Cancer germline predisposing variants and late mortality from subsequent malignant neoplasms among long-term childhood cancer survivors: a report from the St Jude Lifetime Cohort and the Childhood Cancer Survivor Study.

BACKGROUND: Carriers of cancer predisposing variants are at an increased risk of developing subsequent malignant neoplasms among those who have survived childhood cancer. We aimed to investigate whether cancer predisposing variants contribute to the risk of subsequent malignant neoplasm-related late mortality (5 years or more after diagnosis). METHODS: In this analysis, data were included from two retrospective cohort studies, St Jude Lifetime Cohort (SJLIFE) and the Childhood Cancer Survivor Study (CCSS), with prospective follow-up of patients who were alive for at least 5 years after diagnosis with childhood cancer (ie, long-term childhood cancer survivors) with corresponding germline whole genome or whole exome sequencing data. Cancer predisposing variants affecting 60 genes associated with well-established autosomal-dominant cancer-predisposition syndromes were characterised. Subsequent malignant neoplasms were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 with modifications. Cause-specific late mortality was based on linkage with the US National Death Index and systematic cohort follow up. Fine-Gray subdistribution hazard models were used to estimate subsequent malignant neoplasm-related late mortality starting from the first biospecimen collection, treating non-subsequent malignant neoplasm-related deaths as a competing risk, adjusting for genetic ancestry, sex, age at diagnosis, and cancer treatment exposures. SJLIFE (NCT00760656) and CCSS (NCT01120353) are registered with ClinicalTrials.gov. FINDINGS: 12 469 (6172 male and 6297 female) participants were included, 4402 from the SJLIFE cohort (median follow-up time since collection of the first biospecimen 7·4 years [IQR 3·1-9·4]) and 8067 from the CCSS cohort (median follow-up time since collection of the first biospecimen 12·6 years [2·2-16·6]). 641 (5·1%) of 12 469 participants carried cancer predisposing variants (294 [6·7%] in the SJLIFE cohort and 347 [4·3%] in the CCSS cohort), which were significantly associated with an increased severity of subsequent malignant neoplasms (CTCAE grade ≥4 vs grade <4: odds ratio 2·15, 95% CI 1·18-4·19, p=0·0085). 263 (2·1%) subsequent malignant neoplasm-related deaths (44 [1·0%] in the SJLIFE cohort; and 219 [2·7%] in the CCSS cohort) and 426 (3·4%) other-cause deaths (103 [2·3%] in SJLIFE; and 323 [4·0%] in CCSS) occurred. Cumulative subsequent malignant neoplasm-related mortality at 10 years after the first biospecimen collection in carriers of cancer predisposing variants was 3·7% (95% CI 1·2-8·5) in SJLIFE and 6·9% (4·1-10·7) in CCSS versus 1·5% (1·0-2·1) in SJLIFE and 2·1% (1·7-2·5) in CCSS in non-carriers. Carrying a cancer predisposing variant was associated with an increased risk of subsequent malignant neoplasm-related mortality (SJLIFE: subdistribution hazard ratio 3·40 [95% CI 1·37-8·43]; p=0·0082; CCSS: 3·58 [2·27-5·63]; p<0·0001). INTERPRETATION: Identifying participants at increased risk of subsequent malignant neoplasms via genetic counselling and clinical genetic testing for cancer predisposing variants and implementing early personalised cancer surveillance and prevention strategies might reduce the substantial subsequent malignant neoplasm-related mortality burden. FUNDING: American Lebanese Syrian Associated Charities and US National Institutes of Health.

Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia.

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 (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late 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.

Latent cellular analysis robustly reveals subtle diversity in large-scale single-cell RNA-seq data.

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.

C11orf95-RELA fusions drive oncogenic NF-κB signalling in ependymoma.

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.

Associations between mitochondrial copy number, exercise capacity, physiologic cost of walking, and cardiac strain in young adult survivors of childhood cancer.

