Publications by authors named "Ryan D Morin"

88 Publications

PRPS-ST: A protocol-agnostic self-training method for gene expression-based classification of blood cancers.

Blood Cancer Discov 2020 Nov 10;1(3):244-257. Epub 2020 Sep 10.

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

Gene expression classifiers are gaining increasing popularity for stratifying tumors into subgroups with distinct biological features. A fundamental limitation shared by current classifiers is the requirement for comparable training and testing data sets. Here, we describe a self-training implementation of our robability atio-based classification rediction core method (PRPS-ST), which facilitates the porting of existing classification models to other gene expression data sets. In comparison to gold standards, we demonstrate favorable performance of PRPS-ST in gene expression-based classification of DLBCL and B-ALL using a diverse variety of gene expression data types and pre-processing methods, including in classifications with a high degree of class imbalance. Tumors classified by our method were significantly enriched for prototypical genetic features of their respective subgroups. Interestingly, this included cases that were unclassifiable by established methods, implying the potential enhanced sensitivity of PRPS-ST.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774870PMC
November 2020

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

Blood 2020 Oct 29. Epub 2020 Oct 29.

BC Cancer, Vancouver, Canada.

When the WHO 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" entity has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than co-occurring translocations - i.e. copy number variations (CNVs). While the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a shared 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 (DLBCL) morphology. While 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, while MYC IHC has been proposed as a screening tool for FISH testing, two mechanisms were observed that uncoupled MYC rearrangement from IHC positivity. 1) low MYC mRNA expression and 2) false-negative immunohistochemistry (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
October 2020

Evaluating the quantity, quality and size distribution of cell-free DNA by multiplex droplet digital PCR.

Sci Rep 2020 07 28;10(1):12564. Epub 2020 Jul 28.

Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, South Sciences Building (SSB) 7157, Burnaby, BC, V5A 1S6, Canada.

Cell-free DNA (cfDNA) has become a comprehensive biomarker in the fields of non-invasive cancer detection and monitoring, organ transplantation, prenatal genetic testing and pathogen detection. While cfDNA samples can be obtained using a broad variety of approaches, there is an urgent need to standardize analytical tools aimed at assessing its basic properties. Typical methods to determine the yield and fragment size distribution of cfDNA samples are usually either blind to genomic DNA contamination or the presence of enzymatic inhibitors, which can confound and undermine downstream analyses. Here, we present a novel droplet digital PCR assay to identify suboptimal samples and aberrant cfDNA size distributions, the latter typically associated with high levels of circulating tumour DNA (ctDNA). Our assay was designed to promiscuously cross-amplify members of the human olfactory receptor (OR) gene family and includes a customizable diploid locus for the determination of absolute cfDNA concentrations. We demonstrate here the utility of our assay to estimate the yield and quality of cfDNA extracts and deduce fragment size distributions that correlate well with those inferred by capillary electrophoresis and high throughput sequencing. The assay described herein is a powerful tool to establish quality controls and stratify cfDNA samples based on presumed ctDNA levels, then facilitating the implementation of robust, cost-effective and standardized analytical workflows into clinical practice.
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http://dx.doi.org/10.1038/s41598-020-69432-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387491PMC
July 2020

IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms.

Nat Commun 2020 07 7;11(1):3390. Epub 2020 Jul 7.

Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.
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http://dx.doi.org/10.1038/s41467-020-17095-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341758PMC
July 2020

TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate.

Cell 2020 Jul 2;182(2):297-316.e27. Epub 2020 Jul 2.

Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY 10021, USA. Electronic address:

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
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http://dx.doi.org/10.1016/j.cell.2020.05.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384961PMC
July 2020

Genetic and evolutionary patterns of treatment resistance in relapsed B-cell lymphoma.

Blood Adv 2020 07;4(13):2886-2898

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

Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
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http://dx.doi.org/10.1182/bloodadvances.2020001696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362366PMC
July 2020

DLBCL subclassification: divide and conquer?

