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Oncogenic fusions formed through chromosomal rearrangements are hallmarks of childhood cancer that define cancer subtype, predict outcome, persist through treatment, and can be ideal therapeutic targets. However, mechanistic understanding of the etiology of oncogenic fusions remains elusive. Here we report a comprehensive detection of 272 oncogenic fusion gene pairs by using tumor transcriptome sequencing data from 5190 childhood cancer patients. We identify diverse factors, including translation frame, protein domain, splicing, and gene length, that shape the formation of oncogenic fusions. Our mathematical modeling reveals a strong link between differential selection pressure and clinical outcome in CBFB-MYH11. We discover 4 oncogenic fusions, including RUNX1-RUNX1T1, TCF3-PBX1, CBFA2T3-GLIS2, and KMT2A-AFDN, with promoter-hijacking-like features that may offer alternative strategies for therapeutic targeting. We uncover extensive alternative splicing in oncogenic fusions including KMT2A-MLLT3, KMT2A-MLLT10, C11orf95-RELA, NUP98-NSD1, KMT2A-AFDN and ETV6-RUNX1. We discover neo splice sites in 18 oncogenic fusion gene pairs and demonstrate that such splice sites confer therapeutic vulnerability for etiology-based genome editing. Our study reveals general principles on the etiology of oncogenic fusions in childhood cancer and suggests profound clinical implications including etiology-based risk stratification and genome-editing-based therapeutics.

Studies focusing on genetic susceptibility of childhood Hodgkin lymphoma (HL) are limited.To identify genetic variants associated with childhood-onset HL vs adult-onset HL.This genetic association study was performed with 3 cohorts: the St Jude Lifetime Cohort Study (SJLIFE), initiated in 2007 with ongoing follow-up, and the original and expansion cohorts of the Childhood Cancer Survivor Study (CCSS), initiated in the 1990s with ongoing follow-up. Results of these genome-wide association studies (GWASs) were combined via meta-analysis. Data were analyzed from June 2021 to June 2022.Childhood HL was the focused outcome. Single-nucleotide variant (SNV, formerly single-nucleotide polymorphism) array genotyping and imputation were conducted for the CCSS original cohort, and whole-genome sequencing was performed for the SJLIFE and CCSS expansion cohort.A total of 1286 HL cases (mean diagnosis [SD] age, 14.6 [3.9] years), 6193 non-HL childhood cancer cases, and 369 noncancer controls, all of European ancestry, were included in the analysis. Using step-wise conditional logistic regression, the odds ratios (ORs) for each of the 3 independent SNVs identified in the human leukocyte antigen (HLA) locus were 1.80 (95% CI, 1.59-2.03; P = 2.14 × 10-21) for rs28383311, 1.53 (95% CI, 1.37-1.70; P = 2.05 × 10-14) for rs3129198, and 1.51 (95% CI, 1.35-1.69; P = 6.21 × 10-13) for rs3129890. Further HLA imputation revealed 9 alleles and 55 amino acid changes that potentially conferred HL susceptibility. In addition, 5 non-HLA loci were identified: (1) rs1432297 (OR, 1.29; 95% CI, 1.18-1.41; P = 2.5 × 10-8; r2 = 0.55; D' = 0.75 with previously reported rs1432295, REL); (2) rs2757647 (OR, 1.30; 95% CI, 1.18-1.42; P = 3.5 × 10-8; r2 = 0.59; D' = 0.83 with previously reported rs6928977, AHI1); (3) rs13279159 (OR, 1.33; 95% CI, 1.20-1.47; P = 1.7 × 10-8; r2 = 0.75; D' = 1.00 with previously reported rs2019960, PVT1); (4) rs3824662 (OR, 1.52; 95% CI, 1.33-1.73; P = 3.9 × 10-10; r2 = 0.91; D' = 1.00 with previously reported rs3781093, GATA3); and (5) rs117953624 (OR, 1.98; 95% CI, 1.56-2.51; P = 1.5 × 10-8; minor allele frequency, 0.02), a novel uncommon SNV mapped to PDGFD. Twelve of 18 previously reported genome-wide significant non-HLA SNVs (67%) were replicated with statistically significant results.In this genetic association study, a predominantly common and potentially unique genetic etiology was found between childhood-onset and adulthood-onset HL.