PURPOSE: Childhood cancer survivors are at risk for cardiac dysfunction and impaired physical performance, though underlying cellular mechanisms are not well studied. In this cross-sectional study, we examined the association between peripheral blood mitochondrial DNA copy number (mtDNA-CN, a proxy for mitochondrial function) and markers of performance impairment and cardiac dysfunction. METHODS: Whole-genome sequencing, validated by quantitative polymerase chain reaction, was used to estimate mtDNA-CN in 1720 adult survivors of childhood cancer (48.5% female; mean age = 30.7 years, standard deviation (SD) = 9.0). Multivariable logistic regression was performed to evaluate the associations between mtDNA-CN and exercise intolerance, walking inefficiency, and abnormal global longitudinal strain (GLS), adjusting for treatment exposures, age, sex, and race and ethnicity. RESULTS: The prevalence of exercise intolerance, walking inefficiency, and abnormal GLS among survivors was 25.7%, 10.7%, and 31.7%, respectively. Each SD increase of mtDNA-CN was associated with decreased odds of abnormal GLS (adjusted odds ratio (OR) = 0.88, p = 0.04) but was not associated with exercise intolerance (OR = 1.02, p = 0.76) or walking inefficiency (OR = 1.06, p = 0.46). Alkylating agent exposure was associated with increased odds of exercise intolerance (OR = 2.25, p < 0.0001), walking inefficiency (OR = 2.37, p < 0.0001), and abnormal GLS (OR = 1.78, p = 0.0002). CONCLUSIONS: Increased mtDNA-CN is associated with decreased odds of abnormal cardiac function in childhood cancer survivors. IMPLICATIONS FOR CANCER SURVIVORS: These findings demonstrate a potential role for mtDNA-CN as a biomarker of early cardiac dysfunction in this population.

Race and Ethnicity, Socioeconomic Factors, and Epigenetic Age Acceleration in Survivors of Childhood Cancer.

Importance: Current research in epigenetic age acceleration (EAA) is limited to non-Hispanic White individuals. It is imperative to improve inclusivity by considering racial and ethnic minorities in EAA research. Objective: To compare non-Hispanic Black with non-Hispanic White survivors of childhood cancer by examining the associations of EAA with cancer treatment exposures, potential racial and ethnic disparity in EAA, and mediating roles of social determinants of health (SDOH). Design, Setting, and Participants: In this cross-sectional study, participants were from the St Jude Lifetime Cohort, which was initiated in 2007 with ongoing follow-up. Eligible participants included non-Hispanic Black and non-Hispanic White survivors of childhood cancer treated at St Jude Children's Research Hospital between 1962 and 2012 who had DNA methylation data. Data analysis was conducted from February 2023 to May 2024. Exposure: Three treatment exposures for childhood cancer (chest radiotherapy, alkylating agents, and epipodophyllotoxin). Main Outcomes and Measures: DNA methylation was generated from peripheral blood mononuclear cell-derived DNA. EAA was calculated as residuals from regressing Levine or Horvath epigenetic age on chronological age. SDOH included educational attainment, annual personal income, and the socioeconomic area deprivation index (ADI). General linear models evaluated cross-sectional associations of EAA with race and ethnicity (non-Hispanic Black and non-Hispanic White) and/or SDOH, adjusting for sex, body mass index, smoking, and cancer treatments. Adjusted least square means (ALSM) of EAA were calculated for group comparisons. Mediation analysis treated SDOH as mediators with average causal mediation effect (ACME) calculated for the association of EAA with race and ethnicity. Results: Among a total of 1706 survivors including 230 non-Hispanic Black survivors (median [IQR] age at diagnosis, 9.5 [4.3-14.3] years; 103 male [44.8%] and 127 female [55.2%]) and 1476 non-Hispanic White survivors (median [IQR] age at diagnosis, 9.3 [3.9-14.6] years; 766 male [51.9%] and 710 female [48.1%]), EAA was significantly greater among non-Hispanic Black survivors (ALSM = 1.41; 95% CI, 0.66 to 2.16) than non-Hispanic White survivors (ALSM = 0.47; 95% CI, 0.12 to 0.81). Among non-Hispanic Black survivors, EAA was significantly increased among those exposed to chest radiotherapy (ALSM = 2.82; 95% CI, 1.37 to 4.26) vs those unexposed (ALSM = 0.46; 95% CI, -0.60 to 1.51), among those exposed to alkylating agents (ALSM = 2.33; 95% CI, 1.21 to 3.45) vs those unexposed (ALSM = 0.95; 95% CI, -0.38 to 2.27), and among those exposed to epipodophyllotoxins (ALSM = 2.83; 95% CI, 1.27 to 4.40) vs those unexposed (ALSM = 0.44; 95% CI, -0.52 to 1.40). The association of EAA with epipodophyllotoxins differed by race and ethnicity (ß for non-Hispanic Black survivors, 2.39 years; 95% CI, 0.74 to 4.04 years; ß for non-Hispanic White survivors, 0.68; 95% CI, 0.05 to 1.31 years) and the difference was significant (1.77 years; 95% CI, 0.01 to 3.53 years; P for interaction = .049). Racial and ethnic disparities in EAA were mediated by educational attainment (

