Nature Methods, 2020
Authors
Jasleen K. Grewal, Martin Krzywinski, Naomi Altman
Publication Abstract

In the previous column we showed how hidden states driving observable changes in a cell can be modeled as a hidden Markov model (HMM). To confidently use the HMM for inference or prediction, we must first train it to accurately represent observed data. 

The Journal of molecular diagnostics : JMD, 2020
Authors
Moore, Richard A, Zeng, Thomas, Docking, T Roderick, Bosdet, Ian, Butterfield, Yaron S, Munro, Sarah, Li, Irene, Swanson, Lucas, Starks, Elizabeth R, Tse, Kane, Mungall, Andrew J, Holt, Robert A, Karsan, Aly
Publication Abstract
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.

Microbial genomics, 2020
Authors
Cochrane, Kyla, Robinson, Avery V, Holt, Robert A, Allen-Vercoe, Emma
Publication Abstract
Here, we report comprehensive transcriptomic profiles from under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in , allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of tolerization.

Cold Spring Harbor Molecular Case Studies, 2019
Authors
Elisa Majounie, Kathleen Wee, Laura M Williamson, Martin R Jones, Erin Pleasance, Howard J Lim, Cheryl Ho, Daniel J Renouf, Stephen Yip, Steven J M Jones, Marco A Marra, Janessa Laskin
Publication Abstract

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and represents a heterogeneous group of tumours, the majority of which are treated with a combination of surgery, radiation and chemotherapy. Fluoropyrimidine (5-FU) and its oral pro-drug, capecitabine, are commonly prescribed treatments for several solid tumour types including HNSCC. 5-FU-associated toxicity is observed in approximately 30% of treated patients and is largely caused by germline polymorphisms in DPYD which encodes dihydropyrimidine dehydrogenase (DPD), a key enzyme of 5-FU catabolism and deactivation. Although the association of germline DPYD alterations with toxicity is well-described, the potential contribution of somatic DPYD alterations to 5-FU sensitivity has not been explored. In a patient with metastatic HNSCC, in-depth genomic and transcriptomic integrative analysis on a biopsy from a metastatic neck lesion revealed alterations in genes that are associated with 5-FU uptake and metabolism. These included a novel somatic structural variant resulting in a partial deletion affecting DPYD, a variant of unknown significance affecting SLC29A1 and homozygous deletion of MTAP. There was no evidence of deleterious germline polymorphisms that have been associated with 5-FU toxicity, indicating a potential vulnerability of the tumour to 5-FU therapy. The discovery of the novel DPYD variant led to the initiation of 5-FU treatment that resulted in a rapid response lasting 17 weeks, with subsequent relapse due to unknown resistance mechanisms. This suggests that somatic alterations present in this tumour may serve as markers for tumour sensitivity to 5-FU, aiding in selection of personalized treatment strategies.

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Investigational New Drugs, 2020
Authors
Jean-Michel Lavoie, Teresa Mitchell, Sung-Eun Lee, Balvir Deol, Stephen K Chia, Karen A Gelmon, Christian K Kollmannsberger, Anna V Tinker, Steven J M Jones, Marco Marra, Janessa Laskin, Daniel J Renouf
Publication Abstract

Introduction Given the high level of uncertainty surrounding the outcomes of early phase clinical trials, whole genome and transcriptome analysis (WGTA) can be used to optimize patient selection and study assignment. In this retrospective analysis, we reviewed the impact of this approach on one such program. Methods Patients with advanced malignancies underwent fresh tumor biopsies as part of our personalized medicine program (NCT02155621). Tumour molecular data were reviewed for potentially clinically actionable findings and patients were referred to the developmental therapeutics program. Outcomes were reviewed in all patients, including those where trial selection was driven by molecular data (matched) and those where there was no clear molecular rationale (unmatched). Results From January 2014 to January 2018, 28 patients underwent WGTA and enrolled in clinical trials, including 2 patients enrolled in two trials. Fifteen patients were matched to a treatment based on a molecular target. Five patients were matched to a trial based upon single-gene DNA changes, all supported by RNA data. Ten cases were matched on the basis of genome-wide data (n = 4) or RNA gene expression only (n = 6). With a median follow-up of 6.7 months, the median time on treatment was 8.2 weeks. Discussion When compared to single-gene DNA-based data alone, WGTA led to a 3-fold increase in treatment matching. In a setting where there is a high level of uncertainty around both the investigational agents and the biomarkers, more data are needed to fully evaluate the impact of routine use of WGTA.

