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.

Estimated number of adult survivors of childhood cancer in United States with cancer-predisposing germline variants.

PURPOSE: To estimate the absolute number of adult survivors of childhood cancer in the U.S. population who carry a pathogenic or likely pathogenic variant in a cancer predisposition gene. METHODS: Using the Surveillance, Epidemiology, and End Results (SEER) Program, we estimated the number of childhood cancer survivors on December 31, 2016 for each childhood cancer diagnosis, multiplied this by the proportion of carriers of pathogenic/likely pathogenic variants in the St. Jude Lifetime Cohort (SJLIFE) study, and projected the resulting number onto the U.S. RESULTS: Based on genome sequence data, 11.8% of 2450 SJLIFE participants carry a pathogenic/likely pathogenic variant in one of 156 cancer predisposition genes. Given this information, we estimate that 21 800 adult survivors of childhood cancer in the United States carry a pathogenic/likely pathogenic variant in one of these genes. The highest estimated absolute number of variant carriers are among survivors of central nervous system tumors (n = 4300), particularly astrocytoma (n = 1800) and other gliomas (n = 1700), acute lymphoblastic leukemia (n = 4300), and retinoblastoma (n = 3500). The most frequently mutated genes are RB1 (n = 3000), NF1 (n = 2300), and BRCA2 (n = 800). CONCLUSION: Given the increasing number of childhood cancer survivors in the United States, clinicians should counsel survivors regarding their potential genetic risk, consider referral for genetic counseling and testing, and, as appropriate, implement syndrome-specific cancer surveillance or risk-reducing measures.

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.

Structure and evolution of double minutes in diagnosis and relapse brain tumors.

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.

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.).

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology.

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 15 years 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.

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.

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.

Health-related quality of life and DNA methylation-based aging biomarkers among survivors of childhood cancer.

BACKGROUND: Childhood cancer survivors are at high risk for morbidity and mortality and poor patient-reported outcomes, typically health-related quality of life (HRQOL). However, associations between DNA methylation-based aging biomarkers and HRQOL have not been evaluated. METHODS: DNA methylation was generated with Infinium EPIC BeadChip on blood-derived DNA (median for age at blood draw = 34.5 years, range = 18.5-66.6 years), and HRQOL was assessed with age at survey (mean = 32.3 years, range = 18.4-64.5 years) from 2206 survivors in the St Jude Lifetime Cohort. DNA methylation-based aging biomarkers, including epigenetic age using multiple clocks (eg, GrimAge) and others (eg, DNAmB2M: beta-2-microglobulin; DNAmADM: adrenomedullin), were derived from the DNAm Age Calculator (https://dnamage.genetics.ucla.edu). HRQOL was assessed using the Medical Outcomes Study 36-Item Short-Form Health Survey to capture 8 domains and physical and mental component summaries. General linear models evaluated associations between HRQOL and epigenetic age acceleration (EAA; eg, EAA_GrimAge) or other age-adjusted DNA methylation-based biomarkers (eg, ageadj_DNAmB2M) after adjusting for age at blood draw, sex, cancer treatments, and DNA methylation-based surrogate for smoking pack-years. All P values were 2-sided. RESULTS: Worse HRQOL was associated with greater EAA_GrimAge (physical component summaries: ß = -0.18 years, 95% confidence interval [CI] = -0.251 to -0.11 years; P = 1.85 × 10-5; and 4 individual HRQOL domains), followed by ageadj_DNAmB2M (physical component summaries: ß = -0.08 years, 95% CI = -0.124 to -0.037 years; P = .003; and 3 individual HRQOL domains) and ageadj_DNAmADM (physical component summaries: ß = -0.082 years, 95% CI = -0.125 to -0.039 years; P = .002; and 2 HRQOL domains). EAA_Hannum (Hannum clock) was not associated with any HRQOL. CONCLUSIONS: Overall and domain-specific measures of HRQOL are associated with DNA methylation measures of biological aging. Future longitudinal studies should test biological aging as a potential mechanism underlying the association between poor HRQOL and increased risk of clinically assessed adverse health outcomes.

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.

NSD2 E1099K drives relapse in pediatric acute lymphoblastic leukemia by disrupting 3D chromatin organization.

