Clinical Cancer Research
Authors
Matti Annala, Sinja Taavitsainen, Daniel J Khalaf, Gillian Vandekerkhove, Kevin Beja, Joonatan Sipola, Evan W Warner, Cameron Herberts, Amanda Wong, Simon Fu, Daygen L Finch, Conrad D Oja, Joanna Vergidis, Muhammad Zulfiqar, Bernhard J Eigl, Christian K Kollmansberger, Matti Nykter, Martin E Gleave, Kim N Chi, Alexander W Wyatt
Publication Abstract

Cross-resistance renders multiple lines of androgen receptor (AR) signaling inhibitors increasingly futile in metastatic castration-resistant prostate cancer (mCRPC). We sought to determine acquired genomic contributors to cross-resistance.

Frontiers in Genetics
Authors
Simon Haile, Richard D. Corbett, Veronique G. LeBlanc, Lisa Wei, Stephen Pleasance, Steve Bilobram, Ka Ming Nip, Kirstin Brown, Eva Trinh, Jillian Smith, Diane L. Trinh, Miruna Bala, Eric Chuah, Robin J. N. Coope, Richard A. Moore, Andrew J. Mungall, Karen L. Mungall, Yongjun Zhao, Martin Hirst, Samuel Aparicio, Inanc Birol, Steven J. M. Jones, Marco A. Marra
Publication Abstract

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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Cancer Discovery, 2021
Authors
Hai-Feng Zhang, Christopher S Hughes, Wei Li, Jian-Zhong He, Didier Surdez, Amal M El-Naggar, Hongwei Cheng, Anna Prudova, Alberto Delaidelli, Gian Luca Negri, Xiaojun Li, Maj Sofie Orum-Madsen, Michael M Lizardo, Htoo Zarni Oo, Shane Colborne, Taras Shyp, Renata Scopim-Ribeiro, Colin A Hammond, Anne-Chloe Dhez, Sofya Langman, Jonathan Km Lim, Sonia Hy Kung, Amy Li, Anne Steino, Mads Daugaard, Seth J Parker, Ramon I Klein Geltink, Rimas J Orentas, Li-Yan Xu, Gregg B Morin, Olivier Delattre, Dimiter S Dimitrov, Poul H Sorensen
Publication Abstract

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL-1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma (EwS), a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of EwS cells. Mechanistically, IL1RAP binds the cell surface system Xc- transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore IL1RAP maintains cyst(e)ine and glutathione pools which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of EwS cells. Therefore, we define IL1RAP as a new cell surface target in EwS, which is potentially exploitable for immunotherapy.

American Journal of Medical Genetics, 2021
Authors
Hui-Lin Chin, Kieran O'Neill, Kristal Louie, Lindsay Brown, Kamilla Schlade-Bartusiak, Patrice Eydoux, Rosemarie Rupps, Ali Farahani, Cornelius F Boerkoel, Steven J M Jones
Publication Abstract

Prenatal detection of structural variants of uncertain significance, including copy number variants (CNV), challenges genetic counseling, and creates ambiguity for expectant parents. In Duchenne muscular dystrophy, variant classification and phenotypic severity of CNVs are currently assessed by familial segregation, prediction of the effect on the reading frame, and precedent data. Delineation of pathogenicity by familial segregation is limited by time and suitable family members, whereas analytical tools can rapidly delineate potential consequences of variants. We identified a duplication of uncertain significance encompassing a portion of the dystrophin gene (DMD) in an unaffected mother and her male fetus. Using long-read whole genome sequencing and alignment of short reads, we rapidly defined the precise breakpoints of this variant in DMD and could provide timely counseling. The benign nature of the variant was substantiated, more slowly, by familial segregation to a healthy maternal uncle. We find long-read whole genome sequencing of clinical utility in a prenatal setting for accurate and rapid characterization of structural variants, specifically a duplication involving DMD.

