Hyperdiploid Acute Lymphoblastic Leukemia Research Articles

Mutational Landscape and Temporal Evolution during Treatment of Relapsed Acute Lymphoblastic Leukemia

▪IntroductionRelapsed childhood acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and has poor prognosis due to acquired drug resistance. However, the clonal evolution leading to drug resistance at ALL relapse is incompletely understood.MethodsWe performed whole-genome sequencing (WGS) of samples at diagnosis and relapse from 103 Chinese patients, most of whom were enrolled on the Shanghai Children's Medical Center (SCMC) ALL2005 frontline treatment protocol. We also performed ultra-deep sequencing at 5,000-50,000X coverage of 211 serial bone-marrow samples from 17 of these patients (3-23 per case) collected during ALL therapy. Fifteen ALL subtypes were identified based on copy number variation, structural re-arrangement and gene fusion by WGS and RNA-Seq analysis. Cases were selected based on sample availability with no bias on age, gender, cytogenetics, or immunophenotype compared to the characteristics of all relapsed cases at SCMC; however only one CRLF2-rearranged case was observed suggesting that CRLF2-rearrangement could be a rare event in Asian patients. Functional impact of novel mutations was assessed by ectopic expression in ALL cell lines.ResultsRelapse-specific somatic alterations were significantly enriched in 12 genes (NT5C2, NR3C1/2, PRPS1/2, TP53, CREBBP, MSH2/6, PMS2, WHSC1, and FPGS) predominantly involved in drug metabolism and response pathways. These somatic alterations were present in over 50% (56/103) of relapsed ALLs and were enriched in patients with early relapse (9-36 months from diagnosis) compared to very early (<9 months) or late relapse (>36 months) patients. NR3C1 mutations resulted in loss of glucocorticoid receptor transcriptional activity and severely impaired glucocorticoid response in vitro, while FPGS mutations led to reduced enzymatic activation of methotrexate. Genome-wide analysis identified 9 mutational signatures, including two novel ones exclusive to relapse. Novel signature 1 was characterized by C>G mutations and was present in 15% of the relapsed cases with an enrichment for hyperdiploid ALL, while novel signature 2, present in 14% of the relapsed cases, was characterized by C mutations (C>T in particular) followed by a G. These novel signatures gave rise to relapse-specific drug resistance mutations, including PRPS1, TP53, NT5C2, and KRAS mutations.The majority (59%) of the cases underwent a selective sweep as the relapse clone arose from a subclone detectable at diagnosis; however, very early relapse cases were less likely to experience a clonal sweep and had multiple lineages present at relapse. In patients tracked serially through multiple relapses, the clonal composition of drug resistance variants evolved substantially in response to chemotherapy. For example, in one patient, two independent NT5C2-mutant clones appeared at relapse, manifesting convergent evolution (Fig. 1a). One of the NT5C2-mutant clones expanded after subsequent treatment while the other diminished in prevalence. Sequential acquisition of multiple drug resistance mutations was also evident, with one patient sequentially acquiring KRAS, FBXW7, NR3C1 and FPGS mutations over a period of 5 years through multiple relapses (Fig. 1b). Indeed, 17% (18/103) of patients acquired multiple drug resistance mutations at relapse. The median time from detecting resistant clones to relapse was 41 days, suggesting that ultra-deep sequencing offers a means for early detection of genetic lesions that could serve as an indicator of relapse to enable earlier therapeutic intervention.ConclusionsVery early relapses likely have different resistance mechanisms than early or late relapses, perhaps due to increased intrinsic resistance or the presence of multiple drug-resistant clones. At least a subset of relapse-specific drug resistance mutations may be acquired de novo, rather than pre-existing, as evidenced by resistance mutations bearing the novel relapse-specific mutational signatures which are likely to be therapy-induced. Relapsed ALL can acquire multiple drug resistance mutations targeting different drug classes. Frequent monitoring of disease, such as via cell-free DNA sequencing, may enable earlier detection of relapse and facilitate timely treatment to avoid selection for drug-resistant clones. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Functional Characterization of Germline ETV6 Variants Associated with Inherited Predisposition to Acute Lymphoblastic Leukemia in Children