Blood 2020 05;135(20):1722-1724

British Columbia Cancer Centre for Lymphoid Cancer.

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http://dx.doi.org/10.1182/blood.2020005335DOI Listing
May 2020

A Probabilistic Classification Tool for Genetic Subtypes of Diffuse Large B Cell Lymphoma with Therapeutic Implications.

Cancer Cell 2020 04;37(4):551-568.e14

Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

The development of precision medicine approaches for diffuse large B cell lymphoma (DLBCL) is confounded by its pronounced genetic, phenotypic, and clinical heterogeneity. Recent multiplatform genomic studies revealed the existence of genetic subtypes of DLBCL using clustering methodologies. Here, we describe an algorithm that determines the probability that a patient's lymphoma belongs to one of seven genetic subtypes based on its genetic features. This classification reveals genetic similarities between these DLBCL subtypes and various indolent and extranodal lymphoma types, suggesting a shared pathogenesis. These genetic subtypes also have distinct gene expression profiles, immune microenvironments, and outcomes following immunochemotherapy. Functional analysis of genetic subtype models highlights distinct vulnerabilities to targeted therapy, supporting the use of this classification in precision medicine trials.
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http://dx.doi.org/10.1016/j.ccell.2020.03.015DOI Listing
April 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

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

The double-hit signature identifies double-hit diffuse large B-cell lymphoma with genetic events cryptic to FISH.

Blood 2019 10;134(18):1528-1532

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

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit-like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.
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http://dx.doi.org/10.1182/blood.2019002600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839951PMC
October 2019

Integrative genomic analysis of matched primary and metastatic pediatric osteosarcoma.

J Pathol 2019 11 28;249(3):319-331. Epub 2019 Aug 28.

Department of Molecular Oncology, British Columbia Cancer Agency, Vancouver, Canada.

Despite being the most common childhood bone tumor, the genomic characterization of osteosarcoma remains incomplete. In particular, very few osteosarcoma metastases have been sequenced to date, critical to better understand mechanisms of progression and evolution in this tumor. We performed an integrated whole genome and exome sequencing analysis of paired primary and metastatic pediatric osteosarcoma specimens to identify recurrent genomic alterations. Sequencing of 13 osteosarcoma patients including 13 primary, 10 metastatic, and 3 locally recurring tumors revealed a highly heterogeneous mutational landscape, including cases of hypermutation and microsatellite instability positivity, but with virtually no recurrent alterations except for mutations involving the tumor suppressor genes RB1 and TP53. At the germline level, we detected alterations in multiple cancer related genes in the majority of the cohort, including those potentially disrupting DNA damage response pathways. Metastases retained only a minimal number of short variants from their corresponding primary tumors, while copy number alterations showed higher conservation. One recurrently amplified gene, KDR, was highly expressed in advanced cases and associated with poor prognosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5319DOI Listing
November 2019

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Proc Natl Acad Sci U S A 2019 04 18;116(14):6969-6974. Epub 2019 Mar 18.

Molecular Cardiac Physiology Group, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation ( R37C) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca-binding sensitivity due to an increased Ca off-rate constant. Furthermore, we generated R37C mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in R37C at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.
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http://dx.doi.org/10.1073/pnas.1819023116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452669PMC
April 2019

Ultrasensitive Detection of Circulating Tumor DNA in Lymphoma via Targeted Hybridization Capture and Deep Sequencing of Barcoded Libraries.

