High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD‐ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer‐relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (<0.01% allele frequency in the Exome Aggregation Consortium, ExAC) and predicted functional (scaled CADD score ≥ 20) in known or potential ALL predisposition genes (SH2B3, CREBBP, PMS2, MLL, ABL1, and MYH9). Three additional patients carried rare and predicted damaging germline mutations in GAB2, a known activator of the ERK/MAPK and PI3K/AKT pathways and binding partner of PTPN11‐encoded SHP2. The frequency of rare and predicted functional germline GAB2 mutations was significantly higher in our patients (2.6%) than in ExAC (0.28%, P = 4.4 × 10−3), an observation that was replicated in ALL patients from the TARGET project (P = .034). We cloned patient GAB2 mutations and expressed mutant proteins in HEK293 cells and found that frameshift mutation P621fs led to reduced SHP2 binding and ERK1/2 phosphorylation but significantly increased AKT phosphorylation, suggesting possible RAS‐independent leukemogenic effects. Our results support a significant contribution of rare, high penetrance germline mutations to HD‐ALL etiology, and pinpoint GAB2 as a putative novel ALL predisposition gene.
Recent studies using next-generation sequencing of selected individuals, such as those with familial leukemia or congenital syndromes, have identified rare and highly penetrant germline mutations that predispose to childhood acute lymphoblastic leukemia (ALL). High hyperdiploidy (HD), the most common cytogenetic subtype of childhood ALL, is enriched in children with RASopathies who develop ALL and, similarly, a high proportion of ALL patients with germline ETV6 or IKZF1 mutations presented with the HD subtype. Here, we aimed to identify novel predisposition genes in a selected group of HD-ALL patients. Targeted sequencing of 538 cancer-relevant genes was carried out using the UCSF500 Cancer Gene Panel in diagnostic bone marrow (i.e. tumor) DNA from 57 HD-ALL patients from the California Childhood Leukemia Study (CCLS). HD-ALL patients were selected based on absence of somatic KRAS or NRAS hotspot mutations detectable by Sanger sequencing, and absence of somatic copy number deletions from multiplex ligation-dependent probe amplification (MLPA) assays. After filtering out likely somatic mutations (mutant allele fraction <0.44), and restricting to variants with low frequency in unselected individuals (allele frequency <0.01% in the Exome Aggregation Consortium, ExAC) and with predicted functional effects (Combined Annotation Dependent Depletion, CADD score ≥20), we identified 151 putative predisposing mutations. Of 41 mutations of interest selected for validation, 37 (90.2%) were confirmed as germline in origin via Sanger sequencing of remission or newborn bloodspot DNA. Rare and predicted functional germline mutations in known (NBN, SH2B3, ETV6, CREBBP, MSH6) or suspected (MLL, ABL1, FLT3, MYH9) ALL predisposition genes were identified in nine out of 57 patients (15.8%). Three additional patients harbored germline mutations in the GRB2-associated binding protein 2 (GAB2), a known binding partner of PTPN11-encoded SHP2 and activator of the ERK/MAPK and PI3K/AKT pathways. Two GAB2 mutations, a missense mutation S592F and frameshift mutation P621fs, were predicted to be highly functional (CADD scores = 34 and 36 respectively) and absent in ExAC. Frequency of rare and damaging GAB2 mutations was significantly higher in our patient set (2.6%) than in ExAC (0.28%, P = 2.70 x 10-6). We replicated this finding in sequencing data from 309 ALL patients in the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project (0.81% vs. 0.28%, P = 0.015). Patient GAB2 mutations were cloned into HEK293 cells and, following EGF stimulation, we found that the P621fs mutation reduced SHP2 binding and ERK1/2 phosphorylation but increased AKT phosphorylation. This suggested possible Ras-independent leukemogenic effects, supported by a lack of somatic Ras pathway mutations in the three GAB2 mutant patients. Additional functional analyses and sequencing of larger patient cohorts will be required to elucidate the role of germline GAB2 mutations in childhood ALL. Disclosures No relevant conflicts of interest to declare.
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 <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 <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 (<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.
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