▪There is increasing evidence for inherited susceptibility to acute lymphoblastic leukemia (ALL) in children. Both common and rare germline genetic variants can significantly influence the risk of developing ALL, often in an age- and/or leukemia subtype-dependent manner (e.g., ARID5B, IKZF1, GATA3, PIP4K2A, PAX5, and TP53). Recently, we and others reported a novel genetic syndrome characterized by damaging germline ETV6 mutations that co-segregate with hereditary thrombocytopenia and increased propensity for hematologic malignancies (especially ALL). Sequencing 4,405 childhood ALL cases, we subsequently identified 30 germline coding ETV6 variants that were potentially responsible for disease predisposition in 35 patients (Lancet Oncol 2015). These findings unequivocally point to ETV6 as a critical ALL risk gene; however, the exact functional consequences of the ALL-related ETV6 variants are largely unknown and the mechanism by which they contribute to leukemogenesis is unclear. To this end, we first systematically evaluated these 30 ETV6 risk variants for their transcriptional repressor activity, ability to bind to target DNA sequences, localize to the nucleus, and also to homo-dimerize. Using PF4 and MMP3 promoter-driven luciferase transcription in HEK293T cells as the model system, we showed that 18 of these 30 ETV6 variants had loss of transcriptional repression activity (median of 53.2% of wildtype ETV6 activity [range 7.1% to 83.9%]). These include 4 of the 5 frameshift variants, all 4 nonsense variants, and also 10 of 11 missense variants in the ETS/DNA binding domain. None of the missense variants outside the ETS domain had significant effects on transcriptional repression function, and surprisingly the E44fs variant showed enhanced activity. Co-expression of wildtype and each of the 18 loss-of-function variants consistently impaired the normal repressor activity of wildtype ETV6, indicating dominant-negative effects. In cells expressing both wildtype and variant ETV6, immunoprecipitation of variant protein pulled down wildtype ETV6, in line with the dominant negative mode of action of variant ETV6 via dimerization with wildtype protein. Cellular fractionation showed significantly increased cytoplasmic localization of 18 loss-of-function ETV6 variants when expressed in HEK293T cells with concomitant reduction in nuclear ETV6. Analysis using EMSA revealed that all 14 variants in the ETS domain resulted in impaired binding to the consensus sequence in ETV6 target genes in vitro, as was also true for all 4 loss-of-function frameshift variants. To examine the role of ETV6 variants on hematopoietic stem and progenitor cell function, we lentivirally transduced murine LSK cells with wildtype or variant murine Etv6 (R355X or R395C). These cells were cultured ex vivo and analyzed for myeloid and pre-B cell differentiation, using colony formation assay. We found no change in the formation of myeloid colonies, whereas the number of pre-B colonies increased slightly with mutant Etv6, although we observed no effects on cell cycle or apoptosis. Focusing on the 18 ETV6 variants with experimentally confirmed deleterious effects, we then evaluated the association of germline ETV6 status with ALL clinical features in 3,906 children from frontline ALL protocols at St. Jude Children’s Research Hospital and Children’s Oncology Group. Patients with these ETV6 variants were significantly older at ALL diagnosis than those without (13.3 [2.6-21.7] vs 6.8 [0.08-30.7] years, P=0.026) but were more likely to have leukocyte count <50x109/L at presentation (100% vs. 68.6%, P=9.5x10-4). 80% of patients with deleterious ETV6 variants had hyperdiploid ALL compared to 22.2% of those with normal ETV6 (P=9.3x10-8), strongly suggesting interactions between germline and somatic variations during leukemogenesis. Finally, whole genome sequencing of matched ALL blast and germline samples from 5 familial ALLs in 2 kindreds with pathologic ETV6 variants identified somatic lesions as plausible second events to promote leukemogenesis in these cases. In conclusion, we comprehensively characterized 30 ALL risk variants in ETV6, the majority of which function by disrupting DNA binding and impairing its transcriptional repressor activity in a dominant negative fashion. ETV6 variants also affect hematopoiesis but most likely require additional somatic lesions for overt leukemogenesis. DisclosuresMartin:Novartis: Other: Support of clinical trials; Jazz Pharmaceuticals: Other: One time discussion panel. Evans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Hunger:Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Erytech: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Honoraria; Merck: Equity Ownership; Pfizer: Equity Ownership; Amgen: Equity Ownership; Spectrum Pharmaceuticals: Honoraria. Mullighan:Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity’s Board of Directors or advisory committees.

Gene Expression Profiles and Methylation Analysis in Down Syndrome Related Acute Lymphoblastic Leukemia

BackgroundChildren with Down syndrome (DS), which caused by an extra copy of chromosome 21, are predisposed to develop acute lymphoblastic leukemia (ALL). On the other hand, in non-DS children, acquisition of chromosome 21 gain is observed in 95% of hyperdiploid (HD) ALL, which is the most common cytogenetic abnormality pattern in childhood ALL. These may suggest that gain of chromosome 21 relates molecular pathogenesis of ALL. Genetic aberrations of RUNX1 locus on chromosome 21 including iAMP21 or t(12; 21)(p13; q22)/ETV6-RUNX1 were often found in pediatric ALL. While recent studies implicated that HMGN1 or DYRK1A on chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Compared with ALL of non-DS children, DS-ALL have uncommon genetic alterations such as mutations in JAK2 and RAS, mutations or overexpression of CRLF2. These suggest that DS-ALL may have unique biological features compared with ALL of non-DS children. Difference of biological basis between them may correlate to worse prognosis of DS-ALL. Although microarray transcript profiling provided some characteristic gene expression in DS-ALL, no study showed comprehensive transcriptome analysis in DS-ALL.PurposeThis study was conducted to elucidate comprehensive transcriptomic landscape in DS-ALL and to reveal biological features through clustering by gene expression profiling.MethodsOur cohort includes 72 pediatric B-cell precursor ALL samples (48 DS-ALL, 13 HD-ALL, and 11 euploid (EP) ALL). All HD-ALL samples gained chromosome 21. Three cases of DS-ALL and one case of EP-ALL had ETV6-RUNX1 rearrangement. We applied genome-wide analysis using whole-transcriptome sequencing (WTS) to 55 samples, Illumina 450k methylation array to 12 samples, and Illumina EPIC methylation array to 26 samples. For gene expression profiling, we conducted consensus clustering algorithms. Data from methylation arrays were normalized by beta-mixture quantile normalization and merged by common probes.ResultsOur consensus clustering analysis of gene expression stratified samples into three groups such as DS-ALL (cluster 1), DS-ALL and HD-ALL (cluster 2), and EP-ALL (cluster 3). Eight cases with DS-ALL having RAS mutations were classified into cluster 1. DYRK1A, HMGN1, ETS2 on chromosome 21 were highly expressed in cluster 1 and cluster 2 compared with cluster 3. Expression of RUNX1 was lower in cluster 1 and cluster 2 although it was not significant. In addition, FLT3, ELK1, and ETV6 were more highly expressed in cluster 1 and cluster 2 compared with cluster 3. Comparing cluster 1 with cluster 2, NRAS, MAP2K1, JUN, FOS, WT1, EPOR, and PDGFRB were highly expressed in cluster 1. Methylation analysis indicated that methylation pattern of DS-ALL was distinct from that of HD-ALL. Using methylation analysis, we detected differentially methylated regions (DMRs) between DS-ALL and HD-ALL. In DMRs of promoter-associated regions, RUNX1, IGF2BP1, and ETV6 were included. These genes were significantly hypermethylated in DS-ALL. Next, we elucidated methylation profiling of DS-ALL compared with HD-ALL and found hypermethylated genes in DS-ALL. Although these genes may participate pathogenesis of DS-ALL, we could not find any association of gene expression pattern.ConclusionWe identified specific gene expression profiling of DS-ALL and significantly upregulated genes in DS-ALL compared with HD-ALL and EP-ALL. Overexpression of these genes was characteristic gene profiling common to ALL with gaining extra chromosome 21. While ETS2, FLT3, ELK1, NRAS, MAP2K, JUN, FOS, and WT1 were highly expressed in AML, overexpression of these genes may also be related to pathogenesis of DS-ALL. In addition, DS-ALL highly expressed PDGFRB and EPOR, to which these genes are targetable by TKIs and may improve prognosis of DS-ALL. Intriguingly, RUNX1 was hypermethylated in DS-ALL compared with HD-ALL and EP-ALL, indicating that underexpression of RUNX1 was a unique gene profiling of DS-ALL and was due to hypermethylation of RUNX1. In DS patient, hyprmethylation of RUNX1 in their normal blood cells has been reported. This aberrant hypermethylation may be related to pathogenesis of DS-ALL. In this study, we unveiled gene expression profiling and methylation pattern of DS-ALL. However, to elucidate the whole picture of molecular pathogenesis of DS-ALL, we may need further analysis. DisclosuresKataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding.