Background Adult childhood cancer survivors are at risk for frailty, including low muscle mass and weakness (sarcopenia). Using peripheral blood mitochondrial DNA copy number (mtDNAcn) as a proxy for functional mitochondria, this study describes cross-sectional associations between mtDNAcn and sarcopenia among survivors. Methods Among 1762 adult childhood cancer survivors (51.6% male; median age = 29.4 years, interquartile range [IQR] = 23.3-36.8), with a median of 20.6 years from diagnosis (IQR = 15.2-28.2), mtDNAcn estimates were derived from whole-genome sequencing. A subset was validated by quantitative polymerase chain reaction and evaluated cross-sectionally using multivariable logistic regression for their association with sarcopenia, defined by race-, age-, and sex-specific low lean muscle mass or weak grip strength. All statistical tests were 2-sided. Results The prevalence of sarcopenia was 27.0%, higher among female than male survivors (31.5% vs 22.9%; P Conclusions A growing body of evidence supports peripheral blood mtDNAcn as a biomarker for adverse health outcomes; however, this study is the first to report an association between mtDNAcn and sarcopenia among childhood cancer survivors.

The majority of diffuse midline gliomas, H3 K27-altered (DMG-H3 K27-a), are infiltrating pediatric brain tumors that arise in the pons with no effective treatment. To understand how clonal evolution contributes to the tumor’s invasive spread, we performed exome sequencing and SNP array profiling on 49 multi-region autopsy samples from 11 patients with pontine DMG-H3 K27-a enrolled in a phase I clinical trial of PDGFR inhibitor crenolanib. For each patient, a phylogenetic tree was constructed by testing multiple possible clonal evolution models to select the one consistent with somatic mutations and copy number variations across all tumor regions. The tree was then used to deconvolute subclonal composition and prevalence at each tumor region to study convergent evolution and invasion patterns. Somatic variants in the PI3K pathway, a late event, are enriched in our cohort, affecting 70% of patients. Convergent evolution of PI3K at distinct phylogenetic branches was detected in 40% of the patients. 24 (~ 50%) of tumor regions were occupied by subclones of mixed lineages with varying molecular ages, indicating multiple waves of invasion across the pons and extrapontine. Subclones harboring a PDGFRA amplicon, including one that amplified a PDGRFAY849C mutant allele, were detected in four patients; their presence in extrapontine tumor and normal brain samples imply their involvement in extrapontine invasion. Our study expands the current knowledge on tumor invasion patterns in DMG-H3 K27-a, which may inform the design of future clinical trials.

Background Mounting evidence supports the occurrence of accelerating aging among long-term survivors of childhood cancer. We aimed to investigate epigenetic age acceleration (EAA) in survivors and evaluate associations between EAA, treatment exposures, health behaviors, and chronic health conditions (CHCs). Methods Genome-wide methylation data were generated with Infinium EPIC BeadChip on blood-derived DNA from 2139 survivors and 282 frequency matched controls from the St Jude Lifetime Cohort Study. EAAs were estimated as residuals from a linear regression of epigenetic age (Levine’s clock) against chronological age. Adjusted least square mean (ALSM) of EAA was calculated and compared between survivors and controls, across treatment exposures and health behaviors. Associations of EAA with 20 clinically assessed CHCs were evaluated with multivariable piecewise-exponential models. All statistical tests for P values below were 2-sided. Results EAA was statistically significantly higher in survivors than controls (ALSM = 0.63, 95% confidence interval [CI] = 0.26 to 1.01 vs −3.61, 95% CI = −4.43 to 2.80). In a multivariable model among survivors, statistically significantly higher EAA (P Conclusions EAA is statistically significantly higher in survivors of childhood cancer than in noncancer controls and is associated with specific treatment exposures, unfavorable health behaviors, and presence of specific CHCs.

