Andy Mungall

The E3 ubiquitin ligase UBR5 is recurrently mutated in mantle cell lymphoma

We have recently reported the application of RNAseq to mantle cell lymphoma (MCL) transcriptomes revealing recurrent mutations in NOTCH1. Here we describe the targeted resequencing of 18 genes mutated in this discovery cohort using a larger cohort of MCL tumors. In addition to frequent mutations in ATM, CCND1, TP53, and NOTCH1, mutations were also observed recurrently in MEF2B, TRAF2, and TET2. Interestingly, the third most frequently mutated gene was UBR5, a gene encoding a 2799aa protein, with multiple functions, including E3 ligase activity based on a conserved cysteine residue at the C-terminus. Nonsynonymous mutations were detected in 18% (18/102) of tumors, with 61% of the mutations resulting in frameshifts in, or around, exon 58, predicted to result in the loss of this conserved cysteine residue. The recurrence and clustering of deleterious mutations implicate UBR5 mutations as a critical pathogenic event in a subgroup of MCL.

A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer

Background NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown. Patients and methods Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization. Results Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4-NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1-NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib. Conclusion These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin. Clinical trial information Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

Response to angiotensin blockade with irbesartan in a patient with metastatic colorectal cancer.

Personalised oncogenomics analysis revealed potential oncogene addiction of the AP-1 transcriptional complex in a chemo-refractory and MMR-deficient tumor from a patient with metastatic colon cancer. Based on this, treatment with the angiotensin receptor agonist irbesartan was initiated to target the renin–angiotensin system upstream of the AP-1 complex, leading to a profound and durable response.

Resource: Scalable whole genome sequencing of 40,000 single cells identifies stochastic aneuploidies, genome replication states and clonal repertoires

Essential features of cancer tissue cellular heterogeneity such as negatively selected genome topologies, sub-clonal mutation patterns and genome replication states can only effectively be studied by sequencing single-cell genomes at scale and high fidelity. Using an amplification-free single-cell genome sequencing approach implemented on commodity hardware (DLP+) coupled with a cloud-based computational platform, we define a resource of 40,000 single-cell genomes characterized by their genome states, across a wide range of tissue types and conditions. We show that shallow sequencing across thousands of genomes permits reconstruction of clonal genomes to single nucleotide resolution through aggregation analysis of cells sharing higher order genome structure. From large-scale population analysis over thousands of cells, we identify rare cells exhibiting mitotic mis-segregation of whole chromosomes. We observe that tissue derived scWGS libraries exhibit lower rates of whole chromosome anueploidy than cell lines, and loss of p53 results in a shift in event type, but not overall prevalence in breast epithelium. Finally, we demonstrate that the replication states of genomes can be identified, allowing the number and proportion of replicating cells, as well as the chromosomal pattern of replication to be unambiguously identified in single-cell genome sequencing experiments. The combined annotated resource and approach provide a re-implementable large scale platform for studying lineages and tissue heterogeneity.

Detection and genomic characterization of a mammary-like adenocarcinoma

Whole-genome and transcriptome sequencing were performed to identify potential therapeutic strategies in the absence of viable treatment options for a patient initially diagnosed with vulvar adenocarcinoma. Genomic events were prioritized by comparison against variant distributions in the TCGA pan-cancer data set and complemented with detailed transcriptome sequencing and copy-number analysis. These findings were considered against published scientific literature in order to evaluate the functional effects of potentially relevant genomic events. Analysis of the transcriptome against a background of 27 TCGA cancer types led to reclassification of the tumor as a primary HER2+ mammary-like adenocarcinoma of the vulva. This revised diagnosis was subsequently confirmed by follow-up immunohistochemistry for a mammary-like adenocarcinoma. The patient was treated with chemotherapy and targeted therapies for HER2+ breast cancer. The detailed pathology and genomic findings of this case are presented herein.

Abstract 5226: Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis

Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient9s personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient9s PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G>A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications. Citation Format: Kasmintan A. Schrader, Carolyn Chu’ng, Eric Zhao, Hui-li Wong, Yaoqing Shen, Martin Jones, Tom Thomson, Howard Lim, Sean Young, Carol Cremin, Robert Holt, Peter Eirew, Joanna Karasinska, Jacquie Schein, Yongjun Zhao, Andy Mungall, Richard Moore, Yussanne Ma, Alexandra Fok, Robyn Roscoe, Stephen Yip, Gillian Mitchell, Aly Karsan, Steven Jones, David Schaeffer, Janessa Laskin, Marco Marra, Daniel Renouf. Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5226.

