Divergent clonal selection dominates medulloblastoma at recurrence.

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing.

PURPOSE: Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. METHODS: Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. RESULTS: Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2. CONCLUSION: Our findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia.

BACKGROUND: Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS: We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS: AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation-related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS: We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.).

Complete genomic landscape of a recurring sporadic parathyroid carcinoma.

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto-oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high-throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well-characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets.

Whole genome and transcriptome integrated analyses guide clinical care of pediatric poor prognosis cancers.

The role for routine whole genome and transcriptome analysis (WGTA) for poor prognosis pediatric cancers remains undetermined. Here, we characterize somatic mutations, structural rearrangements, copy number variants, gene expression, immuno-profiles and germline cancer predisposition variants in children and adolescents with relapsed, refractory or poor prognosis malignancies who underwent somatic WGTA and matched germline sequencing. Seventy-nine participants with a median age at enrollment of 8.8 y (range 6 months to 21.2 y) are included. Germline pathogenic/likely pathogenic variants are identified in 12% of participants, of which 60% were not known prior. Therapeutically actionable variants are identified by targeted gene report and whole genome in 32% and 62% of participants, respectively, and increase to 96% after integrating transcriptome analyses. Thirty-two molecularly informed therapies are pursued in 28 participants with 54% achieving a clinical benefit rate; objective response or stable disease ≥6 months. Integrated WGTA identifies therapeutically actionable variants in almost all tumors and are directly translatable to clinical care of children with poor prognosis cancers.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes.

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers.

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.

The genome sequence of the Loggerhead sea turtle, Caretta caretta Linnaeus 1758.

We present a genome assembly of Caretta caretta (the Loggerhead sea turtle; Chordata, Testudines, Cheloniidae), generated from genomic data from two unrelated females. The genome sequence is 2.13 gigabases in size. The assembly has a busco completion score of 96.1% and N50 of 130.95 Mb. The majority of the assembly is scaffolded into 28 chromosomal representations with a remaining 2% of the assembly being excluded from these.

Genomic structures and regulation patterns at HPV integration sites in cervical cancer.

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

A platform for oncogenomic reporting and interpretation.

Manual interpretation of variants remains rate limiting in precision oncology. The increasing scale and complexity of molecular data generated from comprehensive sequencing of cancer samples requires advanced interpretative platforms as precision oncology expands beyond individual patients to entire populations. To address this unmet need, we introduce a Platform for Oncogenomic Reporting and Interpretation (PORI), comprising an analytic framework that facilitates the interpretation and reporting of somatic variants in cancer. PORI integrates reporting and graph knowledge base tools combined with support for manual curation at the reporting stage. PORI represents an open-source platform alternative to commercial reporting solutions suitable for comprehensive genomic data sets in precision oncology. We demonstrate the utility of PORI by matching 9,961 pan-cancer genome atlas tumours to the graph knowledge base, calculating therapeutically informative alterations, and making available reports describing select individual samples.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

Genome and Transcriptome Biomarkers of Response to Immune Checkpoint Inhibitors in Advanced Solid Tumors.

PURPOSE: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of solid tumors with dramatic and durable responses seen across multiple tumor types. However, identifying patients who will respond to these drugs remains challenging, particularly in the context of advanced and previously treated cancers. EXPERIMENTAL DESIGN: We characterized fresh tumor biopsies from a heterogeneous pan-cancer cohort of 98 patients with metastatic predominantly pretreated disease through the Personalized OncoGenomics program at BC Cancer (Vancouver, Canada) using whole genome and transcriptome analysis (WGTA). Baseline characteristics and follow-up data were collected retrospectively. RESULTS: We found that tumor mutation burden, independent of mismatch repair status, was the most predictive marker of time to progression (P = 0.007), but immune-related CD8+ T-cell and M1-M2 macrophage ratio scores were more predictive for overall survival (OS; P = 0.0014 and 0.0012, respectively). While CD274 [programmed death-ligand 1 (PD-L1)] gene expression is comparable with protein levels detected by IHC, we did not observe a clinical benefit for patients with this marker. We demonstrate that a combination of markers based on WGTA provides the best stratification of patients (P = 0.00071, OS), and also present a case study of possible acquired resistance to pembrolizumab in a patient with non-small cell lung cancer. CONCLUSIONS: Interpreting the tumor-immune interface to predict ICI efficacy remains challenging. WGTA allows for identification of multiple biomarkers simultaneously that in combination may help to identify responders, particularly in the context of a heterogeneous population of advanced and previously treated cancers, thus precluding tumor type-specific testing.

Uncovering Clinically Relevant Gene Fusions with Integrated Genomic and Transcriptomic Profiling of Metastatic Cancers.

PURPOSE: Gene fusions are important oncogenic drivers and many are actionable. Whole-genome and transcriptome (WGS and RNA-seq, respectively) sequencing can discover novel clinically relevant fusions. EXPERIMENTAL DESIGN: Using WGS and RNA-seq, we reviewed the prevalence of fusions in a cohort of 570 patients with cancer, and compared prevalence to that predicted with commercially available panels. Fusions were annotated using a consensus variant calling pipeline (MAVIS) and required that a contig of the breakpoint could be constructed and supported from ≥2 structural variant detection approaches. RESULTS: In 570 patients with advanced cancer, MAVIS identified 81 recurrent fusions by WGS and 111 by RNA-seq, of which 18 fusions by WGS and 19 by RNA-seq were noted in at least 3 separate patients. The most common fusions were EML4-ALK in thoracic malignancies (9/69, 13%), and CMTM8-CMTM7 in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as NTRK2 (novel partners: SHC3, DAPK1), and NTRK3 (novel partners: POLG, PIBF1). CONCLUSIONS: Utilizing WGS/RNA-seq facilitates identification of novel fusions in clinically relevant genes, and detected a greater proportion than commercially available panels are expected to find. A significant benefit of WGS and RNA-seq is the innate ability to retrospectively identify variants that becomes clinically relevant over time, without the need for additional testing, which is not possible with panel-based approaches.

A Distributed Whole Genome Sequencing Benchmark Study.

Population sequencing often requires collaboration across a distributed network of sequencing centers for the timely processing of thousands of samples. In such massive efforts, it is important that participating scientists can be confident that the accuracy of the sequence data produced is not affected by which center generates the data. A study was conducted across three established sequencing centers, located in Montreal, Toronto, and Vancouver, constituting Canada's Genomics Enterprise (www.cgen.ca). Whole genome sequencing was performed at each center, on three genomic DNA replicates from three well-characterized cell lines. Secondary analysis pipelines employed by each site were applied to sequence data from each of the sites, resulting in three datasets for each of four variables (cell line, replicate, sequencing center, and analysis pipeline), for a total of 81 datasets. These datasets were each assessed according to multiple quality metrics including concordance with benchmark variant truth sets to assess consistent quality across all three conditions for each variable. Three-way concordance analysis of variants across conditions for each variable was performed. Our results showed that the variant concordance between datasets differing only by sequencing center was similar to the concordance for datasets differing only by replicate, using the same analysis pipeline. We also showed that the statistically significant differences between datasets result from the analysis pipeline used, which can be unified and updated as new approaches become available. We conclude that genome sequencing projects can rely on the quality and reproducibility of aggregate data generated across a network of distributed sites.

Pan-cancer analysis of advanced patient tumors reveals interactions between therapy and genomic landscapes.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

The Genome of the Steller Sea Lion (Eumetopias jubatus).

The Steller sea lion is the largest member of the Otariidae family and is found in the coastal waters of the northern Pacific Rim. Here, we present the Steller sea lion genome, determined through DNA sequencing approaches that utilized microfluidic partitioning library construction, as well as nanopore technologies. These methods constructed a highly contiguous assembly with a scaffold N50 length of over 14 megabases, a contig N50 length of over 242 kilobases and a total length of 2.404 gigabases. As a measure of completeness, 95.1% of 4104 highly conserved mammalian genes were found to be complete within the assembly. Further annotation identified 19,668 protein coding genes. The assembled genome sequence and underlying sequence data can be found at the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA475770.

Identification and Analyses of Extra-Cranial and Cranial Rhabdoid Tumor Molecular Subgroups Reveal Tumors with Cytotoxic T Cell Infiltration.

Extra-cranial malignant rhabdoid tumors (MRTs) and cranial atypical teratoid RTs (ATRTs) are heterogeneous pediatric cancers driven primarily by SMARCB1 loss. To understand the genome-wide molecular relationships between MRTs and ATRTs, we analyze multi-omics data from 140 MRTs and 161 ATRTs. We detect similarities between the MYC subgroup of ATRTs (ATRT-MYC) and extra-cranial MRTs, including global DNA hypomethylation and overexpression of HOX genes and genes involved in mesenchymal development, distinguishing them from other ATRT subgroups that express neural-like features. We identify five DNA methylation subgroups associated with anatomical sites and SMARCB1 mutation patterns. Groups 1, 3, and 4 exhibit cytotoxic T cell infiltration and expression of immune checkpoint regulators, consistent with a potential role for immunotherapy in rhabdoid tumor patients.

Therapeutic Implication of Genomic Landscape of Adult Metastatic Sarcoma.

PURPOSE: This study investigated therapeutic potential of integrated genome and transcriptome profiling of metastatic sarcoma, a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options. METHODS: Forty-three adult patients with advanced or metastatic non-GI stromal tumor sarcomas of various histology subtypes who were enrolled in the Personalized OncoGenomics program at BC Cancer were included in this study. Fresh tumor tissues along with blood samples underwent whole-genome and transcriptome sequencing. RESULTS: The most frequent genomic alterations in this cohort are large-scale structural variation and somatic copy number variation. Outlier RNA expression as well as somatic copy number variations, structural variations, and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort. Point mutations or deletions in known targetable cancer genes are rare; for example, tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway, resulting in a few patients with exceptional clinical benefit from everolimus. In addition, we observed recurrent 17p11-12 amplifications, which seem to be a sarcoma-specific event. This may suggest that this region harbors an oncogene(s) that is significant for sarcoma tumorigenesis. Furthermore, some sarcoma tumors carrying a distinct mutational signature suggestive of homologous recombination deficiency seem to demonstrate sensitivity to double-strand DNA-damaging agents. CONCLUSION: Integrated large-scale genomic analysis may provide insights into potential therapeutic targets as well as novel biologic features of metastatic sarcomas that could fuel future experimental and clinical research and help design biomarker-driven basket clinical trials for novel therapeutic strategies.