PURPOSE To investigate cancer treatment plus pathogenic germline mutations (PGMs) in DNA repair genes (DRGs) for identification of childhood cancer survivors at increased risk of subsequent neoplasms (SNs). METHODS Whole-genome sequencing was performed on blood-derived DNA from survivors in the St Jude Lifetime Cohort. PGMs were evaluated in 127 genes from 6 major DNA repair pathways. Cumulative doses of chemotherapy and body region–specific radiotherapy (RT) were abstracted from medical records. Relative rates (RRs) and 95% CIs of SNs by mutation status were estimated using multivariable piecewise exponential models. RESULTS Of 4,402 survivors, 495 (11.2%) developed 1,269 SNs. We identified 538 PGMs in 98 DRGs ( POLG, MUTYH, ERCC2, and BRCA2, among others) in 508 (11.5%) survivors. Mutations in homologous recombination (HR) genes were significantly associated with an increased rate of subsequent female breast cancer (RR, 3.7; 95% CI, 1.8 to 7.7), especially among survivors with chest RT ≥ 20 Gy (RR, 4.4; 95% CI, 1.6 to 12.4), or with a cumulative dose of anthracyclines in the second or third tertile (RR, 4.4; 95% CI, 1.7 to 11.4). Mutations in HR genes were also associated with an increased rate of subsequent sarcoma among those who received alkylating agent doses in the third tertile (RR, 14.9; 95% CI, 4.0 to 38.0). Mutations in nucleotide excision repair genes were associated with subsequent thyroid cancer for those treated with neck RT ≥ 30 Gy (RR, 12.9; 95% CI, 1.6 to 46.6) with marginal statistical significance. CONCLUSION Our study provides novel insights regarding the contribution of genetics, in combination with known treatment-related risks, for the development of SNs. These findings have the potential to facilitate identification of high-risk survivors who may benefit from genetic counseling and/or testing of DRGs, which may further inform personalized cancer surveillance and prevention strategies.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Background: Subsequent thyroid cancer (STC) is one of the most common malignancies in childhood cancer survivors. We aimed to evaluate the polygenic contributions to STC risk and potential utility in improving risk prediction. Methods: A polygenic risk score (PRS) was calculated from 12 independent SNPs associated with thyroid cancer risk in the general population. Associations between PRS and STC risk were evaluated among survivors from St. Jude Lifetime Cohort (SJLIFE) and were replicated in survivors from Childhood Cancer Survivor Study (CCSS). A risk prediction model integrating the PRS and clinical factors, initially developed in SJLIFE, and its performance were validated in CCSS. Results: Among 2,370 SJLIFE survivors with a median follow-up of 28.8 [interquartile range (IQR) = 21.9–36.1] years, 65 (2.7%) developed STC. Among them, the standardized PRS was associated with an increased rate of STC [relative rate (RR) = 1.57; 95% confidence interval (CI) = 1.24–1.98; P < 0.001]. Similar associations were replicated in 6,416 CCSS survivors, among whom 121 (1.9%) developed STC during median follow-up of 28.9 (IQR = 22.6–34.6) years (RR = 1.52; 95% CI = 1.25–1.83; P < 0.001). A risk prediction model integrating the PRS with clinical factors showed better performance than the model considering only clinical factors in SJLIFE (P = 0.004, AUC = 83.2% vs. 82.1%, at age 40), which was further validated in CCSS (P = 0.010, AUC = 72.9% vs. 70.6%). Conclusions: Integration of the PRS with clinical factors provided a statistically significant improvement in risk prediction of STC, although the magnitude of improvement was modest. Impact: PRS improves risk stratification and prediction of STC, suggesting its potential utility for optimizing screening strategies in survivorship care.