Canadian Medical Journal, 2021
Authors
Jason A McVicar, Alana Poon, Nadine R Caron, M Dylan Bould, Jason W Nickerson, Nora Ahmad, Donna May Kimmaliardjuk, Chelsey Sheffield, Caitlin Champion, Daniel I McIsaac
Publication Abstract

Background: Substantial health inequities exist for Indigenous Peoples in Canada. The remote and distributed population of Canada presents unique challenges for access to and use of surgery. To date, the surgical outcome data for Indigenous Peoples in Canada have not been synthesized.

Methods: We searched 4 databases to identify studies comparing surgical outcomes and utilization rates of adults of First Nations, Inuit or Métis identity with non-Indigenous people in Canada. Independent reviewers completed all stages in duplicate. Our primary outcome was mortality; secondary outcomes included utilization rates of surgical procedures, complications and hospital length of stay. We performed meta-analysis of the primary outcome using random effects models. We assessed risk of bias using the ROBINS-I tool.

Results: Twenty-eight studies were reviewed involving 1 976 258 participants (10.2% Indigenous). No studies specifically addressed Inuit or Métis populations. Four studies, including 7 cohorts, contributed adjusted mortality data for 7135 participants (5.2% Indigenous); Indigenous Peoples had a 30% higher rate of death after surgery than non-Indigenous patients (pooled hazard ratio 1.30, 95% CI 1.09-1.54; I 2 = 81%). Complications were also higher for Indigenous Peoples, including infectious complications (adjusted OR 1.63, 95% CI 1.13-2.34) and pneumonia (OR 2.24, 95% CI 1.58-3.19). Rates of various surgical procedures were lower, including rates of renal transplant, joint replacement, cardiac surgery and cesarean delivery.

Interpretation: The currently available data on postoperative outcomes and surgery utilization rates for Indigenous Peoples in Canada are limited and of poor quality. Available data suggest that Indigenous Peoples have higher rates of death and adverse events after surgery, while also encountering barriers accessing surgical procedures. These findings suggest a need for substantial re-evaluation of surgical care for Indigenous Peoples in Canada to ensure equitable access and to improve outcomes.

Nature Communications, 2021
Authors
T Roderick Docking, Jeremy D K Parker, Martin Jädersten, Gerben Duns, Linda Chang, Jihong Jiang, Jessica A Pilsworth, Lucas A Swanson, Simon K Chan, Readman Chiu, Ka Ming Nip, Samantha Mar, Angela Mo, Xuan Wang, Sergio Martinez-Høyer, Ryan J Stubbins, Karen L Mungall, Andrew J Mungall, Richard A Moore, Steven J M Jones, İnanç Birol, Marco A Marra, Donna Hogge, Aly Karsan
Publication Abstract

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.

Developmental Cell, 2021
Authors
Bo Zhang, M Yvonne Kim, GiNell Elliot, Yan Zhou, Guangfeng Zhao, Daofeng Li, Rebecca F Lowdon, Matthew Gormley, Mirhan Kapidzic, Joshua F Robinson, Michael T McMaster, Chibo Hong, Tali Mazor, Emily Hamilton, Renee L Sears, Erica C Pehrsson, Marco A Marra, Steven J M Jones, Misha Bilenky, Martin Hirst, Ting Wang, Joseph F Costello, Susan J Fisher
Publication Abstract

The human placenta and its specialized cytotrophoblasts rapidly develop, have a compressed lifespan, govern pregnancy outcomes, and program the offspring's health. Understanding the molecular underpinnings of these behaviors informs development and disease. Profiling the extraembryonic epigenome and transcriptome during the 2nd and 3rd trimesters revealed H3K9 trimethylation overlapping deeply DNA hypomethylated domains with reduced gene expression and compartment-specific patterns that illuminated their functions. Cytotrophoblast DNA methylation increased, and several key histone modifications decreased across the genome as pregnancy advanced. Cytotrophoblasts from severe preeclampsia had substantially increased H3K27 acetylation globally and at genes that are normally downregulated at term but upregulated in this syndrome. In addition, some cases had an immature pattern of H3K27ac peaks, and others showed evidence of accelerated aging, suggesting subtype-specific alterations in severe preeclampsia. Thus, the cytotrophoblast epigenome dramatically reprograms during pregnancy, placental disease is associated with failures in this process, and H3K27 hyperacetylation is a feature of severe preeclampsia.

