The genetic basis and cell of origin of mixed phenotype acute leukaemia.

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.

Modeling and targeting of erythroleukemia by hematopoietic genome editing.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.

Genomic and outcome analyses of Ph-like ALL in NCI standard-risk patients: a report from the Children's Oncology Group.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL; BCR-ABL1-like ALL) in children with National Cancer Institute (NCI) intermediate- or high-risk (HR) ALL is associated with poor outcome. Ph-like ALL is characterized by genetic alterations that activate cytokine receptor and kinase signaling and may be amenable to treatment with tyrosine kinase inhibitors. The prevalence, outcome, and potential for targeted therapy of Ph-like ALL in standard-risk (SR) ALL is less clear. We retrospectively analyzed a cohort of 1023 SR childhood B-ALL consecutively enrolled in the Children's Oncology Group AALL0331 clinical trial. The Ph-like ALL gene expression profile was identified in 206 patients, and 67 patients with either BCR-ABL1 (n = 6) or ETV6-RUNX1 (n = 61) were excluded from downstream analysis, leaving 139 of 1023 (13.6%) as Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing identified kinase-activating alterations in 38.8% of SR Ph-like cases, including CRLF2 rearrangements (29.5% of Ph-like), ABL-class fusions (1.4%), JAK2 fusions (1.4%), an NTRK3 fusion (0.7%), and other sequence mutations (IL7R, KRAS, NRAS; 5.6%). Patients with Ph-like ALL had inferior 7-year event-free survival compared with non-Ph-like ALL (82.4 ± 3.6% vs 90.7 ± 1.0%, P = .0022), with no difference in overall survival (93.2 ± 2.4% vs 95.8 ± 0.7%, P = .14). These findings illustrate the significant differences in the spectrum of kinase alterations and clinical outcome of Ph-like ALL based on presenting clinical features and establish that genomic alterations potentially targetable with approved kinase inhibitors are less frequent in SR than in HR ALL.

Ph-like acute lymphoblastic leukemia: a high-risk subtype in adults.

Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are conflicting data on the incidence and prognosis of Ph-like ALL in adults. Patients with newly diagnosed B-cell ALL (B-ALL) who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells. Of 148 patients, 33.1% had Ph-like, 31.1% had Ph+, and 35.8% had other B-ALL subtypes (B-other). Within the Ph-like ALL cohort, 61% had cytokine receptor-like factor 2 (CRLF2) overexpression. Patients with Ph-like ALL had significantly worse overall survival (OS), and event-free survival compared with B-other with a 5-year survival of 23% (vs 59% for B-other, P = .006). Sixty-eight percent of patients with Ph-like ALL were of Hispanic ethnicity. The following were associated with inferior OS on multivariable analysis: age (hazard ratio [HR], 3.299; P < .001), white blood cell count (HR, 1.910; P = .017), platelet count (HR, 7.437; P = .005), and Ph-like ALL (HR, 1.818; P = .03). Next-generation sequencing of the CRLF2+ group identified mutations in the JAK-STAT and Ras pathway in 85% of patients, and 20% had a CRLF2 mutation. Within the CRLF2+ group, JAK2 mutation was associated with inferior outcomes. Our findings show high frequency of Ph-like ALL in adults, an increased frequency of Ph-like ALL in adults of Hispanic ethnicity, significantly inferior outcomes of adult patients with Ph-like ALL, and significantly worse outcomes in the CRLF2+ subset of Ph-like ALL. Novel strategies are needed to improve the outcome of these patients.

Targetable kinase gene fusions in high-risk B-ALL: a study from the Children's Oncology Group.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS: We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS: Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS: Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia.

Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL.

A genome-wide association study of susceptibility to acute lymphoblastic leukemia in adolescents and young adults.

Acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA) is characterized by distinct presenting features and inferior prognosis compared with pediatric ALL. We performed a genome-wide association study (GWAS) to comprehensively identify inherited genetic variants associated with susceptibility to AYA ALL. In the discovery GWAS, we compared genotype frequency at 635 297 single nucleotide polymorphisms (SNPs) in 308 AYA ALL cases and 6,661 non-ALL controls by using a logistic regression model with genetic ancestry as a covariate. SNPs that reached P ≤ 5 × 10(-8) in GWAS were tested in an independent cohort of 162 AYA ALL cases and 5,755 non-ALL controls. We identified a single genome-wide significant susceptibility locus in GATA3: rs3824662, odds ratio (OR), 1.77 (P = 2.8 × 10(-10)) and rs3781093, OR, 1.73 (P = 3.2 × 10(-9)). These findings were validated in the replication cohort. The risk allele at rs3824662 was most frequent in Philadelphia chromosome (Ph)-like ALL but also conferred susceptibility to non-Ph-like ALL in AYAs. In 1,827 non-selected ALL cases, the risk allele frequency at this SNP was positively correlated with age at diagnosis (P = 6.29 × 10(-11)). Our results from this first GWAS of AYA ALL susceptibility point to unique biology underlying leukemogenesis and potentially distinct disease etiology by age group.

CREBBP mutations in relapsed acute lymphoblastic leukaemia.

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.

Tyrosine kinome sequencing of pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group TARGET Project.

One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1-like or Ph-like because of similarity of the gene expression profile to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that approximately half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with either a Ph-like gene expression profile or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and 1 FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high-risk ALL.

CREST maps somatic structural variation in cancer genomes with base-pair resolution.

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

Outcome modeling with CRLF2, IKZF1, JAK, and minimal residual disease in pediatric acute lymphoblastic leukemia: a Children's Oncology Group study.

As controversy exists regarding the prognostic significance of genomic rearrangements of CRLF2 in pediatric B-precursor acute lymphoblastic leukemia (ALL) classified as standard/intermediate-risk (SR) or high-risk (HR), we assessed the prognostic significance of CRLF2 mRNA expression, CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2 F232C), deletion/mutation in genes frequently associated with high CRLF2 expression (IKZF1, JAK, IL7R), and minimal residual disease (MRD) in 1061 pediatric ALL patients (499 HR and 562 SR) on COG Trials P9905/P9906. Whereas very high CRLF2 expression was found in 17.5% of cases, only 51.4% of high CRLF2 expressors had CRLF2 genomic lesions. The mechanism underlying elevated CRLF2 expression in cases lacking known genomic lesions remains to be determined. All CRLF2 genomic lesions and virtually all JAK mutations were found in high CRLF2 expressors, whereas IKZF1 deletions/mutations were distributed across the full cohort. In multivariate analyses, NCI risk group, MRD, high CRLF2 expression, and IKZF1 lesions were associated with relapse-free survival. Within HR ALL, only MRD and CRLF2 expression predicted a poorer relapse-free survival; no difference was seen between cases with or without CRLF2 genomic lesions. Thus, high CRLF2 expression is associated with a very poor outcome in high-risk, but not standard-risk, ALL. This study is registered at www.clinicaltrials.gov as NCT00005596 and NCT00005603.

SJPedPanel: A pan-cancer gene panel for childhood malignancies.