Spitzoid melanoma is a specific morphologic variant of melanoma that most commonly affects children and adolescents, and ranges on the spectrum of malignancy from low grade to overtly malignant. These tumors are generally driven by fusions of ALK, RET, NTRK1/3, MET, ROS1 and BRAF1,2. However, in approximately 50% of cases no genetic driver has been established2. Clinical whole-genome and transcriptome sequencing (RNA-Seq) of a spitzoid tumor from an adolescent revealed a novel gene fusion of MAP3K8, encoding a serine-threonine kinase that activates MEK3,4. The patient, who had exhausted all other therapeutic options, was treated with a MEK inhibitor and underwent a transient clinical response. We subsequently analyzed spitzoid tumors from 49 patients by RNA-Seq and found in-frame fusions or C-terminal truncations of MAP3K8 in 33% of cases. The fusion transcripts and truncated genes all contained MAP3K8 exons 1–8 but lacked the autoinhibitory final exon. Data mining of RNA-Seq from the Cancer Genome Atlas (TCGA) uncovered analogous MAP3K8 rearrangements in 1.5% of adult melanomas. Thus, MAP3K8 rearrangements—uncovered by comprehensive clinical sequencing of a single case—are the most common genetic event in spitzoid melanoma, are present in adult melanomas and could be amenable to MEK inhibition. Rearrangements in MAP3K8 respond to MEK inhibition and represent the most common genetic driver in pediatric spitzoid melanoma, and in some adult melanomas lacking other MAPK alterations and for which clinical testing is warranted.

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSβ0-thalassemia from Baylor College of Medicine and from the St. Jude Children’s Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion −α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the β-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10−14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10−13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.

BackgroundThere is currently no method to precisely measure the errors that occur in the sequencing instrument/sequencer, which is critical for next-generation sequencing applications aimed at discovering the genetic makeup of heterogeneous cellular populations.ResultsWe propose a novel computational method, SequencErr, to address this challenge by measuring the base correspondence between overlapping regions in forward and reverse reads. An analysis of 3777 public datasets from 75 research institutions in 18 countries revealed the sequencer error rate to be ~ 10 per million (pm) and 1.4% of sequencers and 2.7% of flow cells have error rates > 100 pm. At the flow cell level, error rates are elevated in the bottom surfaces and > 90% of HiSeq and NovaSeq flow cells have at least one outlier error-prone tile. By sequencing a common DNA library on different sequencers, we demonstrate that sequencers with high error rates have reduced overall sequencing accuracy, and removal of outlier error-prone tiles improves sequencing accuracy. We demonstrate that SequencErr can reveal novel insights relative to the popular quality control method FastQC and achieve a 10-fold lower error rate than popular error correction methods including Lighter and Musket.ConclusionsOur study reveals novel insights into the nature of DNA sequencing errors incurred on DNA sequencers. Our method can be used to assess, calibrate, and monitor sequencer accuracy, and to computationally suppress sequencer errors in existing datasets.

Integrating somatic mutation analysis and gene expression profiling distinguishes pediatric AML subtypes with differential prognoses and clinical risks., Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes. To better understand neuroblastoma pathogenesis, here we analyze whole-genome, whole-exome and/or transcriptome data from 702 neuroblastoma samples. Forty percent of samples harbor at least one recurrent driver gene alteration and most aberrations, including MYCN, ATRX, and TERT alterations, differ in frequency by age. MYCN alterations occur at median 2.3 years of age, TERT at 3.8 years, and ATRX at 5.6 years. COSMIC mutational signature 18, previously associated with reactive oxygen species, is the most common cause of driver point mutations in neuroblastoma, including most ALK and Ras-activating variants. Signature 18 appears early and is continuous throughout disease evolution. Signature 18 is enriched in neuroblastomas with MYCN amplification, 17q gain, and increased expression of mitochondrial ribosome and electron transport-associated genes. Recurrent FGFR1 variants in six patients, and ALK N-terminal structural alterations in five samples, identify additional patients potentially amenable to precision therapy., Genomic analysis of neuroblastoma has revealed important disease etiology. In this study, the authors assembled whole genome, exome and transcriptome data from over 700 neuroblastomas and identified molecular signatures correlated with age, and rare, potentially targetable variants overlooked in smaller cohorts.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma., In most cancers, mutations that lead to oncogene activation and tumor suppressor inactivation synergize to promote tumorigenesis. However, in neuroblastomas, MYCN amplification and ATRX mutations are mutually exclusive and incompatible.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for characterizing the cell-to-cell variation and cellular dynamics in populations which appear homogeneous otherwise in basic and translational biological research. However, significant challenges arise in the analysis of scRNA-seq data, including the low signal-to-noise ratio with high data sparsity, potential batch effects, scalability problems when hundreds of thousands of cells are to be analyzed among others. The inherent complexities of scRNA-seq data and dynamic nature of cellular processes lead to suboptimal performance of many currently available algorithms, even for basic tasks such as identifying biologically meaningful heterogeneous subpopulations. In this study, we developed the Latent Cellular Analysis (LCA), a machine learning–based analytical pipeline that combines cosine-similarity measurement by latent cellular states with a graph-based clustering algorithm. LCA provides heuristic solutions for population number inference, dimension reduction, feature selection, and control of technical variations without explicit gene filtering. We show that LCA is robust, accurate, and powerful by comparison with multiple state-of-the-art computational methods when applied to large-scale real and simulated scRNA-seq data. Importantly, the ability of LCA to learn from representative subsets of the data provides scalability, thereby addressing a significant challenge posed by growing sample sizes in scRNA-seq data analysis.

