Karen Mungall

Genetic Alterations Activating Kinase and Cytokine Receptor Signaling in High-Risk Acute Lymphoblastic Leukemia

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co‐deletion to identify new molecular features at base‐pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non‐synonymous somatic mutations in 20 CIC‐mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA‐interacting domain and three were in exon 20 within an annotated protein‐interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co‐deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild‐type tumours, in a background of 1p/19q co‐deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co‐deletion. Copyright

Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

Identification and analysis of murine pancreatic islet enhancers

Aims/hypothesisThe paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression.MethodsWe combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets.ResultsWe identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs).Conclusions/interpretationsOur results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Annotation of Two Large Contiguous Regions from the Haemonchus contortus Genome Using RNA-seq and Comparative Analysis with Caenorhabditis elegans

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes.

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

BackgroundnNRG1 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.nnnPatients and methodsnHere, 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.nnnResultsnBoth 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.nnnConclusionnThese 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.nnnClinical trial informationnPersonalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

MEN1 Mutations in Hürthle Cell (Oncocytic) Thyroid Carcinoma

CONTEXT AND OBJECTIVEnOncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation, and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management.nnnDESIGN AND PATIENTSnInitially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line, and five Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in the multiple endocrine neoplasia I (MEN1) gene.nnnRESULTSnHere we report the identification of MEN1 loss-of-function mutations in 4% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumors.nnnCONCLUSIONnMenin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of MEN1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis, and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

Abstract 3087: Whole genome sequencing of rhabdoid tumors of the kidney

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnRhabdoid tumors (RT) of the kidney (RTK) are aggressive pediatric solid tumors that predominantly affect infants. There is no effective chemotherapy and the overall 4-year survival rate is 23%. RT has a characteristic loss of SMARCB1 function, found in >90% of the patients. SMARCB1 is a conserved core subunit of the SWI/SNF chromatin-remodeling complex, which in turn is responsible for proper chromatin assembly and dynamic regulation of gene expression. Previous studies showed a remarkable paucity of mutations in coding regions of genomes and a highly penetrant cancer susceptibility in a conditional knockout mouse model. These findings support the interpretation that SMARCB1 is a tumor suppressor whose inactivation is the primary driver in RT and that RT follows a tumorigenesis model in which cancer is driven by aberrant epigenetic regulation and gene expression instead of accumulation of somatic mutations. Characterizing interplays of mutations, gene expression and epigenetic regulation will be important in understanding RT development and biology.nnTo achieve this goal, we will comprehensively characterize genetic and epigenetic aberrations in RT using HiSeq sequencing technology. Our research efforts include profiling whole genome, whole transcriptome, promoter methylation and histone modification in 40 primary RTK samples. Here, we report preliminary results from whole genome analyses.nnUsing an amplification-free library construction method, we sequenced whole genomes of 40 RTK and matched normal cases to an average haploid coverage of 39.4X. The RTK genomes were mostly diploid, but we found 35 loci that are either recurrently focally amplified or deleted using GISTIC 2.0 at FDR ≤0.05. Using the Trans-ABySS de novo short-read assembler, we assembled the RT cases’ whole genomes and identified a total of 19 genes that were recurrently rearranged in 8 out of 40 cases. Eleven of the genes were either known tumor suppressors (e.g. CABIN1, BCR) or associated with developmental or neurodegenerative diseases (e.g. UPB1, SPECC1L). The genome-wide single nucleotide variant and indel analysis showed an average somatic mutation rate at 0.37 per Mb in RTK, comparable to the previous finding of 0.19 per Mb in AT/RT. Approximately 99% of the somatic mutations occurred in non-genic regions. SMARCB1 had homozygous loss of function in 83% of cases by somatic homozygous deletion, or heterozygous deletion or truncating point mutations followed by loss of heterozygosity. The remaining cases appeared to have at least 1 copy of the gene unaffected and analyses are ongoing to investigate the inactivation mechanism in these cases.nnCitation Format: Hye-Jung E. Chun, Kelsey Zhu, Jenny Q. Qian, Karen L. Mungall, Yussanne Ma, Yong-Jun Zhao, Andrew J. Mungall, Richard A. Moore, Jacquie E. Schein, Daniela S. Gerhard, Elizabeth J. Perlman, Marco A. Marra. Whole genome sequencing of rhabdoid tumors of the kidney. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2014-3087

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, DCnnBackground: 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.nnMethods: 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.nnSamples 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.nnResults: 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.nnConclusions: 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.nnCitation 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

Abstract 926: Whole genome and transcriptome sequencing defines the spectrum of somatic changes in high-risk neuroblastoma

The NCI9s Therapeutically Applicable Research to Generate Effective Targets (TARGET) initiative uses state-of-the-art genome-wide approaches to identify therapeutic targets for pediatric cancers. Applications of 2 nd -generation sequencing technologies to the analysis of adult solid tumors and hematopoietic malignancies have led to novel, often clinically relevant insights into these diseases. The objective of this study is to utilize 2 nd -generation sequencing approaches on a highly annotated set of ten high-risk neuroblastomas (NBLs). We performed whole genome shotgun sequencing of six stage 4 MYCN-non-amplified and four stage 4 MYCN-amplified NBL TARGET cases and matched peripheral blood, as well as whole transcriptome sequencing (RNA-Seq) of the corresponding tumor RNA. The tumor and normal genomes were sequenced to 30X haploid coverage, and an average 10.3 Gb of aligned sequence was generated for each tumor transcriptome. The level of sequencing redundancy allowed us to achieve at least 10X coverage across 90% of the genome enabling genome-wide detection of sequence and copy number changes in the tumor DNA. We used alignment and de novo assembly approaches to identify somatic and germline SNVs, indels, structural variants, regions of copy number gains and losses, and genome rearrangements. We used RNA-Seq data to determine whether the detected sequence changes were expressed, and to identify transcripts differentially or alternatively expressed between MYCN-amplified and non-amplified cases. Our analysis of tumor and normal genomes identified an average of 1664 candidate somatic mutations per case, the majority of which were SNVs and small indels falling within introns or intergenic sequence. We also detected two candidate germline mutations in the ALK oncogene, one of which was previously characterized in NBL. An average of 10% of candidate somatic mutations in coding sequence was expressed in the transcriptome representing candidate oncogenic events. In addition, we detected and validated using PCR 9 novel genomic rearrangements resulting in expressed products, 5 of which were somatic and 4 of which were germline. We report on two novel somatic gene fusions, between TRIM37 on chromosome 17 and RNF121 on chromosome 11, and between LSAMP and STAG1 on chromosome 3, previously uncharacterized in NBL. The fusions did not recur in our sample set. This work provides an initial genome-wide view of the landscape of somatic changes that occur in high-risk neuroblastoma and highlights novel candidate oncogenic events that may drive the malignancy. Ongoing efforts will catalogue discovered somatic mutation frequencies in a large set of cases and explore the role of germline DNA variations in NBL tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 926. doi:10.1158/1538-7445.AM2011-926