Impact of Age and Treatment Group in Childhood High Hyperdiploid Low Risk B-Cell Acute Lymphoblastic Leukemia (ALL): Results of the CLG-EORTC 58951 Study

Abstract Background: Since the last decades, pediatric ALL classification has integrated new cytogenetic and genomic data, and several genetic risk factors are identified for treatment stratification. Hyperdiploidy, which is the most common cytogenetic abnormality pattern of pediatric B-lineage ALL, is known since the early 80s and is found in 25-30% of cases. It has been recognized as an isolated good prognostic factor, especially High Hyperdiploidy (HeH), defined by a modal chromosome number higher than 50. In addition, treatment descalations were performed based on NCI criteria for low risk ALL (low leucocytosis &lt; 10 x 109/L and/or age from 1 to ²10 years old) and/or lack of high risk features. Aim: The aim of this study was to assess whether age, under or over 10 years old, has a prognostic impact on the outcome in children presenting B-cell ALL with low risk criteria, and whether therapeutic intensification might improve the outcome within each age group. Methods: Among 2039 patients treated in EORTC 58951 (BFM backbone) from 1998 to 2008, 370 children with B-cell ALL and low risk criteria such as HeH (with classical profile of chromosome gains), low leucocytosis (&lt; 10x109/L), a lack of CNS or gonadal involvement and a lack of very high features (poor response to prephase) were included in this study. Hyperdiploidy was determined either by cytogenetics or flow cytometry. Patients were stratified into 2 risk groups: very low risk (VLR) group or standard risk (AR1) group. VLR group was defined by the criteria indicated above. It led to a less intensive treatment, particularly lower number of injections of anthracyclines and alkylating agents. AR1 group included children with surreptitious or hemorrhagic CNS involvement, and/or HeH with mismatch between modal chromosome number and DNA index. The upper limit of age in the EORTC 58951 was less than 18 years. Main endpoints were Event-Free Survival (EFS) and Overall Survival (OS). Results: Overall patients with low risk hyperdiploid B-cell ALL features treated according to a VLR or AR1 protocol had 6-year EFS and OS rates of 90.3% and 96.2% respectively. These results were comparable to previous published studies on hyperdiploidy in childhood B-lineage ALL. All patients but one reached complete remission after the induction phase. There were 315 children aged less than 10 years old and 55 patients aged 10 to 17 years old. Among children aged 10 years and older treated in VLR or AR1 groups, 6-year EFS and OS rates were respectively 86.7% and 96.4%. In comparison, among less than 10 year old childrentreated in VLR or AR1 groups, 6-year EFS and OS rates were respectively 89.8% (p=0.42) and 95.9% (p=0.93). Age did not appear as a significant prognostic factor in the outcome. Furthermore, among children aged 10 years and older, those treated in the standard risk group (AR1) had 6-year EFS and OS rates of 90% and 100% respectively. This seemed better than for children treated in the very low risk group, with respectively 84% and 92% rates. However, no significant gain was found for children who received standard risk treatment (for EFS and OS rates: p=0.47 and p=0.12 respectively). Relapse and treatment toxicity rates were comparable in both groups. Concerning long-term cardiac toxicity, despite the fact that no significant difference was found between the two risk groups of treatment, standard treatment leads to higher dose of anthracyclines and an increased theoretical risk of cardiac toxicity. Conclusion: In conclusion, age did not appear as a significant prognostic factor in outcome of children treated for a low risk B-cell ALL. Moreover, among children aged 10 years and older, therapeutic intensification in standard risk group did not lead to a significant gain in outcome. However, results are not significant, partly due to a low number of patients, and larger studies should be performed to evaluate whether these children could benefit from a less intensive treatment. Disclosures No relevant conflicts of interest to declare.

Somatic and Germline Mutational Heterogeneity in High Hyperdiploid Acute Lymphoblastic Leukemia