Purpose: We aimed to analyze and compare leukocyte telomere length (LTL) and age-dependent LTL attrition between childhood cancer survivors and noncancer controls, and to evaluate the associations of LTL with treatment exposures, chronic health conditions (CHC), and health behaviors among survivors. Experimental Design: We included 2,427 survivors and 293 noncancer controls of European ancestry, drawn from the participants in St. Jude Lifetime Cohort Study (SJLIFE), a retrospective hospital-based study with prospective follow-up (2007–2016). Common nonneoplastic CHCs (59 types) and subsequent malignant neoplasms (5 types) were clinically assessed. LTL was measured with whole-genome sequencing data. Results: After adjusting for age at DNA sampling, gender, genetic risk score based on 9 SNPs known to be associated with telomere length, and eigenvectors, LTL among survivors was significantly shorter both overall [adjusted mean (AM) = 6.20 kb; SE = 0.03 kb] and across diagnoses than controls (AM = 6.69 kb; SE = 0.07 kb). Among survivors, specific treatment exposures associated with shorter LTL included chest or abdominal irradiation, glucocorticoid, and vincristine chemotherapies. Significant negative associations of LTL with 14 different CHCs, and a positive association with subsequent thyroid cancer occurring out of irradiation field were identified. Health behaviors were significantly associated with LTL among survivors aged 18 to 35 years (Ptrend = 0.03). Conclusions: LTL is significantly shorter among childhood cancer survivors than noncancer controls, and is associated with CHCs and health behaviors, suggesting LTL as an aging biomarker may be a potential mechanistic target for future intervention studies designed to prevent or delay onset of CHCs in childhood cancer survivors. See related commentary by Walsh, p. 2281

Background Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. Results By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10−5 to 10−4, which is 10- to 100-fold lower than generally considered achievable (10−3) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10−5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10−4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. Conclusions We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing. Electronic supplementary material The online version of this article (10.1186/s13059-019-1659-6) contains supplementary material, which is available to authorized users.

