Publications by authors named "Andrew J Mungall"

138 Publications

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Nature 2021 Jul 23;595(7868):585-590. Epub 2021 Jun 23.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, British Columbia, Canada.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
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http://dx.doi.org/10.1038/s41586-021-03648-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8396073PMC
July 2021

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

Front Genet 2021 3;12:665888. Epub 2021 Jun 3.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada.

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.
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http://dx.doi.org/10.3389/fgene.2021.665888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209500PMC
June 2021

A clinical transcriptome approach to patient stratification and therapy selection in acute myeloid leukemia.

Nat Commun 2021 04 30;12(1):2474. Epub 2021 Apr 30.

Experimental Medicine Program, Department of Medicine, University of British Columbia, Vancouver, BC, Canada.

As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.
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http://dx.doi.org/10.1038/s41467-021-22625-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087683PMC
April 2021

The transcriptional landscape of Shh medulloblastoma.

Nat Commun 2021 03 19;12(1):1749. Epub 2021 Mar 19.

Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada.

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.
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http://dx.doi.org/10.1038/s41467-021-21883-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979819PMC
March 2021

Tumor infiltrating neutrophils and gland formation predict overall survival and molecular subgroups in pancreatic ductal adenocarcinoma.

Cancer Med 2021 02 28;10(3):1155-1165. Epub 2020 Dec 28.

Division of Anatomic Pathology, Vancouver General Hospital, Vancouver, BC, Canada.

Background: RNA-sequencing-based classifiers can stratify pancreatic ductal adenocarcinoma (PDAC) into prognostically significant subgroups but are not practical for use in clinical workflows. Here, we assess whether histomorphological features may be used as surrogate markers for predicting molecular subgroup and overall survival in PDAC.

Methods: Ninety-six tissue samples from 50 patients with non-resectable PDAC were scored for gland formation, stromal maturity, mucin, necrosis, and neutrophil infiltration. Prognostic PDAC gene expression classifiers were run on all tumors using whole transcriptome sequencing data from the POG trial (NCT02155621). Findings were validated using digital TCGA slides (n = 50). Survival analysis used multivariate Cox proportional-hazards tests and log-rank tests.

Results: The combination of low gland formation and low neutrophil infiltration was significantly associated with the poor prognosis PDAC molecular subgroup (basal-like or squamous) and was an independent predictor of shorter overall survival, in both frozen section (n = 47) and formalin-fixed paraffin-embedded (n = 49) tissue samples from POG patients, and in the TCGA samples. This finding held true in the subgroup analysis of primary (n = 17) and metastatic samples (n = 79). The combination of high gland formation and high neutrophils had low sensitivity but high specificity for favorable prognosis subgroups.

Conclusions: The assessment of gland formation and neutrophil infiltration on routine histological sections can aid in prognostication and allow inferences to be made about molecular subtype, which may help guide patient management decisions and contribute to our understanding of heterogeneity in treatment response.
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http://dx.doi.org/10.1002/cam4.3695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897949PMC
February 2021

A Distributed Whole Genome Sequencing Benchmark Study.

Front Genet 2020 1;11:612515. Epub 2020 Dec 1.

Canada's Michael Smith Genome Sciences Centre, BC Cancer Research Institute, Provincial Health Services Authority, Vancouver, BC, Canada.

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.
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http://dx.doi.org/10.3389/fgene.2020.612515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736078PMC
December 2020

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

Clin Cancer Res 2021 01 4;27(2):522-531. Epub 2020 Nov 4.

Department of Medical Oncology, BC Cancer, Vancouver, Canada.

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 in thoracic malignancies (9/69, 13%), and in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as (novel partners: , and (novel partners: ).

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1900DOI Listing
January 2021

Whole-slide laser microdissection for tumour enrichment.

J Pathol 2021 02 9;253(2):225-233. Epub 2020 Dec 9.

Canada's Michael Smith Genome Sciences Centre at BC Cancer, Vancouver, BC, Canada.

The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5575DOI Listing
February 2021

The impact of MYC and BCL2 structural variants in tumors of DLBCL morphology and mechanisms of false-negative MYC IHC.

Blood 2021 04;137(16):2196-2208

Centre for Lymphoid Cancer, BC Cancer, Vancouver, BC, Canada.

When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.
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http://dx.doi.org/10.1182/blood.2020007193DOI Listing
April 2021

Subtype-Discordant Pancreatic Ductal Adenocarcinoma Tumors Show Intermediate Clinical and Molecular Characteristics.

Clin Cancer Res 2021 01 13;27(1):150-157. Epub 2020 Oct 13.

Pancreas Centre BC, Vancouver, British Columbia, Canada.

Purpose: RNA-sequencing-based subtyping of pancreatic ductal adenocarcinoma (PDAC) has been reported by multiple research groups, each using different methodologies and patient cohorts. "Classical" and "basal-like" PDAC subtypes are associated with survival differences, with basal-like tumors associated with worse prognosis. We amalgamated various PDAC subtyping tools to evaluate the potential of such tools to be reliable in clinical practice.

Experimental Design: Sequencing data for 574 PDAC tumors was obtained from prospective trials and retrospective public databases. Six published PDAC subtyping strategies (Moffitt regression tools, clustering-based Moffitt, Collisson, Bailey, and Karasinska subtypes) were used on each sample, and results were tested for subtype call consistency and association with survival.

Results: Basal-like and classical subtype calls were concordant in 88% of patient samples, and survival outcomes were significantly different ( < 0.05) between prognostic subtypes. Twelve percent of tumors had subtype-discordant calls across the different methods, showing intermediate survival in univariate and multivariate survival analyses. Transcriptional profiles compatible with that of a hybrid subtype signature were observed for subtype-discordant tumors, in which classical and basal-like genes were concomitantly expressed. Subtype-discordant tumors showed intermediate molecular characteristics, including subtyping gene expression ( < 0.0001) and mutant allelic imbalance ( < 0.001).

Conclusions: Nearly 1 in 6 patients with PDAC have tumors that fail to reliably fall into the classical or basal-like PDAC subtype categories, based on two regression tools aimed toward clinical practice. Rather, these patient tumors show intermediate prognostic and molecular traits. We propose close consideration of the non-binary nature of PDAC subtypes for future incorporation of subtyping into clinical practice.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2831DOI Listing
January 2021

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

Clin Cancer Res 2021 01 5;27(1):202-212. Epub 2020 Oct 5.

Department of Medical Oncology, BC Cancer, Vancouver, British Columbia, Canada.

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 ( = 0.007), but immune-related CD8 T-cell and M1-M2 macrophage ratio scores were more predictive for overall survival (OS; = 0.0014 and 0.0012, respectively). While [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 ( = 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1163DOI Listing
January 2021

Complete Chloroplast Genome Sequence of a Black Spruce (Picea mariana) from Eastern Canada.

Microbiol Resour Announc 2020 Sep 24;9(39). Epub 2020 Sep 24.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada

Here, we present the chloroplast genome sequence of black spruce (), a conifer widely distributed throughout North American boreal forests. This complete and annotated chloroplast sequence is 123,961 bp long and will contribute to future studies on the genetic basis of evolutionary change in spruce and adaptation in conifers.
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http://dx.doi.org/10.1128/MRA.00877-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516155PMC
September 2020

Delving into Early-onset Pancreatic Ductal Adenocarcinoma: How Does Age Fit In?

Clin Cancer Res 2021 01 21;27(1):246-254. Epub 2020 Sep 21.

BC Cancer, Vancouver, British Columbia, Canada.

Purpose: With the rising incidence of early-onset pancreatic cancer (EOPC), molecular characteristics that distinguish early-onset pancreatic ductal adenocarcinoma (PDAC) tumors from those arising at a later age are not well understood.

Experimental Design: We performed bioinformatic analysis of genomic and transcriptomic data generated from 269 advanced (metastatic or locally advanced) and 277 resectable PDAC tumor samples. Patient samples were stratified into EOPC (age of onset ≤55 years; = 117), intermediate (age of onset 55-70 years; = 264), and average (age of onset ≥70 years; = 165) groups. Frequency of somatic mutations affecting genes commonly implicated in PDAC, as well as gene expression patterns, were compared between EOPC and all other groups.

Results: EOPC tumors showed significantly lower frequency of somatic single-nucleotide variant (SNV)/insertions/deletions (indel) in ( = 0.0017), and were more likely to achieve biallelic mutation of through homozygous copy loss as opposed to heterozygous copy loss coupled with a loss-of-function SNV/indel mutation, the latter of which was more common for tumors with later ages of onset ( = 1.5e-4). Transcription factor forkhead box protein C2 () was significantly upregulated in EOPC tumors ( = 0.032). Genes significantly correlated with in PDAC samples were enriched for gene sets related to epithelial-to-mesenchymal transition (EMT) and included ( = 1.8e-8), ( = 6.5e-5), and ( = 2.4e-2).

Conclusions: Our comprehensive analysis of sequencing data generated from a large cohort of PDAC patient samples highlights a distinctive pattern of biallelic mutation in EOPC tumors. Increased expression of in EOPC, with the correlation between and EMT pathways, represents novel molecular characteristics of EOPC..
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1042DOI Listing
January 2021

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

Nat Genet 2020 08 3;52(8):800-810. Epub 2020 Aug 3.

Office of Cancer Genomics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

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.
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http://dx.doi.org/10.1038/s41588-020-0673-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498180PMC
August 2020

Epigenomic programming in early fetal brain development.

Epigenomics 2020 06 17;12(12):1053-1070. Epub 2020 Jul 17.

Department of Microbiology & Immunology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.

To provide a comprehensive understanding of gene regulatory networks in the developing human brain and a foundation for interpreting pathogenic deregulation. We generated reference epigenomes and transcriptomes of dissected brain regions and primary neural progenitor cells (NPCs) derived from cortical and ganglionic eminence tissues of four normal human fetuses. Integration of these data across developmental stages revealed a directional increase in active regulatory states, transcription factor activities and gene transcription with developmental stage. Consistent with differences in their biology, NPCs derived from cortical and ganglionic eminence regions contained common, region specific, and gestational week specific regulatory states. We provide a high-resolution regulatory network for NPCs from different brain regions as a comprehensive reference for future studies.
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http://dx.doi.org/10.2217/epi-2019-0319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857341PMC
June 2020

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

Genet Med 2020 11 6;22(11):1892-1897. Epub 2020 Jul 6.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada.

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.
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http://dx.doi.org/10.1038/s41436-020-0880-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605438PMC
November 2020

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

Oncotarget 2020 Jun 9;11(23):2204-2215. Epub 2020 Jun 9.

Department of Oral Medical and Biological Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada.

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.
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http://dx.doi.org/10.18632/oncotarget.27616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289532PMC
June 2020

Endogenous Retrovirus Transcript Levels Are Associated with Immunogenic Signatures in Multiple Metastatic Cancer Types.

Mol Cancer Ther 2020 09 9;19(9):1889-1897. Epub 2020 Jun 9.

Pancreas Centre BC, Vancouver, British Columbia, Canada.

Next-generation sequencing of solid tumors has revealed variable signatures of immunogenicity across tumors, but underlying molecular characteristics driving such variation are not fully understood. Although expression of endogenous retrovirus (ERV)-containing transcripts can provide a source of tumor-specific neoantigen in some cancer models, associations between ERV levels and immunogenicity across different types of metastatic cancer are not well established. We performed bioinformatics analysis of genomic, transcriptomic, and clinical data across an integrated cohort of 199 patients with metastatic breast, colorectal, and pancreatic ductal adenocarcinoma tumors. Within each cancer type, we identified a subgroup of viral mimicry tumors in which increased ERV levels were coupled with transcriptional signatures of autonomous antiviral response and immunogenicity. In addition, viral mimicry colorectal and pancreatic tumors showed increased expression of DNA demethylation gene Taken together, these data demonstrate the existence of an ERV-associated viral mimicry phenotype across three distinct metastatic cancer types, while indicating links between ERV abundance, epigenetic dysregulation, and immunogenicity.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0094DOI Listing
September 2020

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

Nat Neurosci 2020 07 18;23(7):842-853. Epub 2020 May 18.

Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA.

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-β 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.
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http://dx.doi.org/10.1038/s41593-020-0628-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456619PMC
July 2020

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Blood 2020 07;136(5):572-584

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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http://dx.doi.org/10.1182/blood.2019002385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440974PMC
July 2020

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Nat Med 2020 04 24;26(4):577-588. Epub 2020 Feb 24.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.
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http://dx.doi.org/10.1038/s41591-020-0757-zDOI Listing
April 2020

Sample Tracking Using Unique Sequence Controls.

J Mol Diagn 2020 02 16;22(2):141-146. Epub 2019 Dec 16.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, British Columbia; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.
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http://dx.doi.org/10.1016/j.jmoldx.2019.10.011DOI Listing
February 2020

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

Cell 2019 Nov;179(5):1207-1221.e22

Department of Molecular Oncology, BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada.

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.
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http://dx.doi.org/10.1016/j.cell.2019.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912164PMC
November 2019

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

Cell Rep 2019 11 7;29(8):2338-2354.e7. Epub 2019 Nov 7.

Hopp Children's Cancer Center, Heidelberg 69120, Germany; Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Core Center Heidelberg, Heidelberg 69120, Germany; Department of Pediatric Hematology and Oncology, University Hospital Heidelberg, Heidelberg 69120, Germany.

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.
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http://dx.doi.org/10.1016/j.celrep.2019.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905433PMC
November 2019

Evaluation of protocols for rRNA depletion-based RNA sequencing of nanogram inputs of mammalian total RNA.

PLoS One 2019 31;14(10):e0224578. Epub 2019 Oct 31.

Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, British Columbia, Canada.

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224578PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822755PMC
March 2020

Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses.

Genome Biol 2019 10 17;20(1):210. Epub 2019 Oct 17.

Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.

Background: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood.

Results: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues.

Conclusions: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.
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http://dx.doi.org/10.1186/s13059-019-1830-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796327PMC
October 2019

Altered Gene Expression along the Glycolysis-Cholesterol Synthesis Axis Is Associated with Outcome in Pancreatic Cancer.

Clin Cancer Res 2020 01 3;26(1):135-146. Epub 2019 Sep 3.

Pancreas Centre BC, Vancouver, British Columbia, Canada.

Purpose: Identification of clinically actionable molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcome. Intertumoral metabolic heterogeneity contributes to cancer survival and the balance between distinct metabolic pathways may influence PDAC outcome. We hypothesized that PDAC can be stratified into prognostic metabolic subgroups based on alterations in the expression of genes involved in glycolysis and cholesterol synthesis.

Experimental Design: We performed bioinformatics analysis of genomic, transcriptomic, and clinical data in an integrated cohort of 325 resectable and nonresectable PDAC. The resectable datasets included retrospective The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) cohorts. The nonresectable PDAC cohort studies included prospective COMPASS, PanGen, and BC Cancer Personalized OncoGenomics program (POG).

Results: On the basis of the median normalized expression of glycolytic and cholesterogenic genes, four subgroups were identified: quiescent, glycolytic, cholesterogenic, and mixed. Glycolytic tumors were associated with the shortest median survival in resectable (log-rank test = 0.018) and metastatic settings (log-rank test = 0.027). Patients with cholesterogenic tumors had the longest median survival. and -amplified tumors had higher expression of glycolytic genes than tumors with normal or lost copies of the oncogenes (Wilcoxon rank sum test = 0.015). Glycolytic tumors had the lowest expression of mitochondrial pyruvate carriers and . Glycolytic and cholesterogenic gene expression correlated with the expression of prognostic PDAC subtype classifier genes.

Conclusions: Metabolic classification specific to glycolytic and cholesterogenic pathways provides novel biological insight into previously established PDAC subtypes and may help develop personalized therapies targeting unique tumor metabolic profiles..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1543DOI Listing
January 2020

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

Proc Natl Acad Sci U S A 2019 09 30;116(38):19098-19108. Epub 2019 Aug 30.

Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6;

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 expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
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http://dx.doi.org/10.1073/pnas.1813495116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754609PMC
September 2019

Before and After: Comparison of Legacy and Harmonized TCGA Genomic Data Commons' Data.

Cell Syst 2019 07;9(1):24-34.e10

National Cancer Institute, Bethesda, MD 20892, USA.

We present a systematic analysis of the effects of synchronizing a large-scale, deeply characterized, multi-omic dataset to the current human reference genome, using updated software, pipelines, and annotations. For each of 5 molecular data platforms in The Cancer Genome Atlas (TCGA)-mRNA and miRNA expression, single nucleotide variants, DNA methylation and copy number alterations-comprehensive sample, gene, and probe-level studies were performed, towards quantifying the degree of similarity between the 'legacy' GRCh37 (hg19) TCGA data and its GRCh38 (hg38) version as 'harmonized' by the Genomic Data Commons. We offer gene lists to elucidate differences that remained after controlling for confounders, and strategies to mitigate their impact on biological interpretation. Our results demonstrate that the hg19 and hg38 TCGA datasets are very highly concordant, promote informed use of either legacy or harmonized omics data, and provide a rubric that encourages similar comparisons as new data emerge and reference data evolve.
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http://dx.doi.org/10.1016/j.cels.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707074PMC
July 2019
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