Chemotherapeutic agents circumvent emergence of dasatinib-resistant BCR-ABL kinase mutations in a precise mouse model of Philadelphia chromosome-positive acute lymphoblastic leukemia.

The introduction of cultured p185(BCR-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.

Dynamics of Age- versus Therapy-Related Clonal Hematopoiesis in Long-term Survivors of Pediatric Cancer.

We present the first comprehensive investigation of clonal hematopoiesis (CH) in 2,860 long-term survivors of pediatric cancer with a median follow-up time of 23.5 years. Deep sequencing over 39 CH-related genes reveals mutations in 15% of the survivors, significantly higher than the 8.5% in 324 community controls. CH in survivors is associated with exposures to alkylating agents, radiation, and bleomycin. Therapy-related CH shows significant enrichment in STAT3, characterized as a CH gene specific to survivors of Hodgkin lymphoma, and TP53. Single-cell profiling of peripheral blood samples revealed STAT3 mutations predominantly present in T cells and contributed by SBS25, a mutational signature associated with procarbazine exposure. Serial sample tracking reveals that larger clone size is a predictor for future expansion of age-related CH clones, whereas therapy-related CH remains stable decades after treatment. These data depict the distinct dynamics of these CH subtypes and support the need for longitudinal monitoring to determine the potential contribution to late effects. SIGNIFICANCE: This first comprehensive CH analysis in long-term survivors of pediatric cancer presents the elevated prevalence and therapy exposures/diagnostic spectrum associated with CH. Due to the contrasting dynamics of clonal expansion for age-related versus therapy-related CH, longitudinal monitoring is recommended to ascertain the long-term effects of therapy-induced CH in pediatric cancer survivors. See related commentary by Collord and Behjati, p. 811. This article is highlighted in the In This Issue feature, p. 799.

Etiology of oncogenic fusions in 5,190 childhood cancers and its clinical and therapeutic implication.

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.

Association of Single-Nucleotide Variants in the Human Leukocyte Antigen and Other Loci With Childhood Hodgkin Lymphoma.

Importance: Studies focusing on genetic susceptibility of childhood Hodgkin lymphoma (HL) are limited. Objectives: To identify genetic variants associated with childhood-onset HL vs adult-onset HL. Design, Setting, and Participants: 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. Main Outcomes and Measures: 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. Results: 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. Conclusions and Relevance: In this genetic association study, a predominantly common and potentially unique genetic etiology was found between childhood-onset and adulthood-onset HL.

Convergent evolution and multi-wave clonal invasion in H3 K27-altered diffuse midline gliomas treated with a PDGFR inhibitor.

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.

A Novel Locus on 6p21.2 for Cancer Treatment-Induced Cardiac Dysfunction Among Childhood Cancer Survivors.