Purpose: The risk of subsequent breast cancer among female childhood cancer survivors is markedly elevated. We aimed to determine genetic contributions to this risk, focusing on polygenic determinants implicated in breast cancer susceptibility in the general population. Experimental Design: Whole-genome sequencing (30×) was performed on survivors in the St Jude Lifetime Cohort, and germline mutations in breast cancer predisposition genes were classified for pathogenicity. A polygenic risk score (PRS) was constructed for each survivor using 170 established common risk variants. Relative rate (RR) and 95% confidence interval (95% CI) of subsequent breast cancer incidence were estimated using multivariable piecewise exponential regression. Results: The analysis included 1,133 female survivors of European ancestry (median age at last follow-up = 35.4 years; range, 8.4–67.4), of whom 47 were diagnosed with one or more subsequent breast cancers (median age at subsequent breast cancer = 40.3 years; range, 24.5–53.0). Adjusting for attained age, age at primary diagnosis, chest irradiation, doses of alkylating agents and anthracyclines, and genotype eigenvectors, RRs for survivors with PRS in the highest versus lowest quintiles were 2.7 (95% CI, 1.0–7.3), 3.0 (95% CI, 1.1–8.1), and 2.4 (95% CI, 0.1–81.1) for all survivors and survivors with and without chest irradiation, respectively. Similar associations were observed after excluding carriers of pathogenic/likely pathogenic mutations in breast cancer predisposition genes. Notably, the PRS was associated with the subsequent breast cancer rate under the age of 45 years (RR = 3.2; 95% CI, 1.2–8.3). Conclusions: Genetic profiles comprised of small-effect common variants and large-effect predisposing mutations can inform personalized breast cancer risk and surveillance/intervention in female childhood cancer survivors.

Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric patient, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were present in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment. However, the somatic mutation was present at very low frequency at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript isoform absent in EST database, as well as about 700 RNA-seq of normal brains that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis/relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants. Electronic supplementary material The online version of this article (10.1007/s00401-018-1912-1) contains supplementary material, which is available to authorized users.

Purpose Childhood cancer survivors are at increased risk of subsequent neoplasms (SNs), but the germline genetic contribution is largely unknown. We assessed the contribution of pathogenic/likely pathogenic (P/LP) mutations in cancer predisposition genes to their SN risk. Patients and Methods Whole-genome sequencing (30-fold) was performed on samples from childhood cancer survivors who were ≥ 5 years since initial cancer diagnosis and participants in the St Jude Lifetime Cohort Study, a retrospective hospital-based study with prospective clinical follow-up. Germline mutations in 60 genes known to be associated with autosomal dominant cancer predisposition syndromes with moderate to high penetrance were classified by their pathogenicity according to the American College of Medical Genetics and Genomics guidelines. Relative rates (RRs) and 95% CIs of SN occurrence by mutation status were estimated using multivariable piecewise exponential regression stratified by radiation exposure. Results Participants were 3,006 survivors (53% male; median age, 35.8 years [range, 7.1 to 69.8 years]; 56% received radiotherapy), 1,120 SNs were diagnosed among 439 survivors (14.6%), and 175 P/LP mutations were identified in 5.8% (95% CI, 5.0% to 6.7%) of survivors. Mutations were associated with significantly increased rates of breast cancer (RR, 13.9; 95% CI, 6.0 to 32.2) and sarcoma (RR, 10.6; 95% CI, 4.3 to 26.3) among irradiated survivors and with increased rates of developing any SN (RR, 4.7; 95% CI, 2.4 to 9.3), breast cancer (RR, 7.7; 95% CI, 2.4 to 24.4), nonmelanoma skin cancer (RR, 11.0; 95% CI, 2.9 to 41.4), and two or more histologically distinct SNs (RR, 18.6; 95% CI, 3.5 to 99.3) among nonirradiated survivors. Conclusion The findings support referral of all survivors for genetic counseling for potential clinical genetic testing, which should be prioritized for nonirradiated survivors with any SN and for those with breast cancer or sarcoma in the field of prior irradiation.

The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes., The progress in pre-clinical drug discovery for Wilms tumor (WT) is limited by a lack of disease models. Here, the authors develop 45 heterotopic WT patient-derived xenografts including several anaplastic models that recapitulate the biological heterogeneity of WT, and propose this as a resource for evaluating future therapeutics for WT.

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts. While rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML cases. AMKL in individuals without Down syndrome (non-DS-AMKL) is frequently associated with poor clinical outcomes. Previous efforts have identified chimeric oncogenes in a substantial number of non-DS-AMKL cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements, and NUP98-KDM5A. However, the etiology of 30-40% of cases remains unknown. To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult). We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes. These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes. Overall, these data shed light on the etiology of AMKL and provide useful information for the tailoring of treatment.