Authors
Laura Fachal, Hugues Aschard, Jonathan Beesley, Daniel R Barnes, Jamie Allen, Siddhartha Kar, Karen A Pooley, Joe Dennis, Kyriaki Michailidou, Constance Turman, Penny Soucy, Audrey Lemaçon, Michael Lush, Jonathan P Tyrer, Maya Ghoussaini, Mahdi Moradi Marjaneh, Xia Jiang, Simona Agata, Kristiina Aittomäki, M Rosario Alonso, Irene L Andrulis, Hoda Anton-Culver, Natalia N Antonenkova, Adalgeir Arason, Volker Arndt, Kristan J Aronson, Banu K Arun, Bernd Auber, Paul L Auer, Jacopo Azzollini, Judith Balmaña, Rosa B Barkardottir, Daniel Barrowdale, Alicia Beeghly-Fadiel, Javier Benitez, Marina Bermisheva, Katarzyna Białkowska, Amie M Blanco, Carl Blomqvist, William Blot, Natalia V Bogdanova, Stig E Bojesen, Manjeet K Bolla, Bernardo Bonanni, Ake Borg, Kristin Bosse, Hiltrud Brauch, Hermann Brenner, Ignacio Briceno, Ian W Brock, Angela Brooks-Wilson, et al.
Publication Abstract

Genome-wide association studies have identified breast cancer risk variants in over 150 genomic regions, but the mechanisms underlying risk remain largely unknown. These regions were explored by combining association analysis with in silico genomic feature annotations. We defined 205 independent risk-associated signals with the set of credible causal variants in each one. In parallel, we used a Bayesian approach (PAINTOR) that combines genetic association, linkage disequilibrium and enriched genomic features to determine variants with high posterior probabilities of being causal. Potentially causal variants were significantly over-represented in active gene regulatory regions and transcription factor binding sites. We applied our INQUSIT pipeline for prioritizing genes as targets of those potentially causal variants, using gene expression (expression quantitative trait loci), chromatin interaction and functional annotations. Known cancer drivers, transcription factors and genes in the developmental, apoptosis, immune system and DNA integrity checkpoint gene ontology pathways were over-represented among the highest-confidence target genes.

Nature Genetics, 2020
Authors
Michelle Chan-Seng-Yue, Jaeseung C Kim, Gavin W Wilson, Karen Ng, Eugenia Flores Figueroa, Grainne M O'Kane, Ashton A Connor, Robert E Denroche, Robert C Grant, Jessica McLeod, Julie M Wilson, Gun Ho Jang, Amy Zhang, Sheng-Ben Liang, Ayelet Borgida, Dianne Chadwick, Sangeetha Kalimuthu, Ilinca Lungu, John M S Bartlett, Paul M Krzyzanowski, Vandana Sandhu, Hervé Tiriac, Fieke E M Froeling, Joanna M Karasinska, James T Topham, Daniel J Renouf, David F Schaeffer, Steven J M Jones, Marco A Marra, Janessa Laskin, Runjan Chetty, Lincoln D Stein, George Zogopoulos, Benjamin Haibe-Kains, Peter J Campbell, David A Tuveson, Jennifer J Knox, Sandra E Fischer, Steven Gallinger, Faiyaz Notta
Publication Abstract

Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.

Journal of Experimental Medicine, 2020
Authors
Maryam Ghaedi, Zi Yi Shen, Mona Orangi, Itziar Martinez-Gonzalez, Lisa Wei , Xiaoxiao Lu, Arundhoti Das, Alireza Heravi-Moussavi, Marco A Marra, Avinash Bhandoola, Fumio Takei
Publication Abstract

Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor-related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2- population different from conventional IL-18Rα-ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.

EMBO reports, 2019
Authors
El-Naggar, Amal M, Somasekharan, Syam Prakash, Wang, Yemin, Cheng, Hongwei, Negri, Gian Luca, Pan, Melvin, Wang, Xue Qi, Delaidelli, Alberto, Rafn, Bo, Cran, Jordan, Zhang, Fan, Zhang, Haifeng, Colborne, Shane, Gleave, Martin, Mandinova, Anna, Kedersha, Nancy, Hughes, Christopher S, Surdez, Didier, Delattre, Olivier, Wang, Yuzhuo, Huntsman, David G, Morin, Gregg B, Sorensen, Poul H
Publication Abstract
Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.

The Journal of pathology, 2019
Authors
Negri, Gian Luca, Grande, Bruno M, Delaidelli, Alberto, El-Naggar, Amal, Cochrane, Dawn, Lau, Ching C, Triche, Timothy J, Moore, Richard A, Jones, Steven Jm, Montpetit, Alexandre, Marra, Marco A, Malkin, David, Morin, Ryan D, Sorensen, Poul H
Publication Abstract
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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