Methods Mol Biol 2019 ;1956:383-435

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

Liquid biopsies are rapidly emerging as powerful tools for the early detection of cancer, noninvasive genomic profiling of localized or metastatic tumors, prompt detection of treatment resistance-associated mutations, and monitoring of therapeutic response and minimal residual disease in patients during clinical follow-up. Growing evidence strongly supports the utility of circulating tumor DNA (ctDNA) as a biomarker for the stratification and clinical management of lymphoma patients. However, ctDNA is diluted by variable amounts of cell-free DNA (cfDNA) shed by nonneoplastic cells causing a background signal of wild-type DNA that limits the sensitivity of methods that rely on DNA sequencing. Here, we describe an error suppression method for single-molecule counting that relies on targeted sequencing of cfDNA libraries constructed with semi-degenerate barcode adapters. Custom pools of biotinylated DNA baits for target enrichment can be designed to specifically track somatic mutations in one patient, survey mutation hotspots with diagnostic and prognostic value or be comprised of comprehensive gene panels with broad patient coverage in lymphoma. Such methods are amenable to track ctDNA levels during longitudinal liquid biopsy testing with high specificity and sensitivity and characterize, in real time, the genetic profiles of tumors without the need of standard invasive biopsies. The analysis of ultra-deep sequencing data according to the bioinformatics pipelines also described in this chapter affords to harness lower limits of detection for ctDNA below 0.1%.
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http://dx.doi.org/10.1007/978-1-4939-9151-8_20DOI Listing
July 2019

SUBSTRA: Supervised Bayesian Patient Stratification.

Bioinformatics 2019 09;35(18):3263-3272

Computational Systems Immunology, Pfizer Worldwide R&D, Berlin, Germany.

Motivation: Patient stratification methods are key to the vision of precision medicine. Here, we consider transcriptional data to segment the patient population into subsets relevant to a given phenotype. Whereas most existing patient stratification methods focus either on predictive performance or interpretable features, we developed a method striking a balance between these two important goals.

Results: We introduce a Bayesian method called SUBSTRA that uses regularized biclustering to identify patient subtypes and interpretable subtype-specific transcript clusters. The method iteratively re-weights feature importance to optimize phenotype prediction performance by producing more phenotype-relevant patient subtypes. We investigate the performance of SUBSTRA in finding relevant features using simulated data and successfully benchmark it against state-of-the-art unsupervised stratification methods and supervised alternatives. Moreover, SUBSTRA achieves predictive performance competitive with the supervised benchmark methods and provides interpretable transcriptional features in diverse biological settings, such as drug response prediction, cancer diagnosis, or kidney transplant rejection.

Availability And Implementation: The R code of SUBSTRA is available at https://github.com/sahandk/SUBSTRA.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz112DOI Listing
September 2019

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

Biotechniques 2019 02;66(2):85-92

Canada's Michael Smith Genome Sciences Centre, BC Cancer, 675 West 10th Avenue, Vancouver, BC, Canada.

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.
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http://dx.doi.org/10.2144/btn-2018-0148DOI Listing
February 2019

Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 as Therapeutic Target for Enhancing Immune Recognition.

Cancer Discov 2019 04 31;9(4):546-563. Epub 2019 Jan 31.

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

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II-deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of mutations in both MHC-I- and MHC-II-negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant . Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in -mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming. SIGNIFICANCE: We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin-specific context. Specifically, mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1090DOI Listing
April 2019

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

Blood 2019 03 7;133(12):1313-1324. Epub 2019 Jan 7.

Lymphoid Malignancies Branch, Center for Cancer Research and.

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 , , and , 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.
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http://dx.doi.org/10.1182/blood-2018-09-871418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428665PMC
March 2019

Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma.

J Clin Oncol 2019 01 3;37(3):190-201. Epub 2018 Dec 3.

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

Purpose: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression.

Patients And Methods: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data.

Results: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested.

Conclusion: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.
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http://dx.doi.org/10.1200/JCO.18.01583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804880PMC
January 2019

A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens.