Abstract High hyperdiploidy (HD), the most common cytogenetic subtype of acute lymphoblastic leukemia (ALL), is characterized by a nonrandom gain of chromosomes and is thought to arise from a single abnormal mitosis. However, the causes of this leukemia-initiating event remain unknown. A recognized enrichment of HD-ALL among children with RASopathies and with germline ETV6 mutations suggests that germline predisposition underlies a component of HD-ALL risk, in addition to the increased risk of the HD subtype with ALL-associated heritable risk variants in PIP4K2A, ARID5B, and CEBPE. Though cure rates of HD-ALL are high, the significant treatment-related morbidities and mortality warrant more etiologic investigations which may reveal molecularly-targeted therapies for this disease. We carried out deep-sequencing of 538 cancer-related genes using the UCSF500 Cancer Gene Panel in 57 HD-ALL tumors from California Childhood Leukemia Study patients. Selected patients lacked overt KRAS and NRAS hotspot mutations (assessed by Sanger sequencing) and common ALL deletions (assessed by MLPA), to enrich for discovery of novel driver genes. A Combined Annotation Dependent Depletion (CADD) Phred score ≥20 was used to filter predicted damaging mutations. To remove polymorphisms, we retained only mutations with allele frequency &lt;0.01% in the Exome Aggregation Consortium (ExAC). We adjusted the mutant allele fraction (MAF) of each mutation in relation to chromosome copy-number, as determined using the CNVkit tool. Sanger sequencing of remission DNA was used to validate a subset of predicted germline mutations (adjusted MAF≥0.45) of interest, including in known ALL predisposition genes. Novel damaging somatic mutations were discovered in epigenetic regulatory genes, including DOT1L (n=4), with 33% of patients harboring mutations in this pathway. Somatic mutations in the receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway were found in two thirds of patients, including mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. An extraordinary level of tumor heterogeneity was detected, with microclonal (mutant allele fraction &lt;10%) hotspot mutations in KRAS, NRAS, FLT3 or PTPN11 identified in 31/57 (54.4%) patients. Multiple microclonal mutations at KRAS and NRAS codons 12 and 13 significantly co-occurred within tumor samples (P=4.8x10-4), suggesting ongoing formation of, and selection for, Ras mutation. Moreover, 7 patients had multiple microclonal mutations at the same Ras hotspot locus, in adjacent codon 12/13 nucleotides or in adjacent codons. The adjacent mutations occurred on different sequencing reads in all 7 patients (P=0.016), indicating they were part of distinct tumor subclones. We also detected an unexpectedly high frequency of putatively causal germline mutations, which were validated in remission DNA samples by Sanger sequencing. At least 25% of HD-ALL patients carried one or more rare (&lt;0.01% allele frequency in ExAC) and predicted-damaging germline mutations in known ALL predisposition genes, DNA repair genes, or within known hotspot mutation loci that had previously been reported mutated only in tumor genomes. Future work is required to investigate whether tumor microheterogeneity should impact therapeutic regimens and to elucidate the biologic function of epigenetic dysregulation in development of HD-ALL. Whole-exome sequencing of more patients and functional analysis of novel mutations are required to understand the contribution of germline predisposition to HD-ALL etiology, which may be much larger than previously realized. Disclosures No relevant conflicts of interest to declare.

Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8×10−4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches.

Immunophenotypic Characterization of Cytogenetic Subgroups in Egyptian Pediatric Patients With B-Cell Acute Lymphoblastic Leukemia.

Identification of prognostic factors in acute lymphoblastic leukemia (ALL) patients is important for stratifying patients into risk groups and tailoring treatment accordingly. Molecular and cytogenetic abnormalities are the most important prognostic factors. Minimal residual disease (MRD) is also an important predictor of relapse in ALL. However, the correlation of both prognostic variables has not been thoroughly studied. We investigated the correlation between defined cytogenetic abnormalities and selected new MRD markers (CD79b, CD123, and CD200) in 56 newly diagnosed Egyptian pediatric B-cell ALL patients. CD123 found to be expressed in 45% of patients, CD200 in 80.3%, and CD79b in 67.9%. MRD analysis during treatment showed stable expression patterns of CD200. There was significant association of CD123 expression with the hyperdiploid ALL group (P= .017). Another association (P= .029) was found between CD79b negativity and the t(12;21) group. CD200 was widely expressed in all groups. There is a significant correlation between some markers, and certain ALL recurrent cytogenetic subgroups (CD123 and hyperdiploidy, CD79b negativity, and ETV-RUNX1 group) have good prognostic value. CD200 can be used as MRD markers in ALL patients and can also can serve as therapy targets.

Slower early response to treatment and distinct expression profile of childhood high hyperdiploid acute lymphoblastic leukaemia with DNA index < 1.16.

Acute lymphoblastic leukaemias (ALL) with 51-67 chromosomes are defined as high hyperdiploid (HHD) and are generally associated with good prognosis. However, several studies show heterogeneity in HHD ALL and suggest that the favourable prognosis is associated rather with higher ploidy defined by DNA index (DNAi) ≥ 1.16 or with a presence of specific single or combined trisomies. HHD ALL with DNAi < 1.16 are only rarely studied separately. Using single nucleotide polymorphism array, we analysed 89 childhood HHD ALL patients divided into groups with lower (<1.16; n = 34) and higher (≥1.16; n = 55) DNAi. We assessed treatment response, presence of secondary aberrations, mutations in RAS pathway genes and CREBBP and also gene expression profile (GEP) to reveal differences between the two subgroups. Cases with 51-54 chromosomes had DNAi 1.1-1.16 and cases with 55-67 chromosomes had DNAi ≥ 1.16. The groups with lower and higher DNAi had distinct response to early treatment and distinct GEP. The better response of the group with higher DNAi was associated with specific trisomies (trisomy of chromosome 10 or combined with trisomies 4 and/or 17). Our results suggest that cytogenetically defined HHD ALL can in fact be divided into two biologically distinguishable subgroups and that DNAi 1.16 is a relevant value to separate between the two. © 2016 Wiley Periodicals, Inc.

