To discover driver fusions beyond canonical exon-to-exon chimeric transcripts, we develop CICERO, a local assembly-based algorithm that integrates RNA-seq read support with extensive annotation for ...candidate ranking. CICERO outperforms commonly used methods, achieving a 95% detection rate for 184 independently validated driver fusions including internal tandem duplications and other non-canonical events in 170 pediatric cancer transcriptomes. Re-analysis of TCGA glioblastoma RNA-seq unveils previously unreported kinase fusions (KLHL7-BRAF) and a 13% prevalence of EGFR C-terminal truncation. Accessible via standard or cloud-based implementation, CICERO enhances driver fusion detection for research and precision oncology. The CICERO source code is available at https://github.com/stjude/Cicero.
Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer ...cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.
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 prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, ...direct patient care, and enable genetic counseling of patients and families.
In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism).
Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer.
Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American Lebanese Syrian Associated Charities and the National Cancer Institute.).
Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB).
Adult patients ...enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available.
A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH-MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses.
Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH-MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.
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 recent identification of compounds that interact with the spliceosome (sudemycins, spliceostatin A, and meayamycin) indicates that these molecules modulate aberrant splicing via SF3B1 inhibition. ...Through whole transcriptome sequencing, we have demonstrated that treatment of Rh18 cells with sudemycin leads to exon skipping as the predominant aberrant splicing event. This was also observed following reanalysis of published RNA-seq data sets derived from HeLa cells after spliceostatin A exposure. These results are in contrast to previous reports that indicate that intron retention was the major consequence of SF3B1 inhibition. Analysis of the exon junctions up-regulated by these small molecules indicated that these sequences were absent in annotated human genes, suggesting that aberrant splicing events yielded novel RNA transcripts. Interestingly, the length of preferred downstream exons was significantly longer than the skipped exons, although there was no difference between the lengths of introns flanking skipped exons. The reading frame of the aberrantly skipped exons maintained a ratio of 2:1:1, close to that of the cassette exons (3:1:1) present in naturally occurring isoforms, suggesting negative selection by the nonsense-mediated decay (NMD) machinery for out-of-frame transcripts. Accordingly, genes involved in NMD and RNAs encoding proteins involved in the splicing process were enriched in both data sets. Our findings, therefore, further elucidate the mechanisms by which SF3B1 inhibition modulates pre-mRNA splicing.
To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), ...whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97-99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing.
We developed cis-X, a computational method for discovering regulatory noncoding variants in cancer by integrating whole-genome and transcriptome sequencing data from a single cancer sample. cis-X ...first finds aberrantly cis-activated genes that exhibit allele-specific expression accompanied by an elevated outlier expression. It then searches for causal noncoding variants that may introduce aberrant transcription factor binding motifs or enhancer hijacking by structural variations. Analysis of 13 T-lineage acute lymphoblastic leukemias identified a recurrent intronic variant predicted to cis-activate the TAL1 oncogene, a finding validated in vivo by chromatin immunoprecipitation sequencing of a patient-derived xenograft. Candidate oncogenes include the prolactin receptor PRLR activated by a focal deletion that removes a CTCF-insulated neighborhood boundary. cis-X may be applied to pediatric and adult solid tumors that are aneuploid and heterogeneous. In contrast to existing approaches, which require large sample cohorts, cis-X enables the discovery of regulatory noncoding variants in individual cancer genomes.
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