDM5A were assigned to the extremely high group and others to the intermediate group.ConclusionWe propose a new risk stratification comprising five prognostic subgroups based on the genetic aberrations. The whole-transcriptome analysis facilitated further understanding of the whole genetic background of pediatric patients with AML whose genetic background was unclear and was, thus, expected to improve more accurate risk stratification. DisclosuresNo relevant conflicts of interest to declare.

Sex Cord Stromal Tumors in Children and Adolescents: A First Report by The South African Children's Cancer Study Group (1990-2015).

Pediatric sex cord stromal tumors (SCSTs) are extremely rare and there are no reported data from Africa. The authors evaluated the outcomes of children and adolescents with biopsy-proven SCSTs in preparation for the introduction of a national protocol. Retrospective data were collated from 9 South African pediatric oncology units from January 1990 to December 2015. Kaplan-Meier analysis was performed to estimate overall survival (OS) and event-free survival. Twenty-three patients were diagnosed with SCSTs, 3 male and 20 female individuals, during the study period. Histologies included 1 thecoma, 9 Sertoli-Leydig cell tumors, and 13 juvenile granulosa cell tumors. Stage I tumors predominated (n=14; 60.9%), with 2 stage II (8.7%), 5 stage III (21.7%), and 2 stage IV tumors (8.7%). The upfront resection rate was 91.3% with no reported surgical morbidity or mortality and an OS of 82.1%. Chemotherapy approaches were not standardized. Most children (81.8%), except 2, had recognized platinum-based regimens. Chemotherapy-related toxicity was minimal and acceptable. Assessment of glomerular filtration rate and audiology assessments were infrequent and not standardized. Three patients were lost to follow-up. Although the numbers in this cohort are small, this study represents the first national cohort in Africa. The 5-year OS of 82.1% was encouraging. Standardized management of rare tumors like SCSTs is critical to improve ensure OS and address potential long-term sequelae.

Distinctive Demography, Biology, and Outcome of Acute Myeloid Leukemia and Myelodysplastic Syndrome in Children With Down Syndrome: Children's Cancer Group Studies 2861 and 2891

AbstractIn recent pediatric trials of acute myeloid leukemia (AML), children with Down syndrome (DS) have had significantly more megakaryoblastic leukemia and have experienced better outcome than other children. To further characterize AML in DS, Children's Cancer Group Studies 2861 and 2891 prospectively studied demography, biology, and response in AML and myelodysplastic syndrome (MDS) of children with and without DS. These studies evaluated timing of induction therapy and compared postremission chemotherapy with marrow transplantation in 1,206 children. One-hundred eighteen (9.8%) had DS, a fourfold increase in 20 years. DS patients were younger, had lower white blood cell and platelet counts, more antecedent MDS, acute megakaryoblastic leukemia or undifferentiated AML, and an under-representation of chromosomal translocations (P < .001 for each variable). Four-year event-free survival in DS was 69% versus 35% in others (P < .001). Intensively timed induction conferred significantly higher mortality in DS patients; bone marrow transplantation offered no advantage. Conventional induction followed by chemotherapy achieved an 88%, 4-year, disease-free survival in DS patients versus 42% in others (P < .001). Megakaryoblastic leukemia was unfavorable in others but prognostically neutral in DS. AML in DS is demographically and biologically distinct from AML in other children. It is singularly responsive to conventional chemotherapy and may warrant even less therapy. The increasing proportion of DS patients with AML most likely reflects changes in attitudes about entering DS patients on AML trials and possibly increasing ability to distinguish megakaryoblastic leukemia from lymphoid leukemia.

Clinical Features and Treatment Outcome of Children With Biphenotypic CD2+CD19+ Acute Lymphoblastic Leukemia: A Children's Cancer Group Study