Acute lymphoblastic leukemia in the context of RASopathies

Noonan syndrome is associated with a range of malignancies including acute lymphoblastic leukemia (ALL). However, little information is available regarding the frequency, natural history, characteristics and prognosis of ALL in Noonan syndrome or RASopathies in general. Cross-referencing data from a large prospective cohort of 1176 patients having a molecularly confirmed RASopathy with data from the French childhood cancer registry allowed us to identify ALL in 6 (0.5%) patients including 4/778 (0.5%) with a germline PTPN11 mutation and 2/94 (2.1%) with a germline SOS1 mutation. None of the patients of our series with CFC syndrome (with germline BRAF or MAP2K1/MAP2K2 mutation – n = 121) or Costello syndrome (with HRAS mutation – n = 35) had an ALL. A total of 19 Noonan-ALL were gathered by adding our patients to those of the International Berlin-Munster-Frankfurt (I-BFM) study group and previously reported patients. Strikingly, all Noonan-associated ALL were B-cell precursor ALL, and high hyperdiploidy with more than 50 chromosomes was found in the leukemia cells of 13/17 (76%) patients with available genetics data. Our data suggest that children with Noonan syndrome are at higher risk to develop ALL. Like what is observed for somatic PTPN11 mutations, NS is preferentially associated with the development of hyperdiploid ALL that will usually respond well to chemotherapy. However, Noonan syndrome patients seem to have a propensity to develop post therapy myelodysplasia that can eventually be fatal. Hence, one should be particularly cautious when treating these patients.

Childhood Hyperdiploid Acute Lymphoblastic Leukemia

Background:Acute lymphoblastic leukemias (ALL) with 51-67 chromosomes in leukemic cells are defined as high-hyperdiploid (HHD). In childhood, these leukemias comprise 25-30% of all cases, typically arise from B lymphocyte precursors and are generally associated with good prognosis. Besides the number of chromosomes, the hyperdiploid ALLs can be determined also by DNA index (established by flow cytometry), representing ratio of DNA content in leukemic vs. normal diploid cell. Cases with DNA index >=1.16 and <1.6 are often considered as "typical" high hyperdiploid ALLs. Leukemias with >50 chromosomes and DNA index <1.16 are only rarely studied separately and here we assign these cases as "low DNA index HHD" (LDI-HHD). Favorable prognosis is associated mainly with the "high DNA index HHD" (HDI-HHD) cases (DNA index >= 1.16) and, according to some studies, particularly with cases characterized by "triple-trisomy", i.e. concurrent gain of chromosomes 4, 10 and 17. However, as the triple-trisomy significantly overlaps with HDI-HHD, it is not clear whether the favorable prognosis is driven rather by the triple-trisomy itself or by the higher ploidy of these patients (with most cases having DNA index >= 1.16). In this study we aimed to analyze biological and clinical features of HHD leukemias, compare LDI-HHD and HDI-HHD cases and verify prognostic role of triple-trisomy of chromosomes 4, 10 and 17.Patients and methods:We tested 75 patients with hyperdiploid childhood ALL defined by the presence of 51-67 chromosomes (46 HDI-HHD, 29 LDI-HHD). To determine the type of hyperdiploidy we used flow cytometry (DNA index) and whole genome single nucleotide polymorphism (SNP) array. SNP array also enabled precise determination of amplified chromosomes as well as partial gains and losses and calculation of a SNP array-based "theoretical" DNA index. From clinical features we analyzed final risk stratification of patients (based in this subgroup on early treatment response measured at day +8 by morphology ("prednisone response"), at day +15 by flow cytometry and at day +33 and week +12 by PCR quantification of immunoglobulin a T-cell receptor rearrangements) and their outcome.Results:Our data show that correlation of DNA content in leukemic cells determined by flow cytometry and by SNP-array is very high (Spearman correlation: rho = 0.96; p-value < 2,2e-16). Besides the HHD patients we analyzed DNA index and SNP array in 53 non-high hyperdiploid patients (<=50 chromosomes); all the non-HHD cases had DNA index <1.1. In HHD patients we found negative correlation between DNA index and final risk stratification (decrease of DNA index with increasing risk (standard - medium - high), p=0.004). As expected, patients with triple-trisomy have higher DNA index and number of chromosomes (p<0.0001); however, 4/38 were found among the LDI-HHD patients. None of the triple-trisomy patients was stratified into the high-risk treatment, while in patients without the triple-trisomy the distribution of high risk vs. non-high risk therapy was 13 vs. 24 (p<0.0001) with 10/25 (40%) HR cases among LDI-HHD and 3/12 (25%) HR cases among HDI-HHD patients. Patients with triple-trisomy have better very early response to treatment measured at day +15 (p=0.009) and this difference remains significant also when only patients with HDI-HHD are analyzed separately (p=0.014). There is no significant difference in event free survival analysis as overall outcome of this group is very good - only 4 events emerged within the whole cohort so far (1 secondary AML in patient with triple-trisomy and one relapse and two deaths in 3 patients without triple-trisomy, one of those from the LDI-HHD group).Conclusion:High hyperdiploidy can be determined by karyotype, SNP array and also by flow cytometry, where cases with DNA index > 1.1 are highly likely to carry > 50 chromosomes. Patients with high hyperdiploidy over 50 chromosomes form a subgroup of childhood ALL with a generally very good prognosis. However, some heterogeneity within this group is present. Our data suggest that patients with LDI-HHD are more often stratified into high risk treatment. On the other hand, patients with triple-trisomy of chromosomes 4, 10 and 17 are characterized by a rapid response to initial therapy, which is not just a result of coexisting HDI-HHD status.Supported by grant IGA MZ NT14350/3. DisclosuresNo relevant conflicts of interest to declare.