Background: Large scale genomics projects have identified driver alterations for most childhood cancers that provide reliable biomarkers for clinical diagnosis and disease monitoring using targeted sequencing. However, there is lack of a comprehensive panel that matches the list of known driver genes. Here we fill this gap by developing SJPedPanel for childhood cancers. Results: SJPedPanel covers 5,275 coding exons of 357 driver genes, 297 introns frequently involved in rearrangements that generate fusion oncoproteins, commonly amplified/deleted regions (e.g., MYCN for neuroblastoma, CDKN2A and PAX5 for B-/T-ALL, and SMARCB1 for AT/RT), and 7,590 polymorphism sites for interrogating tumors with aneuploidy, such as hyperdiploid and hypodiploid B-ALL or 17q gain neuroblastoma. We used driver alterations reported from an established real-time clinical genomics cohort (n=253) to validate this gene panel. Among the 485 pathogenic variants reported, our panel covered 417 variants (86%). For 90 rearrangements responsible for oncogenic fusions, our panel covered 74 events (82%). We re-sequenced 113 previously characterized clinical specimens at an average depth of 2,500X using SJPedPanel and recovered 354 (91%) of the 389 reported pathogenic variants. We then investigated the power of this panel in detecting mutations from specimens with low tumor purity (as low as 0.1%) using cell line-based dilution experiments and discovered that this gene panel enabled us to detect ∼80% variants with allele fraction of 0.2%, while the detection rate decreases to ∼50% when the allele fraction is 0.1%. We finally demonstrate its utility in disease monitoring on clinical specimens collected from AML patients in morphologic remission. Conclusions: SJPedPanel enables the detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions for childhood cancers by targeted sequencing of ∼0.15% of human genome. It will enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Evaluation of cytarabine against Ewing sarcoma xenografts by the pediatric preclinical testing program.

Treatment with the nucleoside analog cytarabine has been shown to mimic changes in gene expression associated with downregulation of the EWS-FLI1 oncogene in Ewing sarcoma cell lines, selectively inhibit their growth in vitro, and cause tumor regression in athymic nude mice. For this report cytarabine was studied in vitro against a panel of 23 pediatric cancer cell lines and in vivo against 6 Ewing sarcoma xenografts. Acute lymphoblastic leukemia cell lines were the most sensitive to cytarabine in vitro (median IC(50) 9 nM), while Ewing sarcoma cell lines showed intermediate sensitivity (median IC(50) 232 nM). Cytarabine at a dose of 150 mg/kg administered daily 5× failed to significantly inhibit growth of five xenograft models, but reduced growth rate of the A673 xenograft by 50%. Cytarabine shows no differential in vitro activity against Ewing sarcoma cell lines and is ineffective in vivo against Ewing sarcoma xenografts at the dose and schedule studied.

Relapse-Fated Latent Diagnosis Subclones in Acute B Lineage Leukemia Are Drug Tolerant and Possess Distinct Metabolic Programs.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.

Mutational landscape and patterns of clonal evolution in relapsed pediatric acute lymphoblastic leukemia.

Relapse of acute lymphoblastic leukemia (ALL) remains a leading cause of childhood death. Prior studies have shown clonal mutations at relapse often arise from relapse-fated subclones that exist at diagnosis. However, the genomic landscape, evolutionary trajectories and mutational mechanisms driving relapse are incompletely understood. In an analysis of 92 cases of relapsed childhood ALL, incorporating multimodal DNA and RNA sequencing, deep digital mutational tracking and xenografting to formally define clonal structure, we identify 50 significant targets of mutation with distinct patterns of mutational acquisition or enrichment. CREBBP, NOTCH1, and Ras signaling mutations rose from diagnosis subclones, whereas variants in NCOR2, USH2A and NT5C2 were exclusively observed at relapse. Evolutionary modeling and xenografting demonstrated that relapse-fated clones were minor (50%), major (27%) or multiclonal (18%) at diagnosis. Putative second leukemias, including those with lineage shift, were shown to most commonly represent relapse from an ancestral clone rather than a truly independent second primary leukemia. A subset of leukemias prone to repeated relapse exhibited hypermutation driven by at least three distinct mutational processes, resulting in heightened neoepitope burden and potential vulnerability to immunotherapy. Finally, relapse-driving sequence mutations were detected prior to relapse using deep digital PCR at levels comparable to orthogonal approaches to monitor levels of measurable residual disease. These results provide a genomic framework to anticipate and circumvent relapse by earlier detection and targeting of relapse-fated clones.

Germline deletion of ETV6 in familial acute lymphoblastic leukemia.

Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.