BACKGROUND: The NSD2 p.E1099K (EK) mutation is shown to be enriched in patients with relapsed acute lymphoblastic leukemia (ALL), indicating a role in clonal evolution and drug resistance. RESULTS: To uncover 3D chromatin architecture-related mechanisms underlying drug resistance, we perform Hi-C on three B-ALL cell lines heterozygous for NSD2 EK. The NSD2 mutation leads to widespread remodeling of the 3D genome, most dramatically in terms of compartment changes with a strong bias towards A compartment shifts. Systematic integration of the Hi-C data with previously published ATAC-seq, RNA-seq, and ChIP-seq data show an expansion in H3K36me2 and a shrinkage in H3K27me3 within A compartments as well as increased gene expression and chromatin accessibility. These results suggest that NSD2 EK plays a prominent role in chromatin decompaction through enrichment of H3K36me2. In contrast, we identify few changes in intra-topologically associating domain activity. While compartment changes vary across cell lines, a common core of decompacting loci are shared, driving the expression of genes/pathways previously implicated in drug resistance. We further perform RNA sequencing on a cohort of matched diagnosis/relapse ALL patients harboring the relapse-specific NSD2 EK mutation. Changes in patient gene expression upon relapse significantly correlate with core compartment changes, further implicating the role of NSD2 EK in genome decompaction. CONCLUSIONS: In spite of cell-context-dependent changes mediated by EK, there appears to be a shared transcriptional program dependent on compartment shifts which could explain phenotypic differences across EK cell lines. This core program is an attractive target for therapeutic intervention.

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.

Epigenetic Age in Peripheral Blood Among Children, Adolescent, and Adult Survivors of Childhood Cancer.

Importance: Certain cancer therapies are risk factors for epigenetic age acceleration (EAA) among survivors of childhood cancer, and EAA is associated with chronic health conditions (CHCs). However, small numbers of younger survivors (aged <20 years) previously evaluated have limited the ability to calculate EAA among this age group. Objective: To evaluate the change rate of epigenetic age (EA) and EAA in younger compared with older survivors and the possible association of EAA with early-onset obesity (aged <20 years), severity/burden of CHCs, and late mortality (>5 years from cancer diagnosis). Design, Setting, and Participants: Study participants were from the St Jude Lifetime Cohort, initiated in 2007 with ongoing follow-up. The present study was conducted from April 17, 2022, to March 23, 2023. Survivors in this cohort of European ancestry with DNA methylation data were included. Cross-sectional annual changes in EA and EAA were compared across 5 different chronologic age groups: age 0 to 9 (children), 10 to 19 (adolescents), 20 to 34 (younger adults), 35 to 49 (middle-aged adults), and greater than or equal to 50 (older adults) years. Logistic regression evaluated the association between EAA and early-onset obesity or severity/burden of CHCs. Cox proportional hazards regression assessed the association between EAA and late mortality. Main Outcomes and Measures: Early-onset obesity, severity/burden of CHCs (graded using the Common Terminology Criteria for Adverse Events (grade 1, mild; 2, moderate; 3, severe/disabling; 4, life-threatening) and were combined into high vs low severity/burden based on frequency and grade), and late mortality were the outcomes based on follow-up until April 2020. Expanded DNA methylation profiling increased the number of survivors younger than 20 years (n = 690). Epigenetic age was calculated primarily using the Levine clock, and EAA was derived from least squares regression of EA against chronologic age and was standardized to a z score (Levine EEA). Results: Among 2846 participants (median age, 30.3 [IQR, 9.3-41.5] years; 53% males), the cross-sectional annual change in EA_Levine was higher in children (1.63 years) and adolescents (1.14 years), and the adjusted least-squares mean of Levine EEA was lower in children (-0.22 years) and older adults (-1.70 years). Each 1-SD increase in Levine EEA was associated with increased risk of developing early-onset obesity (odds ratio [OR], 1.46; 95% CI, 1.19-1.78), high severity/burden of CHCs (OR, 1.13; 95% CI, 1.03-1.24), and late mortality (hazard ratio, 1.75; 95% CI, 1.35-2.26). Conclusions and Relevance: The findings of this study suggest that EAA measured in children and adolescent survivors of childhood cancer is associated with early-onset obesity, severity/burden of all CHCs, and late mortality. Evaluating EAA may help identify survivors of childhood cancer at increased risk for early-onset obesity, morbidity in general, and mortality.

Distinct DNA methylation signatures associated with blood lipids as exposures or outcomes among survivors of childhood cancer: a report from the St. Jude lifetime cohort.