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

SummaryRhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving subunits of the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n=87) and adult (n=78) samples, including cases with RUNX1-RUNX1T1 (n=85) or CBFB-MYH11 (n=80) rearrangements, by whole-genome or whole-exome sequencing. In addition to previously reported somatic mutations in the Ras signaling pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a recurrent and previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated a remarkably different spectrum of cooperating mutations as RUNX1-RUNX1T1 cases harbored recurrent somatic mutations in DHX15 and ZBTB7A, as well as an enrichment of somatic mutations in epigenetic regulators, including ASXL2, and in components of the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscape of cooperating mutations between these related AML subtypes.

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n=22), diffuse oligodendroglial tumors (d-OTs; n=20), diffuse astrocytomas (DAs; n=17), angiocentric gliomas (n=15), and gangliogliomas (n=17). Most LGNTs (84%) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82% of DNETs and 40% of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87%), and MYB fusion genes were the most common genetic alteration in DAs (41%). A BRAF:p.V600E mutation was present in 35% of gangliogliomas and 18% of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78% of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

The introduction of cultured p185BCR-ABL-expressing (p185+) Arf−/− pre-B cells into healthy syngeneic mice induces aggressive acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease. We adapted this high-throughput Philadelphia chromosome–positive (Ph+) ALL animal model for in vivo luminescent imaging to investigate disease progression, targeted therapeutic response, and ALL relapse in living mice. Mice bearing high leukemic burdens (simulating human Ph+ ALL at diagnosis) entered remission on maximally intensive, twice-daily dasatinib therapy, but invariably relapsed with disseminated and/or central nervous system disease. Although relapse was frequently accompanied by the eventual appearance of leukemic clones harboring BCR-ABL kinase domain (KD) mutations that confer drug resistance, their clonal emergence required prolonged dasatinib exposure. KD P-loop mutations predominated in mice receiving less intensive therapy, whereas high-dose treatment selected for T315I “gatekeeper” mutations resistant to all 3 Food and Drug Administration–approved BCR-ABL kinase inhibitors. The addition of dexamethasone and/or L-asparaginase to reduced-intensity dasatinib therapy improved long-term survival of the majority of mice that received all 3 drugs. Although non–tumor-cell–autonomous mechanisms can prevent full eradication of dasatinib-refractory ALL in this clinically relevant model, the emergence of resistance to BCR-ABL kinase inhibitors can be effectively circumvented by the addition of “conventional” chemotherapeutic agents with alternate antileukemic mechanisms of action.

Summary Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis. Pediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here the authors analyse the genomes, exomes and transcriptomes of 37 such tumours and identify genetic alterations whose nature, timing and potential interactions are key events with prognostic significance in pediatric adrenocortical tumorigenesis.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

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.)

Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and medulloblastoma, and a paucity of mutations in low-grade glioma and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A and ZMYM3. We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.

Osteosarcoma is a neoplasm of mesenchymal origin with features of osteogenic differentiation. Patients with recurrent or metastatic disease have a very poor prognosis. To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue (obtained from 19 patients) in the discovery cohort as well as 14 samples from 13 patients in the validation cohort. Our results demonstrate that pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). Moreover, single nucleotide variations (SNVs) exhibit a pattern of localized hypermutation called “kataegis” in 50% of the tumors. Despite these regions of kataegis across the osteosarcoma genomes, we detected relatively few recurrent SNVs, and only when SVs were included did we identify the major pathways that are mutated in osteosarcoma. We identified p53 pathway lesions in all 19 patient’s tumors in the discovery cohort, 9 of which were translocations in the first intron of the TP53 gene, leading to gene inactivation. This mechanism of p53 gene inactivation is unique to osteosarcoma among pediatric cancers. In an additional cohort of 32 patients, TP53 gene alterations were identified in 29 of those tumors. Beyond TP53, the RB1, ATRX and DLG2 genes showed recurrent somatic alterations (SNVs and/or SVs) in 29–53% of the tumors. These data highlight the power of whole-genome sequencing in identifying recurrent somatic alterations in cancer genomes that may be missed using other methods.