J Mol Diagn 2019 03 23;21(2):214-227. Epub 2018 Nov 23.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. Electronic address:

Recurrent activating point mutations in KRAS are critical drivers in pancreatic cancer and have been attributed to resistance to anti-epidermal growth factor receptor therapy in colorectal cancer. Although KRAS genotyping provides limited clinical utility in the diagnosis and management of pancreatic cancer patients at present, inferences about the fractional abundance of KRAS mutations may inform on tumor purity in traditionally challenging clinical specimens and their potential use in precision medicine. KRAS genetic testing has indeed become an essential tool to guide treatment decisions in colorectal cancer, but an unmet need for methods standardization exists. Here, we present a unique droplet digital PCR method that enables the simultaneous detection and quantification of KRAS exon 2, 3, and 4 point mutations and copy number alterations. We have validated 13 mutations (G12S, G12R, G12D, G12A, G12V, G12C, G13D, G60V, Q61H, Q61L, A146V, A146T, and A146P) and focal KRAS amplifications by conducting this assay in a cohort of 100 DNA samples extracted from fresh frozen tumor biopsies, formaldehyde-fixed, paraffin-embedded tissue, and liquid biopsy specimens. Despite its modest lower limit of detection (approximately 1%), this assay will be a rapid cost-effective means to infer the purity of biopsy specimens carrying KRAS mutations and can be used in noninvasive serial monitoring of circulating tumor DNA to evaluate clinical response and/or detect early signs of relapse.
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http://dx.doi.org/10.1016/j.jmoldx.2018.09.007DOI Listing
March 2019

High-resolution architecture and partner genes of rearrangements in lymphoma with DLBCL morphology.

Blood Adv 2018 10;2(20):2755-2765

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

Genomic rearrangements in the locus occur in ∼12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of , , , and the immunoglobulin () loci in 112 tumors with DLBCL morphology harboring rearrangement. We characterized the location of the rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the coding region and in intron 1 (the "genic cluster"). Genic cluster rearrangements were enriched for translocations involving (80%), whereas nongenic rearrangements occurred mostly downstream of the gene with a variety of partners, including and Other recurrent partners included , , and , which has not previously been described as a partner. We compared 2 commercially available FISH break-apart assays for the locus and observed discordant results in 32% of cases examined, including some with - and - rearrangements. In cases of high-grade B-cell lymphoma with and and/or rearrangement (HGBL-DH), so-called "double-hit" lymphomas, the majority of rearrangements had non- partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, - rearrangements showed a trend toward inferior time to progression and overall survival compared with -non- rearrangements. Our data reveal clinically relevant architecture of rearrangements in lymphomas with DLBCL morphology.
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http://dx.doi.org/10.1182/bloodadvances.2018023572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199666PMC
October 2018

Temporal Dynamics of Genomic Alterations in a Germline-Mutated Pancreatic Cancer With Low Genomic Instability Burden but Exceptional Response to Fluorouracil, Oxaliplatin, Leucovorin, and Irinotecan.

JCO Precis Oncol 2018 19;2. Epub 2018 Oct 19.

, , , , , , , , , , , , and , British Columbia Cancer Agency; , , , , and , Pancreas Centre BC; and , Simon Fraser University; , , , , , and , University of British Columbia; and , Vancouver General Hospital, Vancouver, British Columbia, Canada.

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http://dx.doi.org/10.1200/PO.18.00057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446469PMC
October 2018

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma.

Nat Commun 2018 10 1;9(1):4001. Epub 2018 Oct 1.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.
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http://dx.doi.org/10.1038/s41467-018-06354-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167379PMC
October 2018

Novel Multiplexing Strategies for Quantification of Rare Alleles Using ddPCR.

Methods Mol Biol 2018 ;1768:275-301

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

Droplet digital PCR (ddPCR) has come to be regarded as the gold standard for the ultrasensitive detection and absolute quantification of closely related DNA sequences within complex mixtures. Most ddPCR assays to date, however, rely on sets of hydrolysis probes conjugated with dyes having different emission spectra to allow independent counting of rare mutant and wild-type alleles. Here, we describe a set of novel strategies that leverage the simultaneous detection and quantification of both mutant and wild-type alleles with a single hydrolysis probe. Variants of these strategies empower multiplexing and a more cost-effective approach for concurrent screening of multiple genetic variants.
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http://dx.doi.org/10.1007/978-1-4939-7778-9_16DOI Listing
February 2019

Disruption of the Gut Microbiota With Antibiotics Exacerbates Acute Vascular Rejection.