BACKGROUND: DNA methylation (DNAm) plays an important role in lipid metabolism, however, no epigenome-wide association study (EWAS) of lipid levels has been conducted among childhood cancer survivors. Here, we performed EWAS analysis with longitudinally collected blood lipid data from survivors in the St. Jude lifetime cohort study. METHODS: Among 2052 childhood cancer survivors of European ancestry (EA) and 370 survivors of African ancestry (AA), four types of blood lipids, including high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglycerides (TG), were measured during follow-up beyond 5-years from childhood cancer diagnosis. For the exposure EWAS (i.e., lipids measured before blood draw for DNAm), the DNAm level was an outcome variable and each of the blood lipid level was an exposure variable; vice versa for the outcome EWAS (i.e., lipids measured after blood draw for DNAm). RESULTS: Among EA survivors, we identified 43 lipid-associated CpGs in the HDL (n = 7), TC (n = 3), and TG (n = 33) exposure EWAS, and 106 lipid-associated CpGs in the HDL (n = 5), LDL (n = 3), TC (n = 4), and TG (n = 94) outcome EWAS. Among AA survivors, we identified 15 lipid-associated CpGs in TG exposure (n = 6), HDL (n = 1), LDL (n = 1), TG (n = 5) and TC (n = 2) outcome EWAS with epigenome-wide significance (P < 9 × 10-8). There were no overlapping lipids-associated CpGs between exposure and outcome EWAS among EA and AA survivors, suggesting that the DNAm changes of different CpGs could be the cause or consequence of blood lipid levels. In the meta-EWAS, 12 additional CpGs reached epigenome-wide significance. Notably, 32 out of 74 lipid-associated CpGs showed substantial heterogeneity (Phet < 0.1 or I2 > 70%) between EA and AA survivors, highlighting differences in DNAm markers of blood lipids between populations with diverse genetic ancestry. Ten lipid-associated CpGs were cis-expression quantitative trait methylation with their DNAm levels associated with the expression of corresponding genes, out of which seven were negatively associated. CONCLUSIONS: We identified distinct signatures of DNAm for blood lipids as exposures or outcomes and between EA and AA survivors, revealing additional genes involved in lipid metabolism and potential novel targets for controlling blood lipids in childhood cancer survivors.

The Genetic and Epigenetic Features of Bilateral Wilms Tumor Predisposition: A Report from the Children's Oncology Group AREN18B5-Q Study.

This study comprehensively evaluated the landscape of genetic and epigenetic events that predispose to synchronous bilateral Wilms tumor (BWT). We performed whole exome or whole genome sequencing, total-strand RNA-seq, and DNA methylation analysis using germline and/or tumor samples from 68 patients with BWT from St. Jude Children's Research Hospital and the Children's Oncology Group. We found that 25/61 (41%) of patients evaluated harbored pathogenic or likely pathogenic germline variants, with WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%) and the BRCA-related genes (5%) BRCA1, BRCA2, and PALB2 being most common. Germline WT1 variants were strongly associated with somatic paternal uniparental disomy encompassing the 11p15.5 and 11p13/WT1 loci and subsequent acquired pathogenic CTNNB1 variants. Somatic coding variants or genome-wide copy number alterations were almost never shared between paired synchronous BWT, suggesting that the acquisition of independent somatic variants leads to tumor formation in the context of germline or early embryonic, post-zygotic initiating events. In contrast, 11p15.5 status (loss of heterozygosity, loss or retention of imprinting) was shared among paired synchronous BWT in all but one case. The predominant molecular events for BWT predisposition include pathogenic germline variants or post-zygotic epigenetic hypermethylation at the 11p15.5 H19/ICR1 locus (loss of imprinting). This study demonstrates that post-zygotic somatic mosaicism for 11p15.5 hypermethylation/loss of imprinting is the single most common initiating molecular event predisposing to BWT. Evidence of somatic mosaicism for 11p15.5 loss of imprinting was detected in leukocytes of a cohort of BWT patients and long-term survivors, but not in unilateral Wilms tumor patients and long-term survivors or controls, further supporting the hypothesis that post-zygotic 11p15.5 alterations occurred in the mesoderm of patients who go on to develop BWT. Due to the preponderance of BWT patients with demonstrable germline or early embryonic tumor predisposition, BWT exhibits a unique biology when compared to unilateral Wilms tumor and therefore warrants continued refinement of its own treatment-relevant biomarkers which in turn may inform directed treatment strategies in the future.

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.