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.

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.

Analysis of Ugandan cervical carcinomas identifies human papillomavirus clade-specific epigenome and transcriptome landscapes.

Cervical cancer is the most common cancer affecting sub-Saharan African women and is prevalent among HIV-positive (HIV+) individuals. No comprehensive profiling of cancer genomes, transcriptomes or epigenomes has been performed in this population thus far. We characterized 118 tumors from Ugandan patients, of whom 72 were HIV+, and performed extended mutation analysis on an additional 89 tumors. We detected human papillomavirus (HPV)-clade-specific differences in tumor DNA methylation, promoter- and enhancer-associated histone marks, gene expression and pathway dysregulation. Changes in histone modification at HPV integration events were correlated with upregulation of nearby genes and endogenous retroviruses.

Tumor microRNA profile and prognostic value for lymph node metastasis in oral squamous cell carcinoma patients.

Neck lymph node metastasis (LN+) is one of the most significant prognostic factors affecting 1-in-2 patients diagnosed with oral squamous cell carcinoma (OSCC). The different LN outcomes between clinico-pathologically similar primary tumors suggest underlying molecular signatures that could be associated with the risk of nodal disease development. MicroRNAs (miRNAs)are short non-coding molecules that regulate the expression of their target genes to maintain the balance of cellular processes. A plethora of evidence has indicated that aberrantly expressed miRNAs are involved in cancers with either an antitumor or oncogenic role. In this study, we characterized miRNA expression among OSCC fresh-frozen tumors with known outcomes of nodal disease (82 LN+, 76 LN0). We identified 49 differentially expressed miRNAs in tumors of the LN+ group. Using penalized lasso Cox regression, we identified a group of 10 miRNAs of which expression levels were highly associated with nodal-disease free survival. We further reported a 4-miRNA panel (miR-21-5p, miR-107, miR-1247-3p, and miR-181b-3p) with high accuracy in discriminating LN status, suggesting their potential application as prognostic biomarkers for nodal disease.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

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.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Comprehensive genomic profiling of glioblastoma tumors, BTICs, and xenografts reveals stability and adaptation to growth environments.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma.

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.

NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma.

PURPOSE: Gene fusions involving neuregulin 1 (NRG1) have been noted in multiple cancer types and have potential therapeutic implications. Although varying results have been reported in other cancer types, the efficacy of the HER-family kinase inhibitor afatinib in the treatment of NRG1 fusion-positive pancreatic ductal adenocarcinoma is not fully understood. EXPERIMENTAL DESIGN: Forty-seven patients with pancreatic ductal adenocarcinoma received comprehensive whole-genome and transcriptome sequencing and analysis. Two patients with gene fusions involving NRG1 received afatinib treatment, with response measured by pretreatment and posttreatment PET/CT imaging. RESULTS: Three of 47 (6%) patients with advanced pancreatic ductal adenocarcinoma were identified as KRAS wild type by whole-genome sequencing. All KRAS wild-type tumors were positive for gene fusions involving the ERBB3 ligand NRG1. Two of 3 patients with NRG1 fusion-positive tumors were treated with afatinib and demonstrated a significant and rapid response while on therapy. CONCLUSIONS: This work adds to a growing body of evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in pancreatic cancers. Based on the clinical outcomes described here, patients with KRAS wild-type tumors harboring NRG1 gene fusions may benefit from treatment with afatinib.See related commentary by Aguirre, p. 4589.

Application of a Neural Network Whole Transcriptome-Based Pan-Cancer Method for Diagnosis of Primary and Metastatic Cancers.

Importance: A molecular diagnostic method that incorporates information about the transcriptional status of all genes across multiple tissue types can strengthen confidence in cancer diagnosis. Objective: To determine the practical use of a whole transcriptome-based pan-cancer method in diagnosing primary and metastatic cancers and resolving complex diagnoses. Design, Setting, and Participants: This cross-sectional diagnostic study assessed Supervised Cancer Origin Prediction Using Expression (SCOPE), a machine learning method using whole-transcriptome RNA sequencing data. Training was performed on publicly available primary cancer data sets, including The Cancer Genome Atlas. Testing was performed retrospectively on untreated primary cancers and treated metastases from volunteer adult patients at BC Cancer in Vancouver, British Columbia, from January 1, 2013, to March 31, 2016, and testing spanned 10 822 samples and 66 output classes representing untreated primary cancers (n = 40) and adjacent normal tissues (n = 26). SCOPE's performance was demonstrated on 211 untreated primary mesothelioma cancers and 201 treatment-resistant metastatic cancers. Finally, SCOPE was used to identify the putative site of origin in 15 cases with initial presentation as cancers with unknown primary of origin. Results: A total of 10 688 adult patient samples representing 40 untreated primary tumor types and 26 adjacent-normal tissues were used for training. Demographic data were not available for all data sets. Among the training data set, 5157 of 10 244 (50.3%) were male and the mean (SD) age was 58.9 (14.5) years. Testing was performed on 211 patients with untreated primary mesothelioma (173 [82.0%] male; mean [SD] age, 64.5 [11.3] years); 201 patients with treatment-resistant cancers (141 [70.1%] female; mean [SD] age, 55.6 [12.9] years); and 15 patients with cancers of unknown primary of origin; among the treatment-resistant cancers, 168 were metastatic, and 33 were the primary presentation. An accuracy rate of 99% was obtained for primary epithelioid mesotheliomas tested (125 of 126). The remaining 85 mesotheliomas had a mixed etiology (sarcomatoid mesotheliomas) and were correctly identified as a mixture of their primary components, with potential implications in resolving subtypes and incidences of mixed histology. SCOPE achieved an overall mean (SD) accuracy rate of 86% (11%) and F1 score of 0.79 (0.12) on the 201 treatment-resistant cancers and matched 12 of 15 of the putative diagnoses for cancers with indeterminate diagnosis from conventional pathology. Conclusions and Relevance: These results suggest that machine learning approaches incorporating multiple tumor profiles can more accurately identify the cancerous state and discriminate it from normal cells. SCOPE uses the whole transcriptomes from normal and tumor tissues, and results of this study suggest that it performs well for rare cancer types, primary cancers, treatment-resistant metastatic cancers, and cancers of unknown primary of origin. Genes most relevant in SCOPE's decision making were examined, and several are known biological markers of respective cancers. SCOPE may be applied as an orthogonal diagnostic method in cases where the site of origin of a cancer is unknown, or when standard pathology assessment is inconclusive.

Intratumoral Genetic and Functional Heterogeneity in Pediatric Glioblastoma.

Pediatric glioblastoma (pGBM) is a lethal cancer with no effective therapies. To understand the mechanisms of tumor evolution in this cancer, we performed whole-genome sequencing with linked reads on longitudinally resected pGBM samples. Our analyses showed that all diagnostic and recurrent samples were collections of genetically diverse subclones. Clonal composition rapidly evolved at recurrence, with less than 8% of nonsynonymous single-nucleotide variants being shared in diagnostic-recurrent pairs. To track the origins of the mutational events observed in pGBM, we generated whole-genome datasets for two patients and their parents. These trios showed that genetic variants could be (i) somatic, (ii) inherited from a healthy parent, or (iii) de novo in the germlines of pGBM patients. Analysis of variant allele frequencies supported a model of tumor growth involving slow-cycling cancer stem cells that give rise to fast-proliferating progenitor-like cells and to nondividing cells. Interestingly, radiation and antimitotic chemotherapeutics did not increase overall tumor burden upon recurrence. These findings support an important role for slow-cycling stem cell populations in contributing to recurrences, because slow-cycling cell populations are expected to be less prone to genotoxic stress induced by these treatments and therefore would accumulate few mutations. Our results highlight the need for new targeted treatments that account for the complex functional hierarchies and genomic heterogeneity of pGBM. SIGNIFICANCE: This work challenges several assumptions regarding the genetic organization of pediatric GBM and highlights mutagenic programs that start during early prenatal development.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2111/F1.large.jpg.

Base excision repair deficiency signatures implicate germline and somatic MUTYH aberrations in pancreatic ductal adenocarcinoma and breast cancer oncogenesis.

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH-mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases (N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise (N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH-associated polyposis (N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36).

Publisher Correction: The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions.

In the version of this article originally published, the color key in Fig. 1a was wrong. In the Cytogenetics key, the box over t(8;21) originally was green. It should have been red, matching the color of the sections of the pie graphs below the key that were labeled with 15% and 19%.

A high-throughput protocol for isolating cell-free circulating tumor DNA from peripheral blood.

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

MAVIS: merging, annotation, validation, and illustration of structural variants.

Summary: Reliably identifying genomic rearrangements and interpreting their impact is a key step in understanding their role in human cancers and inherited genetic diseases. Many short read algorithmic approaches exist but all have appreciable false negative rates. A common approach is to evaluate the union of multiple tools increasing sensitivity, followed by filtering to retain specificity. Here we describe an application framework for the rapid generation of structural variant consensus, unique in its ability to visualize the genetic impact and context as well as process both genome and transcriptome data. Availability and implementation: http://mavis.bcgsc.ca. Supplementary information: Supplementary data are available at Bioinformatics online.

Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples.

Tissues used in pathology laboratories are typically stored in the form of formalin-fixed, paraffin-embedded (FFPE) samples. One important consideration in repurposing FFPE material for next generation sequencing (NGS) analysis is the sequencing artifacts that can arise from the significant damage to nucleic acids due to treatment with formalin, storage at room temperature and extraction. One such class of artifacts consists of chimeric reads that appear to be derived from non-contiguous portions of the genome. Here, we show that a major proportion of such chimeric reads align to both the 'Watson' and 'Crick' strands of the reference genome. We refer to these as strand-split artifact reads (SSARs). This study provides a conceptual framework for the mechanistic basis of the genesis of SSARs and other chimeric artifacts along with supporting experimental evidence, which have led to approaches to reduce the levels of such artifacts. We demonstrate that one of these approaches, involving S1 nuclease-mediated removal of single-stranded fragments and overhangs, also reduces sequence bias, base error rates, and false positive detection of copy number and single nucleotide variants. Finally, we describe an analytical approach for quantifying SSARs from NGS data.

Clinical outcomes after whole-genome sequencing in patients with metastatic non-small-cell lung cancer.

The Personalized Onco-Genomics (POG) program at BC Cancer integrates whole-genome (DNA) and RNA sequencing into practice for metastatic malignancies. We examined the subgroup of patients with metastatic non-small-cell lung cancer (NSCLC) and report the prevalence of actionable targets, treatments, and outcomes. We identified patients who were enrolled in the POG program between 2012 and 2016 who had a tumor biopsy and blood samples with comprehensive DNA (80×, 40× normal) and RNA sequencing followed by in-depth bioinformatics to identify potential cancer drivers and actionable targets. In NSCLC cases, we compared the progression-free survival (PFS) of "POG-informed therapies" with the PFS of the last regimen prior to POG (PFS ratio). In 29 NSCLC cases, 11 were male (38%), the median age was 60.2 yr (range: 39.4-72.6), and histologies included were adenocarcinoma (93%) and squamous cell carcinoma (7%). Potential molecular targets (i.e., cancer drivers including TP53 mutations) were identified in 26 (90%), and 21 (72%) had actionable targets. Therapies based on standard-of-care mutation analysis, such as EGFR mutations, were not considered POG-informed therapies. Thirteen received POG-informed therapies, of which three had no therapy before POG; therefore a comparator PFS could not be obtained. Of 10 patients with POG-informed therapy, median PFS ratio was 0.94 (IQR 0.2-3.4). Three (30%) had a PFS ratio ≥1.3, and three (30%) had a PFS ratio ≥0.8 and <1.3. In this small cohort of NSCLC, 30% demonstrated longer PFS with POG-informed therapies. Larger studies will help clarify the role of whole-genome analysis in clinical practice.