AbstractLeukemic cells from a subset of children with acute lymphoblastic leukemia (ALL) express lymphoid antigens of both T lineage and B lineage, but the clinical significance of this immunophenotype is unknown. We now report the first comprehensive comparison of treatment outcomes among a large cohort of children with CD2+CD19+ biphenotypic ALL (N = 77), B-lineage ALL (BL) (N = 1,631), or T-lineage ALL (TL) (N = 347) ALL who were treated on risk-adjusted Children's Cancer Group (CCG) protocols. CD2+CD19+ patients were more similar to BL than TL patients with respect to presenting features and antigen expression. The percentages of patients achieving successful induction therapy outcome were 98.7%, 97.8%, and 97.3% for CD2+CD19+, BL, and TL patients, respectively. Univariate comparisons of 4-year event-free survival (83.7%, 72.8%, 75.2% for CD2+CD19+, BL, and TL patients, respectively) achieved borderline significance (CD2+CD19+ B, P = .08; CD2+CD19+v T, P = .07). Relative hazard rate (RHR) estimates for BL and TL compared with CD2+CD19+ were 1.79 and 1.90, respectively, implying a better outcome for biphenotypic patients. However, multivariate adjusted RHRs for BL and TL compared with CD2+CD19+ were 1.43 (P = .29) and 1.16 (P = .76), respectively, suggesting a significant reduction in risk for BL or TL patients once adjustment was made for the more favorable characteristics of the CD2+CD19+ group. Thus, pediatric ALL patients treated on contemporary CCG protocols who present with CD2+CD19+ biphenotypic leukemia generally have good treatment outcomes, due in part to their favorable presenting features.

Clinical Features and Treatment Outcome of Children With Myeloid Antigen Positive Acute Lymphoblastic Leukemia: A Report From the Children's Cancer Group

AbstractLeukemic cells from a significant number of children with acute lymphoblastic leukemia (ALL) express protein antigens characteristic of both lymphoid and myeloid cells, yet the clinical significance of this immunophenotype has remained controversial. In the current study, we have determined relationships between myeloid antigen expression and treatment outcome in a large cohort of children with newly diagnosed ALL. A total of 1,557 children enrolled on risk-adjusted Children's Cancer Group studies were classified as myeloid antigen positive (My+) or myeloid antigen negative (My−) B-lineage ALL (BL) or T-lineage ALL (TL), according to expression of CD7, CD19, CD13, and CD33 antigens on the surface of their leukemic cells. My+ patients in both BL and TL groups were more likely than My− patients to have favorable presenting features. Induction therapy outcome was similar for My+ and My− patients in both the BL and TL categories. Importantly, 4-year event-free survival (EFS) was similar for My+ BL (77.0%, standard deviation [SD] = 4.0%) versus My− BL (75.9%, SD = 1.8%) and for My+ TL (72.7%, SD = 7.1%) versus My− TL (70.1%, SD = 5.7%). An overall relative hazard rate (RHR) of 0.89 (P = .49) was determined by a cross strata analysis for My+ versus My− patients. Moreover, similar EFS and RHR also were found when My+ and My− BL patients were compared according to National Cancer Institute risk classification. Thus, patients with My+ ALL have similar treatment outcomes as My− ALL patients. In contrast to previous studies, this result was independent of treatment risk category, demonstrating that myeloid antigen expression was not an adverse prognostic factor for childhood ALL.

Effects of Race and Ethnicity on Clinical Features, Tumor Genetics and Outcome in Children with KMT2A Rearranged Acute Myeloid Leukemia