BACKGROUND: Adult survivors of childhood cancer are at increased risk of cardiac late effects. METHODS: Using whole-genome sequencing data from 1870 survivors of European ancestry in the St. Jude Lifetime Cohort (SJLIFE) study, genetic variants were examined for association with ejection fraction (EF) and clinically assessed cancer therapy-induced cardiac dysfunction (CCD). Statistically significant findings were validated in 301 SJLIFE survivors of African ancestry and 4020 survivors of European ancestry from the Childhood Cancer Survivor Study. All statistical tests were 2-sided. RESULTS: A variant near KCNK17 showed genome-wide significant association with EF (rs2815063-A: EF reduction = 1.6%; P = 2.1 × 10-8) in SJLIFE survivors of European ancestry, which replicated in SJLIFE survivors of African ancestry (EF reduction = 1.5%; P = .004). The rs2815063-A also showed a 1.80-fold (P = .008) risk of severe or disabling or life-threatening CCD and replicated in 4020 Childhood Cancer Survivor Study survivors of European ancestry (odds ratio = 1.40; P = .04). Notably, rs2815063-A was specifically associated among survivors exposed to doxorubicin only, with a stronger effect on EF (3.3% EF reduction) and CCD (2.97-fold). Whole blood DNA methylation data in 1651 SJLIFE survivors of European ancestry showed statistically significant correlation of rs2815063-A with dysregulation of KCNK17 enhancers (false discovery rate <5%), which replicated in 263 survivors of African ancestry. Consistently, the rs2815063-A was associated with KCNK17 downregulation based on RNA sequencing of 75 survivors. CONCLUSIONS: Leveraging the 2 largest cohorts of childhood cancer survivors in North America and survivor-specific polygenomic functional data, we identified a novel risk locus for CCD, which showed specificity with doxorubicin-induced cardiac dysfunction and highlighted dysregulation of KCNK17 as the likely molecular mechanism underlying this genetic association.

The Association of Mitochondrial Copy Number With Sarcopenia in Adult Survivors of Childhood Cancer.

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 < .001) and associated with age at diagnosis; 51.7% of survivors with sarcopenia were diagnosed ages 4-13 years (P = .01). Sarcopenia was most prevalent (39.0%) among central nervous system tumor survivors. Cranial radiation (odds ratio [OR] = 1.84, 95% confidence interval [CI] = 1.32 to 2.59) and alkylating agents (OR = 1.34, 95% CI = 1.04 to 1.72) increased, whereas glucocorticoids decreased odds (OR = 0.72, 95% CI = 0.56 to 0.93) of sarcopenia. mtDNAcn decreased with age (ß = -0.81, P = .002) and was higher among female survivors (ß = 9.23, P = .01) and among survivors with a C allele at mt.204 (ß = -17.9, P = .02). In adjusted models, every standard deviation decrease in mtDNAcn increased the odds of sarcopenia 20% (OR = 1.20, 95% CI = 1.07 to 1.34). 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.

SequencErr: measuring and suppressing sequencer errors in next-generation sequencing data.

BACKGROUND: There 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Polygenic Risk Score Improves Risk Stratification and Prediction of Subsequent Thyroid Cancer after Childhood Cancer.

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.

Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators.

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.

Shortened Leukocyte Telomere Length Associates with an Increased Prevalence of Chronic Health Conditions among Survivors of Childhood Cancer: A Report from the St. Jude Lifetime Cohort.

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 (P trend = 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.

Pan-neuroblastoma analysis reveals age- and signature-associated driver alterations.

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.

The Clonal Evolution of Metastatic Osteosarcoma as Shaped by Cisplatin Treatment.

To investigate the genomic evolution of metastatic pediatric osteosarcoma, we performed whole-genome and targeted deep sequencing on 14 osteosarcoma metastases and two primary tumors from four patients (two to eight samples per patient). All four patients harbored ancestral (truncal) somatic variants resulting in TP53 inactivation and cell-cycle aberrations, followed by divergence into relapse-specific lineages exhibiting a cisplatin-induced mutation signature. In three of the four patients, the cisplatin signature accounted for >40% of mutations detected in the metastatic samples. Mutations potentially acquired during cisplatin treatment included NF1 missense mutations of uncertain significance in two patients and a KIT G565R activating mutation in one patient. Three of four patients demonstrated widespread ploidy differences between samples from the sample patient. Single-cell seeding of metastasis was detected in most metastatic samples. Cross-seeding between metastatic sites was observed in one patient, whereas in another patient a minor clone from the primary tumor seeded both metastases analyzed. These results reveal extensive clonal heterogeneity in metastatic osteosarcoma, much of which is likely cisplatin-induced. IMPLICATIONS: The extent and consequences of chemotherapy-induced damage in pediatric cancers is unknown. We found that cisplatin treatment can potentially double the mutational burden in osteosarcoma, which has implications for optimizing therapy for recurrent, chemotherapy-resistant disease.