Evidence for Heterogeneous Genetic Associations with Acute Lymphoblastic Leukemia (ALL) By Cytogenetics and Sex in High-Risk Patients Treated with Matched Unrelated Donor Allogeneic Blood or Marrow Transplant (URD-BMT)

Inherited genetic polymorphisms in ARID5B, GATA3, and IKZF1 have been reported to be associated with ALL and vary by cytogenetic subgroups. For example, ARID5B variants are associated with hyperdiploid B-ALL in pediatric patients, which occurs in approximately 30% of ALL patients and is a marker of good prognosis. In contrast, a variant in GATA3 (rs3824662) has been consistently associated with ALL regardless of cytogenetics. While associations by subtype and across age have been explored in ALL patients, the effect of sex on the association of germline variants with ALL has not been tested, despite the higher incidence and worse prognosis of ALL in males than females. We tested the association of previously reported ALL variants in a high-risk ALL population of URD-BMT recipients, assessing the association of these variants with different cytogenetic subgroups and for heterogeneity of effect by sex.Genotyping was performed using the Illumina Omni-Express BeadChip containing approximately 730,000 single nucleotide polymorphisms (SNPs). After quality control, cohort 1 (C1) and cohort 2 (C2) included 364 (C1) and 82 (C2) B-ALL cases of continental European ancestry and 2,229 (C1) and 809 (C2) controls (unrelated healthy donors). The sample size of non-European cases was not adequate for genetic analyses. Cases were a subset of the genome wide association study (GWAS) named Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT (DISCOVeRY-BMT) a parent GWAS study in collaboration with the Center for International Blood and Marrow Transplant Research. We tested SNPs within GATA3, IKZF1 and ARID5B for associations with disease status using logistic regression assuming an additive model adjusted for age. Analyses were done separately for the following B-ALL subgroups: hyperdiploid negative ALL, Ph-negative ALL, Abnormal cytogenetics and Normal cytogenetics. P-values for each cohort were combined using METAL software with weights proportional to the square root of the sample size(Pmeta). Sex-stratified analyses were performed using all B-ALL cases and by subgroup. Tests for heterogeneity using Cochran's Q method and I² statistic were used to calculate the percentage of variation across sex and cytogenetic group, abnormal versus normal).The study population consisted mostly of non-hyperdiploid and Ph negative B-ALL. The GATA3 variant conferred a 78% (P =2.9x10-10) and 82% (P =0.001) increased odds of B-ALL in C1 and C2, respectively, Pmeta=1.7x10-12 (Table 1). The GATA3 SNP was significantly associated, Pmeta < 5x10-8, with all subgroups except abnormal cytogenetics ALL. The SNP in IKZF1 wasassociated (Pmeta < 5x10-8) with B-ALL overall, Ph negative and normal cytogenetics ALL, with some evidence of heterogeneity in odds ratios (ORs) between normal and abnormal cytogenetics B-ALL in both cohorts. The ARID5B variant, previously shown to be strongly associated with pediatric ALL, showed weak or no evidence of association (Table 1). IKZF1 (rs11980379) associations were stronger across all associated subgroups in males (Table 2). When comparing ORs calculated for males and females in there was evidence of significant heterogeneity (Table 2) for B-ALL overall (P =0.02), non-hyperdiploid (P =0.02) and normal cytogenetics (P =0.004).GATA3 showed consistent association, with replication, across the cytogenetic subgroups analyzed. IKZF1 variant demonstrated differences by sex, with significant associations between this SNP and B-ALL, observed primarily in males, regardless of cytogenetics. Thus, sex-specific associations were not an artifact of underlying cytogenetic distributions between males and females. The variation between males and females indicates the IKZF1 variant may impact ALL risk differently in males and females, potentially explaining both the overall greater risk of ALL in males and the risk of ALL with worse outcomes. ARID5B associations have previously been associated with hyperdiploid ALL, however the majority of our high-risk study population is non-hyperdiploid, so ARID5B may be a marker of good risk ALL which is rarely treated with URD-BMT. [Display omitted] [Display omitted] DisclosuresHahn:Novartis: Equity Ownership; NIH/NHLBI: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NHLBI: Research Funding.

Tumor Vs Normal Gene Expression Profiles Identify Deregulated Pathways in ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia: A Microarray-Based Meta-Analysis

Acute lymphoblastic leukemia (ALL), a clinically and biologically heterogeneous disease, is the most common type of childhood cancer. Approximately 25% of B-cell precursor ALL (BCP-ALL) carry the cryptic chromosomal translocation t(12;21)(p13;q22), the most frequent genetic aberration in pediatric ALL. This translocation combines two transcription factors and essential regulators of normal hematopoiesis, ETV6 and RUNX1, generating the fusion oncogene ETV6/RUNX1 (synonym TEL/AML1). Recent studies in various animal models have strengthened the view that ETV6/RUNX1 positive cells give rise to preleukemic clones with a differentiation block in the pro/pre-B stage of B cell development that, after acquisition of additional mutations, may transform into full malignancy. However, the effects triggered by the expression of ETV6/RUNX1 and the associated additional mutated genes are not well understood.We compared global gene expression data from ETV6/RUNX1 positive ALL and normal hematopoietic progenitor cells to identify expression signatures underlying the malignant phenotype. Our meta-analysis comprised Affymetrix HG-U133A und HG-U133 Plus 2.0 data from a total of 321 patients with ETV6/RUNX1 positive ALL, different ETV6/RUNX1 positive cell models, leukemias with 11q23 rearrangements and hyperdiploid ALL, as well as 251 samples from various normal controls including normal bone marrow, hematopoietic stem cells (HSCs) and normal B-(progenitor) cells (multipotent progenitor, pro-, pre-, immature-, naive-, centrocyte-, centroblast-, memory-, plasmablast-B cells). To eliminate potential lab batch effects, we performed RMA normalization of all microarray CEL files and next applied the batch effect removal algorithm ComBat. For determination of differentially expressed genes and pathway analyses, the R/Bioconductor packages limma and SPIA were used, respectively.We generated a gene expression landscape of the normal B-cell development and were able to show that pre-B cells are the closest normal counterpart of ETV6/RUNX1 positive ALL. Furthermore, in comparisons with normal HSCs the p53-, mTOR-, PI3K-AKT-, Apoptosis-, and JAK-STAT signaling pathways were found to be deregulated in those ALL. The combination of multiple global gene expression data sets from ETV6/RUNX1 positive ALL offers the possibility of an integrated large-scale meta-analysis and reveals novel insights into deregulated gene networks in this type of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Functional Diversity of Single Stem Cell Clones in Patients' Acute Lymphoblastic Leukemia Growing in Mice: An Adverse Subclone with Distinct DNA-Methylation Pattern, Slow Growth In Vivo and Drug Resistance