Black and Hispanic children with AML tend to have worse outcomes compared to White non-Hispanic children. Potential contributing factors include higher acuity at presentation, higher infection related mortality, and less availability of hematopoietic stem cell donors (Winestone. Amer J Hematol. 2017; Aplenc. Blood 2006; Gramatges. PBC 2017). We hypothesized that differences in disease biology contribute to inferior outcomes along racial/ethnic lines, and these differences may be apparent among specific cytogenetic subsets. To test this hypothesis, we mined the publicly available TARGET AML dataset which includes 956 newly diagnosed children with AML enrolled on Children's Cancer Group study CCG-2961 or Children's Oncology Group studies AAML03P1 or AAML0531. There were 557 White, non-Hispanic (WNH), 115 Black (including six Black Hispanic), 170 non-Black Hispanic, 45 Asian, and 69 patients of other races included in our analysis. Overall survival (OS) was defined as time from study entry until death; event free survival (EFS) was time from study entry to first event (induction failure, death, death without remission, or relapse). Patients were censored at the date of last contact. We used the Kaplan-Meier method to estimate OS and EFS; the log-rank statistic tested survival differences. Pearson's chi-squared or Fisher's exact test was used to test for differences between proportions. Welch's t-test, ANOVA, and Wilcoxon rank-sum tests were used for continuous variables. Comparing tumor cytogenetics by race/ethnicity: when compared to WNH children, t(8;21) was more common among Black (OR=2.03; 95% CI: 1.20-3.42) and Hispanic (OR=2.04; 95% CI: 1.29-3.24) children. Black children were also more likely to have either -5/-5q or -7/-7q compared to WNH (OR=2.60; 95% CI: 1.29-5.23). We then interrogated KMT2A rearrangements (KMT2Ar), finding that there was no difference in the percentage of patients with a KMT2Ar on the basis of race/ethnicity. The distribution of various KMT2A fusions in Hispanic patients was not significantly different than WNH, but Black children were more likely to have t(6;11)(q27;q23) both overall (OR=5.78; 95% CI: 1.73-19.28) and among patients with KMT2Ar (OR 7.29; 95% CI: 1.94-27.40). Black KMT2Ar children were older than WNH and Hispanic KMT2Ar children (11.2 vs. 6.5 years, p=0.002). Hispanic children with KMT2Ar had a higher presenting white blood cell count (WBC median 114,700/µL) compared to WNH (median WBC 49,600/µL; p=0.01) and Black (38,300/µL; p=0.006) children. We then investigated survival: similar to prior studies, Black children had inferior EFS and OS compared to WNH (both p&amp;lt;0.0001). Hispanic and Asian children had similar survival to WNH children. However, the EFS/OS of both Black and Hispanic children with KMT2Ar were worse than WNH with KMT2Ar (Fig1A,B), though the reasons for poor outcome were different for Black and Hispanic children. Eight of 21 Hispanic children had induction failure compared to 0/19 Black and 1/85 WNH children (p&amp;lt;0.001), whereas Black children tended to suffer death or relapse as first event. Higher WBC at presentation did not explain the difference in outcome for Hispanic children: when we restricted our analysis to those with a presenting WBC &amp;gt;100,000/µL, Hispanic children still had an inferior EFS compared to WNH with a trend towards inferior OS (Fig 1C, D). The poor outcome of Black children with KMT2Ar may be driven by the higher proportion with the known poor prognostic t(6;11) and older age; but even when restricted to age &amp;gt;10yrs, Black children tended to have worse outcomes compared to WNH patients. In conclusion, using the TARGET data set, we identified differences in tumor genetics, clinical features and outcome by race/ethnicity. Specifically, among KMT2Ar patients, we found that Black and Hispanic children had significantly worse outcomes than WNH - but for different reasons with potential biologic implications. The difference in age and distribution of KMT2A fusion among Black children suggest distinct AML pathogenesis, while higher presenting WBC and increased induction failure risk among Hispanic children suggest that genetic factors may contribute to disease phenotype and response to therapy. In ongoing work, we are investigating racial/ethnic differences in tumor biology and pharmacogenomics to better understand the causes of poor outcome with the ultimate aim of eliminating long-standing survival disparities. Disclosures Rau: Jazz Pharmaceuticals, Inc.: Consultancy, Other: Travel Fees.

Impact of immunophenotypic characteristics on genetic subgrouping in childhood acute lymphoblastic leukemia: Tokyo Children's Cancer Study Group (TCCSG) study L04-16.

Immunophenotyping was performed in 1044 consecutive childhood acute lymphoblastic leukemia (ALL) patients enrolled in the Tokyo Children's Cancer Study Group L04-16 trial, revealing novel findings associated with genetic abnormalities. In addition to TCF3-PBX1 and MEF2D fusions, the CD10(+) subtype of KMT2A-MLLT3-positive ALL frequently exhibited the cytoplasmic-μ(+) pre-B ALL immunophenotype. Although ETV6-RUNX1 was significantly correlated with myeloid antigen expression, more than half of patients expressed neither CD33 nor CD13, while the CD27(+) /CD44(-) immunophenotype was maintained. Expression of CD117 and CD56 in B-cell precursor-ALL was limited to certain subtypes including ETV6-RUNX1 and KMT2A-MLLT3. Besides BCR-ABL1, CRLF2, hyperdiploidy, and hypodiploidy, CD66c was also expressed in Ph-like kinase fusion-, PAX5 fusion-, and DUX4 fusion-positive ALL, but not in MEF2D fusion-positive ALL, indicating constant selectivity of CD66c expression. In T-ALL, SIL-TAL1-positive patients were likely to exhibit a more mature immunophenotype. Expression of CD21 and CD10 was not rare in T-ALL, while lack of CD28 was an additional feature of early T-cell precursor-ALL. Considering the immunophenotype as a prognostic maker, MEF2D fusion-positive ALL with CD5 expression may be associated with a poorer prognosis in comparison with those lacking CD5 expression. In cases with characteristic marker expression, the presence of certain fusion transcripts could be predicted accurately.