1570ODEMONSTRATION OF TEMPORAL HETEROGENEITY IDENTIFIED BY GENOME SEQUENCING AND THE POTENTIAL EFFECT ON TREATMENT DECISIONS FOR ADVANCED CANCER PATIENTS

ABSTRACT Aim: Tumour heterogeneity poses a significant challenge to the success of treatment; tumours with similar histological features may have substantially different biological drivers. This analysis is part of a study in which we use whole genome and transcriptome sequencing to aid in therapeutic decision-making in patients (pts) with advanced cancers. Analysis of tumours at different time points is an integral feature to our study design. Methods: Eligible pts with incurable cancers with limited or no standard chemotherapy options, have several samples analyzed: a fresh tumour biopsy; a blood sample for normal comparison; and archival tumours if available. Samples undergo comprehensive DNA (40X-80X) and RNA sequencing (fresh biopsy) and in-depth bioinformatic analysis to identify somatic mutations, copy number alterations, structural rearrangements, and corresponding gene expression changes that may provide informative (diagnostic) or actionable targets. Results: Between July 2012- May 2014, 80 pts have consented, representing 28 different tumour types. Median number of lines of chemo prior to sequencing was 3. Of the 51 pts analyzed, 21 have matched archival and fresh biopsy material. Typically the archival material is from the primary and the fresh tissue is from a metastatic site. We compared the genetic variants between the primary tumors and metastases, and those identified in both samples. The number of shared non-synonymous single nucleotide variants (SNV) between the archival and fresh samples varied considerably from 0 to 117 with the most overlap observed in the 3 colorectal cancer pts. In the pts with no shared SNV, germline variants, copy number profiles and other variants clearly demonstrated the metastatic biopsy was from the same primary. Non-synonymous SNV were detected in individual pt DNA in both the fresh biopsy (range 21-423) and the archival (range 7-338). These included actionable variants in genes such as KRAS, TP53 and BRCA2. There was the suggestion that some genes may be more stable over time (TP53). Conclusions: Our data demonstrates the large and variable heterogeneity of advanced cancer when compared to archival samples. This included important actionable and druggable targets that would influence treatment decisions. This highlights the importance of repeat biopsies to guide treatment decisions wherever possible. Disclosure: All authors have declared no conflicts of interest.

Abstract LB-173: Genome analysis informs chemotherapy decision-making in patients with advanced malignancies.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Tumor heterogeneity poses a significant challenge to the success of treatment; tumors with similar histological features may have substantially different underlying biological drivers. Applying personal genomic information prospectively to assist in chemotherapy decision-making could result in more effective and efficient cancer treatment. Methods: Eligible subjects with incurable cancers for whom there are limited or no standard chemotherapy options, have a tumor biopsy and when possible a tissue matched normal sample plus “normal” blood samples taken specifically for genomic analysis. Archival specimens are concurrently analyzed to look for changes over time and with chemo and/or radiation. Samples are subject to both an Ampliseq amplicon cancer panel analysis and whole genome DNA and RNA sequencing. State-of-the-art bioinformatics approaches are used to identity genes with somatic variants, copy number variants, and expression changes. All variants are integrated into a pathway analysis to identify tumor specific biological processes that may be driving the tumor. These pathways are matched to the known drug databases and to manual literature reviews to identify drugs that may be useful or drugs that may be counter-indicated and a report is generated and discussed. Results: Between July 2012 and January 2013, 10 subjects (of 30 planned) have been enrolled; 3 cases of colorectal and 2 of breast, 1 each of squamous skin, squamous ethmoid sinus cancer, nasopharynx, lung, and one CLL-peripheral mantle cell cancer. The Ampliseq panel results have generally correlated well with whole genome and RNA sequencing results, although the panel, providing less comprehensive information albeit more rapidly, has not been as informative a modality for identifying candidate druggable driver events. And the case of ethmoid cancer was discovered to be a rare pediatric tumor, this was not identified by the panel. Each case had extensive pathway mapping and uncovered potential drug targets that would not have necessarily been considered without this analyses. To date, 3 subjects have initiated treatment based on the reports generated; the analyses are in process for the remaining cases. We also note that we observe significant genomic differences between the archival and fresh tumor materials. Conclusions: Initial results suggest that the information garnered from detailed genomic analysis can inform chemotherapy decision-making. The panel is adept at profiling the common abnormalities that are the target of many of the current generation of molecular targeted drugs; however the whole genome approach provides a comprehensive view of the genomic landscape providing more information on particular aberrant pathways affected and ideas for drug repositioning. For now we have elected to employ whole genome and transcriptome analysis in addition to an “oncogene panel” to both compare the relative utility of these two approaches and provide as comprehensive a view of candidate druggable driver events across patients to inform on the next generation of rapid panel designs. Citation Format: Janessa J. Laskin, Karen Gelmon, Howard Lim, Daniel Renouf, Stephen Yip, David Huntsman, Anna Tinker, Cheryl Ho, Erin D. Pleasance, Yvonne Li, Yaoqing Shen, Katayoon Kasaian, Richard Corbett, Karen Mungall, Yongjun Zhao, Andy Mungall, Jacquie Schein, Robyn Roscoe, Steven Jones, Marco Marra. Genome analysis informs chemotherapy decision-making in patients with advanced malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-173. doi:10.1158/1538-7445.AM2013-LB-173