Blood Advances, 2021
Authors
Ryan D Morin, Sarah E Arthur, Sarit Assouline
Publication Abstract

Tazemetostat represents the first epigenetic therapy approved for the treatment of follicular lymphoma (FL). It inhibits the activity of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase, the first of a multitude of epigenetic regulators that have been identified as recurrently mutated in FL and germinal center diffuse large B-cell lymphoma. In this review, we discuss the initial discovery and ongoing exploration of the functional role of EZH2 mutations in lymphomagenesis. We also explore the path from the preclinical development of tazemetostat to its approval for the treatment of relapsed FL, and potential future therapeutic applications. We discuss the clinical data that led to the approval of tazemetostat and ongoing research into the function of EZH2 and of tazemetostat in lymphomas that derive from the germinal center, which could increase the applicability of this drug in the future.

Journal of Medical Genetics, 2021
Authors
Chloe Mighton, Amanda C Smith, Justin Mayers, Robert Tomaszewski, Sherryl Taylor, Stacey Hume, Ron Agatep, Elizabeth Spriggs, Harriet E Feilotter, Laura Semenuk, Henry Wong, Lorena Lazo de la Vega, Christian R Marshall, Michelle M Axford, Talia Silver, George S Charames, Vanessa Di Gioacchino, Nicholas Watkins, William D Foulkes, Marcos Clavier, Nancy Hamel, George Chong, Ryan E Lamont, Jillian Parboosingh, Aly Karsan, Ian Bosdet, Sean S Young, Tracy Tucker, Mohammad Reza Akbari, Marsha D Speevak, Andrea K Vaags, Matthew S Lebo, Jordan Lerner-Ellis, Canadian Open Genetics Repository Working Group
Publication Abstract

Background: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation.

Methods: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin.

Results: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants.

Conclusions: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Cancers, 2021
Authors
Bram De Laere, Alessio Crippa, Ashkan Mortezavi, Christophe Ghysel, Prabhakar Rajan, Martin Eklund, Alexander Wyatt, Luc Dirix, Piet Ost, Henrik Grönberg, Johan Lindberg
Publication Abstract

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease, characterized by common and rare driver gene alterations that provide a selective growth advantage for progressing tumour cells. We hypothesized that the number of distinct gene driver alteration-affected pathways or gene classes was associated with poor prognosis in patients initiating androgen receptor signalling inhibitors (ARSi). We performed a post hoc analysis of an amalgamated baseline circulating tumour DNA (ctDNA) mutational landscape dataset of ARSi-treated men with mCRPC (n = 342). We associated the detected hotspot, pathogenic, and/or high impact protein function-affecting perturbations in 39 genes into 13 pathways. Progression-free (PFS) and overall survival (OS) were analysed using Kaplan-Meier curves and multivariate Cox regression models. Driver gene alterations were detected in 192/342 (56.1%) evaluable patients. An increased number of affected pathways, coined pathway complexity index (PCI), resulted in a decremental PFS and OS, and was independently associated with prognosis once ≥3 pathway or gene classes were affected (PFS HR (95%CI): 1.7 (1.02-2.84), p = 0.04, and OS HR (95%CI): 2.5 (1.06-5.71), p = 0.04). Additionally, visceral disease and baseline PSA and plasma ctDNA levels were independently associated with poor prognosis. Elevated PCI is associated with poor ARSi outcome and supports comprehensive genomic profiling to better infer mCRPC prognosis.

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