An 8-year retrospective study of adult and paediatric Burkitt’s lymphoma at Tygerberg Hospital, South Africa

Background: Burkitt lymphoma(BL) is a high grade non-Hodgkin lymphoma, which may be underdiagnosed in South Africa, due to a high burden of infectious diseases such as HIV and TB which may present with similar clinical features. Aim: To describe demographics and clinico-pathological characteristics of patients diagnosed with BL. Setting: Tygerberg Hospital (TBH), South Africa between 2007-2014. Methods: We performed a retrospective descriptive and survival analysis of patients diagnosed with BL at TBH between 01 January 2007 and 31 December 2014 with at least 24-month follow-up. Data was collected from the Tygerberg Lymphoma Study Group database and the South African Children Cancer Study Group Tumour Registry. Results: There were 73 patients with BL, of whom 68 were admitted to TBH and whose data was further analysed. The majority of patients were adults (74%). There was a female predominance in adults and a male predominance in children ( p = 0.002). Various regimens were used in adults while a single treatment protocol was used in children. The proportion of patients with HIV and advanced BL was higher in adults than in children. The 2-year overall survival of the treatment group was 45%. The outcome of patients with BL in adults (34%) was poorer than that of children (69%) ( p = 0.022). HIV negative patients had a non-significant survival advantage (57%) over HIV positive patients with 41% 2-year overall survival ( p = 0.2876). Conclusion: This study demonstrates a better cure rate in children treated for BL compared to adults, with HIV-infection being a risk factor for poor outcome.

First person profile: Leslie L. Robison, PhD: A world-renowned epidemiologist, Dr. Robison played key roles in developing both the national Childhood Cancer Survivor Study and the St. Jude Lifetime Cohort Study.

First person profile: Leslie L. Robison, PhD: A world-renowned epidemiologist, Dr. Robison played key roles in developing both the national Childhood Cancer Survivor Study and the St. Jude Lifetime Cohort Study.

NUDT15 Variants Confer High Incidence of Secondary Malignancies of ALL in Children

Background: Secondary or subsequent malignant neoplasms (SMNs) are one of the most serious late complications in children and adolescents after treatment for acute lymphoblastic leukemia (ALL). Several studies have reported that excess dosing of 6-mercaptopurine (6-MP) therapy and polymorphisms of 6-MP metabolizing enzyme coding gene might influence the risk of SMNs. Recently, nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) variants have been identified as a cause of high sensitivity to 6-MP, which reflects a high sensitivity to 6MP in East Asians. Therefore, we investigated an association between NUDT15 genotype and incidence of SMNs in pediatric ALL. Methods: Germline samples including bone marrow samples, peripheral blood samples and buccal swab samples during remission were obtained from 13 cases who suffered from SMN after childhood ALL. The prevalence of major NUDT15 non-functional alleles (*2 and *3), which is categorized intermediate or poor metabolizer of 6-MP in Clinical Pharmacogenetics Implementation Consortium Guideline (Relling M et al. Clin Pharmacol Ther 2019), was evaluated by using Sanger sequencing and/or whole exome sequencing. The NUDT15 variants prevalence in SMN patients with primary ALL was compared to those without SMN, who were registered in the TCCSG L04-16 study, which was conducted by the Tokyo Children's Cancer Study Group (TCCSG). Results: Patients' profiles and NUDT15 genotypes were summarized in table 1. Median ages at diagnoses of ALL and SMNs were 4 (1-15) and 13 (5-44) years, respectively. Median duration between ALL and SMNs was 8 (1-30) years. SMNs included acute myeloblastic leukemia (AML) / myelodysplastic syndrome (MDS) (n = 8), brain tumor (n = 1), basal cell carcinoma (n = 1), bladder cancer (n = 1), Ewing sarcoma (n = 1) and osteosarcoma (n = 1). Among the 13 SMN cases, six patients (46.2%) possessed NUDT15 non-functional alleles. The SMNs of these cases included AML / MDS (n = 4), bladder cancer (n = 1) and osteosarcoma (n = 1). Among them, 3 patients, whose SMNs were AML, bladder cancer and osteosarcoma, were treated with radiation therapy against primary ALL. All NUDT15 genotypes were heterozygous variants (3 were *1/*2 and 3 were *1/*3) and seemed to be intermediate metabolizers. In patients without known SMNs in TCCSG ALL L04-16 cohort study, 73 of 437 (16.7%) possessed NUDT15 non-functional alleles. Three of them had bi-allelic variants (2 cases were *3/*3, 1 case was *2/*3), and the others were all heterozygous. The prevalence of NUDT15 non-functional alleles was significantly higher in SMNs compared to that of non-SMN cases (P = 0.012). Conclusion: Our study indicated the NUDT15 hypomorphic variant alleles could be a risk of SMNs after ALL treatment with 6-MP. NUDT15 heterozygotic variants carriers are generally tolerable of 6-MP, and are usually treated with normal or slightly low-dose of 6-MP. However, our results suggested that standard adjustment of 6-MP in maintenance therapy might cause potential overdose of 6-MP for cases with NUDT15 variants, leading to increased risk of SMNs. To certify it, a higher number of study samples and somatic samples analysis of 6-MP induced SMNs are required. To obtain the best long-term event-free survival probability, it might be necessary to reduce the risk of SMNs development without compromising therapeutic effect by further optimal adjustment of 6-MP dose (or alternative agents). Disclosures No relevant conflicts of interest to declare.

Association of Germline Variants of TCF3 and PAX5 with Pediatric Acute Lymphoblastic Leukemia Development

Background: The development of B-lymphocyte is controlled by the coordinated action of transcription factors networks, such as IKZF1, TCF3, EBF1 or PAX5. It is increasingly recognized that germline variants of those genes which disrupt their function are responsible for inborn errors of humoral immunity. Meanwhile, it is well understood genetic alterations of those molecules are frequently altered in B-lineage acute lymphoblastic leukemia (ALL) and are associated with leukemogenesis. However, those involvements are usually attributed to somatic mutation. Germline variations may also trigger cancer development in some cases. For instance, genome-wide association studies (GWAS) have identified B-cell precursor (BCP-) ALL susceptibility variants within IKZF1. Furthermore, germline variants of IKZF1, TCF3, and PAX5 are reported to be associated with B-ALL development. Therefore, we hypothesize that attenuated function due to germline variation in those genes may predispose to ALL development in Japanese population to some extent. Method: Saliva DNA from 583 pediatric ALL patients in remission registered in Tokyo Children's Cancer Study Group (TCCSG) were analyzed in this study. Targeted sequencing for exons and exon-intron boundaries of IKZF1, TCF3, and PAX5 was performed using next-generation sequencer. Frequency of the detected variant alleles was compared to data from the Exome Aggregation Consortium (ExAC) East Asian Cohort (n=4327), Tohoku Medical Megabank Organization (ToMMo) cohort (n=3554), and the Human Genetic Variation Database (HGVD) cohort (n=1208) by chi-square test or Fisher's exact test. Results: In total, 113 variants (28 nonsynonymous, 19 synonymous, 56 intronic, 8 untranslated regions, and 2 deletions) were detected. We could not detect any nonsynonymous variations, insertion or deletion in IKZF1. On the other hand, we detected 8 rare variations in TCF3 and 7 of them are found to be deleterious by in silico analysis. Among them, 1 variant, TCF3 p.S514L, was significantly more common in ALL patients (0.26% in patients vs 0% in ExAC, 0.01% in ToMMO and 0% in HGVD, p=0.0017, 0.0115 and 0.0343 respectively). In PAX5, we found 6 rare variations and 3 of them are found to be deleterious by in silico analysis, However, we could not identify a statistically significant difference in frequency among these variants between TCCSG and normal cohorts. On the other hand, a patient who harbored heterozygous PAX5 p.V26G, which had been reported as a somatic mutation in B-ALL, was diagnosed with primary antibody deficiency after treatment of B-ALL. He is receiving immunoglobulin replacement therapy without relapse of ALL for 15 years. Discussion: Our study implied TCF3 p.S514L is associated with ALL development. Besides, we identified a case of heterozygous PAX5 p.V26G variant who exhibited antibody deficiency and B-ALL. These findings suggest those rare variants impair the normal B-cell development and may predispose to ALL development. GWAS is effective in evaluating common genetic variants. However, it is hard to identify the genes involved Mendelian inheritance or functionally defective rare variants associated with disease susceptibility. Although it is rare, our study suggests that rare variants of genes involved in B cell development could be associated with ALL susceptibility and immunodeficiency, and we revealed the frequency of functionally defective rare variants in Japanese childhood ALL cohort. Disclosures No relevant conflicts of interest to declare.