Acute lymphoblastic leukemia (ALL) consists of genetically heterogeneous cell subpopulations, but little is known about how genetic differences lead to functional differences between the clones. Of major clinical importance, aggressive, treatment-resistant and putatively relapse-inducing subclones need to be identified and require effective eradication by treatment. The most aggressive subpopulation likely determines prognosis and outcome in each patient.We aimed at characterizing on a functional as well as on a genetic level single stem cell clones derived from patients' samples growing in mice and to combine the results of both levels in order to learn which genetic characteristics are associated with adverse functional behavior.We transplanted primary tumor cells from a 5-year old girl with hyperdiploid ALL involving a trisomic X chromosome at first relapse into severely immune-compromised mice and lentivirally modified them to express the fluorochromes red, blue and green at different amounts and combinations (RGB marking, Weber et al., 2012). Eight single stem cell clones were generated by limiting dilution transplantation and their uniqueness was verified by ligation-mediated (LM) PCR.We functionally compared the single stem cell clones between each other by re-mixing them in a single mouse for in vivo assays; analysis was performed one-by-one for each clone by flow cytometry where they could be distinguished from each other using their unique color codes. Clones showed clear differences in proliferation rate with faster and slower growing clones, independently whether 2 or 5 clones were mixed. When mice harboring clone mixtures were treated with conventional chemotherapy, clonal composition changed markedly and resistant clones overgrew sensitive clones indicating selective clonal responses and clonal advantage. A clone which showed especially slow growth in vivo was most resistant to in vivo treatment with Glucocorticoids.The slowly proliferating, Glucocorticoid-resistant clone had lost the additional X chromosome, which was present in all other clones and the bulk and showed a distinct DNA-methylation pattern analyzed by 450K arrays (illumina). In exome analysis, the clone showed 11 unique alterations including a single nucleotide variant in the oncogene USP6. We are currently performing RNA sequencing analysis to assess the differential gene expression in the clones.Taken together, genetic multicolor marking PDX ALL cells in the individualized xenograft mouse model allowed generating viable single cell clones for genetic functional characterization in vivo. Within the heterogeneous tumor bulk, an subclone existed which showed slow tumor growth and drug resistance which was associated with distinct genetic characteristics. Our studies allow the challenging functional characterization of subclones in vivo in order to develop efficient novel treatment approaches to eliminate aggressive stem cell clones in ALL.Weber K, Thomaschewski M, Benten D, Fehse B., RGB marking with lentiviral vectors for multicolor clonal cell tracking. Nat Protoc. 2012 Apr 5;7(5):839-49. doi: 10.1038/nprot.2012.026. DisclosuresNo relevant conflicts of interest to declare.

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18).In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.

Methotrexate resistance in relation to treatment outcome in childhood acute lymphoblastic leukemia.

BackgroundMethotrexate (MTX) eradicates leukemic cells by disrupting de novo nucleotide biosynthesis and DNA replication, resulting in cell death. Since its introduction in 1947, MTX-containing chemotherapeutic regimens have proven instrumental in achieving curative effects in acute lymphoblastic leukemia (ALL). However, drug resistance phenomena pose major obstacles to efficacious ALL chemotherapy. Moreover, clinically relevant molecular mechanisms underlying chemoresistance remain largely obscure. Several alterations in MTX metabolism, leading to impaired accumulation of this cytotoxic agent in tumor cells, have been classified as determinants of MTX resistance. However, the relation between MTX resistance and long-term clinical outcome of ALL has not been shown previously.MethodsWe have collected clinical data for 235 childhood ALL patients, for whom samples taken at the time of diagnosis were also broadly characterized with respect to MTX resistance. This included measurement of concentrations of MTX polyglutamates in leukemic cells, mRNA expression of enzymes involved in MTX metabolism (FPGS, FPGH, RFC, DHFR, and TS), MTX sensitivity as determined by the TS inhibition assay, and FPGS activity.ResultsHerein we demonstrate that higher accumulation of long-chain polyglutamates of MTX is strongly associated with better overall (10-year OS: 90.6 vs 64.1 %, P = 0.008) and event-free survival (10-year EFS: 81.2 vs 57.6 %, P = 0.029) of ALL patients. In addition, we assessed both the association of several MTX resistance-related parameters determined in vitro with treatment outcome as well as clinical characteristics of pediatric ALL patients treated with MTX-containing combination chemotherapy. High MTX sensitivity was associated with DNA hyperdiploid ALL (P < 0.001), which was linked with increased MTX accumulation (P = 0.03) and elevated reduced folate carrier (RFC) expression (P = 0.049) in this subset of ALL patients. TEL-AML1 fusion was associated with increased MTX resistance (P = 0.023). Moreover, a low accumulation of MTX polyglutamates was observed in MLL-rearranged and TEL-AML1 rearranged ALL (P < 0.05).ConclusionsThese findings emphasize the central role of MTX in ALL treatment thereby expanding our understanding of the molecular basis of clinical differences in treatment response between ALL individuals. In particular, the identification of patients that are potentially resistant to MTX at diagnosis may allow for tailoring novel treatment strategies to individual leukemia patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0158-9) contains supplementary material, which is available to authorized users.

The genomic landscape of high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploid (51-67 chromosomes) acute lymphoblastic leukemia (ALL) is one of the most common childhood malignancies, comprising 30% of all pediatric B cell-precursor ALL. Its characteristic genetic feature is the nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18 and 21, with individual trisomies or tetrasomies being seen in over 75% of cases, but the pathogenesis remains poorly understood. We performed whole-genome sequencing (WGS) (n = 16) and/or whole-exome sequencing (WES) (n = 39) of diagnostic and remission samples from 51 cases of high hyperdiploid ALL to further define the genomic landscape of this malignancy. The majority of cases showed involvement of the RTK-RAS pathway and of histone modifiers. No recurrent fusion gene-forming rearrangement was found, and an analysis of mutations on trisomic chromosomes indicated that the chromosomal gains were early events, strengthening the notion that the high hyperdiploid pattern is the main driver event in this common pediatric malignancy.