Transplantation 2018 07;102(7):1085-1095

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

Background: The gut microbiota influences many immunological processes but how its disruption affects transplant rejection is poorly understood.

Methods: Interposition grafting of aortic segments was used to examine vascular rejection. The gut microbiota was disrupted in graft recipients using an antibiotic cocktail (ampicillin, vancomycin, metronidazole, neomycin sulfate) in their drinking water.

Results: Treatment of mice with antibiotics severely reduced total bacterial content in the intestine and disrupted the bacterial composition. Short-term treatment of mice for only the first 3 weeks of life resulted in the population of the intestine in mature mice with bacterial communities that were mildly different from untreated mice, containing slightly more Clostridia and less Bacteroides. Antibiotic disruption of the gut microbiota of graft recipients, either for their entire life or only during the first 3 weeks of life, resulted in increased medial injury of allograft arteries that is reflective of acute vascular rejection but did not affect intimal thickening reflective of transplant arteriosclerosis. Exacerbated vascular rejection resulting from disruption of the gut microbiota was related to increased infiltration of allograft arteries by neutrophils.

Conclusions: Disruption of the gut microbiota early in life results in exacerbation of immune responses that cause acute vascular rejection.
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http://dx.doi.org/10.1097/TP.0000000000002169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228629PMC
July 2018

The genomic landscape of two Burkitt lymphoma cases and derived cell lines: comparison between primary and relapse samples.

Leuk Lymphoma 2018 09 3;59(9):2159-2174. Epub 2018 Jan 3.

a Department of Medicine , McGill University, Lady Davis Institute, Jewish General Hospital , Montreal , Canada.

Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.
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http://dx.doi.org/10.1080/10428194.2017.1413186DOI Listing
September 2018

Targeted error-suppressed quantification of circulating tumor DNA using semi-degenerate barcoded adapters and biotinylated baits.

Sci Rep 2017 09 5;7(1):10574. Epub 2017 Sep 5.

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

Ultrasensitive methods for rare allele detection are critical to leverage the full potential offered by liquid biopsies. Here, we describe a novel molecular barcoding method for the precise detection and quantification of circulating tumor DNA (ctDNA). The major benefits of our design include straightforward and cost-effective production of barcoded adapters to tag individual DNA molecules before PCR and sequencing, and better control over cross-contamination between experiments. We validated our approach in a cohort of 24 patients with a broad spectrum of cancer diagnoses by targeting and quantifying single-nucleotide variants (SNVs), indels and genomic rearrangements in plasma samples. By using personalized panels targeting a priori known mutations, we demonstrate comprehensive error-suppression capabilities for SNVs and detection thresholds for ctDNA below 0.1%. We also show that our semi-degenerate barcoded adapters hold promise for noninvasive genotyping in the absence of tumor biopsies and monitoring of minimal residual disease in longitudinal plasma samples. The benefits demonstrated here include broad applicability, flexibility, affordability and reproducibility in the research and clinical settings.
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http://dx.doi.org/10.1038/s41598-017-10269-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585219PMC
September 2017

Mapping the human T cell repertoire to recurrent driver mutations in MYD88 and EZH2 in lymphoma.

Oncoimmunology 2017;6(7):e1321184. Epub 2017 Apr 28.

Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada.

Oncogenic "driver" mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: , and . Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17-26 HLA class I alleles and 3 × 10-3 × 10 T cells per donor, we identified CD4 T cells against EFISECGEII from EZH2 (presented by HLA-DRB1*13:02) and CD8 T cells against RIPIKYKA from MYD88 (presented by HLA-B*07:02). We failed to detect RIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.
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http://dx.doi.org/10.1080/2162402X.2017.1321184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543822PMC
April 2017