NUDT15 Genetic Variation Is the Strongest Predictive Marker of Tolerance to 6-Mercaptopurine in Japanese Childhood ALL Patients: A Genome-Wide Association Study

Introduction In Asians, NUDT15 genetic variants have been reported to be associated with low tolerance to 6-mercaptopurine (6-MP). However, variation in 6-MP tolerance is still observed among patients without the NUDT15 variants. Some patients required high doses of 6-MP to obtain treatment effect. We performed a genome-wide association study (GWAS) in Japanese to confirm reported associations as well as to identify additional genetic loci associated with variation of 6-MP tolerance in childhood acute lymphoblastic leukemia (ALL) patients. Methods We included ALL patients enrolled into a Tokyo Children's Cancer Study Group (TCCSG) trial who started maintenance therapy by normal protocol dose (30-50 mg/m2/day). Whole genome single nucleotide polymorphism (SNP) microarray genotyping was performed followed by whole genome SNP imputation (Japanese reference genome). Data for over six million SNPs were available for analysis. Association analysis was performed adjusting for age at diagnosis and considering average 6-MP dose during 168 days from maintenance therapy initiation as the outcome. Results A total of 224 patients were included in the analysis. The median age at diagnosis was 4.7 (range = 0.9-15.7) years old and boys comprised 56.3%. The average 6-MP dose during the 168 days of maintenance therapy was 41.1 (range = 13.3-78.5) mg/m2/day. Variants representing 10 genomic regions showed potential association (P &amp;lt; 5 x 10-6) with average 6-MP dose, in which one locus was genome-wide significant (13q14.2, rs116855232). Variant rs116855232 is located in the previously identified NUDT15 gene and represents the strongest association with average 6-MP dose within our Japanese study (β = -11.45, P = 8.5 x 10-10). In contrast, the TPMT locus, which is predictive of 6-MP intolerance in populations of European ancestry, was not associated with 6-MP dose in Japanese. Among the suggestive loci requiring further follow-up is a locus residing in AFF3 (2q11.2, P = 2.1 x 10-6), a gene previously implicated in childhood leukemia. Conclusion We have validated that NUDT15 rs116855232 is a locus with strong association with 6-MP tolerable dose, and it currently represents the most meaningful clinically predictive marker in Japanese. These findings indicated that NUDT15 genotyping should be considered prior to initiation of 6-MP treatment. Prospects for the identification of additional loci of 6-MP tolerance are high after further validation of suggestive loci observed in this Japanese study. Figure Disclosures No relevant conflicts of interest to declare.

Is radiotherapy required in first-line treatment of stage I diffuse anaplastic Wilms tumor? A report of SIOP-RTSG, AIEOP, JWiTS, and UKCCSG.

As a significant proportion of relapses occurred in the tumor bed or abdomen on patients with the fifth National Wilms Tumor Study stage I anaplastic Wilms tumor (WT), flank radiotherapy was added for stage I anaplastic WT in the subsequent study of the Children's Oncology Group (AREN0321). Preliminary results revealed reduction of relapse rate and improved survival. In cases treated with preoperative chemotherapy, such as in International Society of Pediatric Oncology (SIOP), the value of radiotherapy has never been studied. The aim of this observational study is to describe the pattern of recurrence and survival of patients with stage I diffuse anaplastic WT (DAWT) after induction chemotherapy. Retrospective data analysis of the pattern of relapse and survival of all patients with stage I DAWT were included in recent SIOP, L'Associazone Italiana Ematologica Oncologia Pediatrica (AIEOP), Japan Wilms Tumor Study Group (JWiTS), United Kingdom Children's Cancer Study Group (UKCCSG) renal tumor registries. Postoperative treatment consisted of actinomycin D, vincristine, and doxorubicin for 28weeks without local irradiation. One hundred nine cases with stage I DAWT were identified, of which 95 cases received preoperative chemotherapy. Of these, seven patients underwent preoperative true-cut biopsy. Sixteen of the 95 patients relapsed (17%), six locally, four at distant site, and six combined, and all treated according to SIOP 2001 relapse protocol, which resulted in a 5-year overall survival of 93%. Despite 13% locoregional relapse rate, an excellent rescue rate was achieved after salvage treatment, in patients with stage I DAWT whose first-line treatment comprised three-drug chemotherapy (including doxorubicin), without flank irradiation. Therefore, we continue not to advocate the use of radiotherapy in first-line treatment after preoperative chemotherapy in stage I DAWT in the next SIOP protocol.

Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionNOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations.MethodsA total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS).ResultsIn total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern.Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors.Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations.ConclusionsWe observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.