A Novel Functional Polymorphism in the CCAAT/Enhancer Binding Protein (C/EBP), Epsilon (CEBPE) Gene Promoter Influences Acute Lymphoblastic Leukemia Risk Via Interaction with IKZF1

Purpose: We sought to identify and characterize the functional polymorphism in the CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE) gene, which is known to harbor a genetic risk modifier for childhood acute lymphoblastic leukemia (ALL).Background: Association between ALL risk and polymorphic variation at the CEBPE gene, a zinc-finger hematopoietic and apoptotic regulator expressed exclusively in myeloid cells, was established in a genome-wide association study and replicated in other populations. The actual functional genetic variation that actually contributes the leukemia risk is not known. The discovery and functional characterization of the functional variant may lead to risk stratification, prevention, and/or therapeutic modalities.Methods: Utilizing California Childhood Leukemia Study genome-wide SNP data from Hispanic subjects, we refine the genetic signal to a specific single nucleotide polymorphism (SNP) within the CEBPE gene promoter (rs2239635). This SNP was identified first using genetic imputation using 1000 Genomes data as a framework, and further validated in additional subjects using Taqman genotyping. Functional work to characterize the impact of the polymorphism was performed using prediction methods (database searches), chromatin immunoprecipitation assays from normal subjects heterozygous for rs2239635, and luciferase expression vectors carrying the SNP.Results: The minor allele of rs2239635 conferred a 1.87-fold increased risk of B-cell ALL (95% CI = 1.51-2.30; P = 6.2x10-9) and a 2.94-fold increased risk of high hyperdiploid B-cell ALL (95% CI = 2.12-4.06; P = 8.2x10-11), but only a 1.51-fold increased risk of non-hyperdiploid B-cell ALL (95% CI = 1.11-2.04; P = 8.8x10-3). Adjusting for rs2239635 attenuated all SNP associations in the region, including the original GWAS allele, strongly suggest that the GWAS signal in CCLS Hispanics is attributable to rs2239635. We also demonstrate, using published functional data and our own chromatin immunoprecipitation assays, that this SNP disrupts a canonical transcription factor binding site for Ikaros (IKZF1), an essential hematopoietic transcription factor. Using expression vectors, we found the polymorphic locus to have transcriptional repressive properties that are diminished in the presence of the ALL risk allele. Additionally, a heritable risk allele within IKZF1 interacts with rs2239635 in a multiplicative fashion (P = 0.02) in case-control association analyses.Discussion: In sum, evidence for a functional CEBPE polymorphism links two hematopoietic developmental transcriptional factors and highlights feedback control between them. The minor, risk-associated variant of CEBPE may alter the ability of IKZF1 to repress CEBPE expression in precursor B-cells, causing lineage confusion and enhanced pre-B cell leukemia risk. This risk primarily impacts risk of pediatric ALL that exhibits the high hyperdiploid phenotype. DisclosuresNo relevant conflicts of interest to declare.

CEBPE polymorphism confers an increased risk of childhood acute lymphoblastic leukemia: a meta-analysis of 11 case-control studies with 5,639 cases and 10,036 controls.

The association between CCAAT/enhancer binding protein-ε (CEBPE) rs2239633 polymorphism and acute lymphoblastic leukemia (ALL) risk has been reported, but results of previous studies remain controversial and ambiguous. To assess the association between CEBPE rs2239633 polymorphism and childhood ALL risk, a meta-analysis was performed. Based on comprehensive searches of the PubMed, Embase, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biomedical Literature Database (CBM), we identified outcome data from all articles estimating the association between CEBPE rs2239633 polymorphism and childhood ALL risk. The pooled odds ratio (OR) with 95% confidence intervals (CIs) were calculated. A significant association between CEBPE rs2239633 polymorphism with childhood ALL was found (OR = 1.19, 95% CI 1.11-1.28, P < 0.01). Subgroup analysis stratified by ethnicity also suggested a significant association between this polymorphism and childhood ALL in the Caucasian subgroup (OR = 1.19, 95% CI 1.09-1.30, P < 0.01) and Hispanic subgroup (OR = 1.39, 95% CI 1.18-1.63, P < 0.01). No significant association was observed in the Asian subgroup (OR = 1.05, 95% CI 0.90-1.22, P = 0.53). The CEBPE rs2239633 polymorphism increased B cell ALL risk (OR = 1.29, 95% CI 1.15-1.44, P < 0.01) and B hyperdiploid ALL risk (OR = 1.84, 95% CI 1.40-2.43, P < 0.01). This meta-analysis demonstrated that the CEBPE rs2239633 polymorphism was significantly associated with childhood ALL risk.

IKZF1 rs4132601 polymorphism and acute lymphoblastic leukemia susceptibility: a meta-analysis

Several studies have been conducted to examine the association between IKZF1 rs4132601 polymorphism and acute lymphoblastic leukemia (ALL) risk. However, the conclusions remain controversial. We therefore performed a meta-analysis. PubMed, Embase, Web of Science, Weipu and Chinese Biomedical Literature (CBM) databases were searched. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. A total of 15 case–control studies with 8333 cases and 36 036 controls were included in this meta-analysis. The results suggested that rs4132601 was associated with an increased ALL risk. Significant associations were found among Caucasians and Hispanics but not among Asians. In subgroup analysis by age group, both adults and children showed increased ALL risk. In subgroup analysis by subtype of ALL, significantly increased risks were observed in B-cell ALL and B hyperdiploid ALL, but not in T-cell ALL. This study suggests that IKZF1 rs4132601polymorphism is a risk factor for ALL.

Evolutionary trajectories of hyperdiploid ALL in monozygotic twins.

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally.