Homologous Recombination Deficiency and Platinum-Based Therapy Outcomes in Advanced Breast Cancer.

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response.Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI).Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04).Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521-30. ©2017 AACR.

Spatial heterogeneity in medulloblastoma.

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Molecular etiology of an indolent lymphoproliferative disorder determined by whole-genome sequencing.

In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing were performed on a patient with an unclassifiable small lymphoproliferative disorder. Variants from genome sequencing were prioritized using a combination of comparative variant distributions in a spectrum of lymphomas, and meta-analyses of gene expression profiling. In this patient, the molecular variants that we believe to be most relevant to the disease presentation most strongly resemble a diffuse large B-cell lymphoma (DLBCL), whereas the gene expression data are most consistent with a low-grade chronic lymphocytic leukemia (CLL). The variant of greatest interest was a predicted NOTCH2-truncating mutation, which has been recently reported in various lymphomas.

Putative BRAF activating fusion in a medullary thyroid cancer.

Medullary thyroid cancer (MTC) is a malignancy of the calcitonin-producing parafollicular cells of the thyroid gland. Surgery is the only curative treatment for this cancer. External beam radiation therapy is reserved for adjuvant treatment of MTC with aggressive features. Targeted therapeutics vandetanib and cabozantinib are approved for the treatment of aggressive and metastatic tumors that are not amenable to surgery. The use of these multikinase inhibitors are supported by the observed overactivation of the RET oncoprotein in a large subpopulation of MTCs. However, not all patients carry oncogenic alterations of this kinase. Hence, there is still a need for comprehensive molecular characterization of MTC utilizing whole-genome and transcriptome-sequencing methodologies with the aim of identifying targetable mutations. Here, we describe the genomic profiles of two medullary thyroid cancers and report the presence of a putative oncogenic BRAF fusion in one. Such alterations, previously observed in other malignancies and known targets of available drugs, can benefit patients who currently have no treatment options.

The genomic and transcriptomic landscape of anaplastic thyroid cancer: implications for therapy.

BACKGROUND: Anaplastic thyroid carcinoma is the most undifferentiated form of thyroid cancer and one of the deadliest of all adult solid malignancies. Here we report the first genomic and transcriptomic profile of anaplastic thyroid cancer including those of several unique cell lines and outline novel potential drivers of malignancy and targets of therapy. METHODS: We describe whole genomic and transcriptomic profiles of 1 primary anaplastic thyroid tumor and 3 authenticated cell lines. Those profiles augmented by the transcriptomes of 4 additional and unique cell lines were compared to 58 pairs of papillary thyroid carcinoma and matched normal tissue transcriptomes from The Cancer Genome Atlas study. RESULTS: The most prevalent mutations were those of TP53 and BRAF; repeated alterations of the epigenetic machinery such as frame-shift deletions of HDAC10 and EP300, loss of SMARCA2 and fusions of MECP2, BCL11A and SS18 were observed. Sequence data displayed aneuploidy and large regions of copy loss and gain in all genomes. Common regions of gain were however evident encompassing chromosomes 5p and 20q. We found novel anaplastic gene fusions including MKRN1-BRAF, FGFR2-OGDH and SS18-SLC5A11, all expressed in-frame fusions involving a known proto-oncogene. Comparison of the anaplastic thyroid cancer expression datasets with the papillary thyroid cancer and normal thyroid tissue transcriptomes suggested several known drug targets such as FGFRs, VEGFRs, KIT and RET to have lower expression levels in anaplastic specimens compared with both papillary thyroid cancers and normal tissues, confirming the observed lack of response to therapies targeting these pathways. Further integrative data analysis identified the mTOR signaling pathway as a potential therapeutic target in this disease. CONCLUSIONS: Anaplastic thyroid carcinoma possessed heterogeneous and unique profiles revealing the significance of detailed molecular profiling of individual tumors and the treatment of each as a unique entity; the cell line sequence data promises to facilitate the more accurate and intentional drug screening studies for anaplastic thyroid cancer.

Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors.

We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation. RESULTS: We identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas. CONCLUSIONS: Our comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

MEN1 mutations in Hürthle cell (oncocytic) thyroid carcinoma.

CONTEXT AND OBJECTIVE: Oncocytic 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. DESIGN AND PATIENTS: Initially, 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. RESULTS: Here 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. CONCLUSION: Menin, 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.

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers.

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted "panel" sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate "drivers." Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.

Diagnostic value of next-generation sequencing in an unusual sphenoid tumor.

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.

Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation.

The immunoglobulin heavy-chain locus (IGH) encodes variable (IGHV), diversity (IGHD), joining (IGHJ), and constant (IGHC) genes and is responsible for antibody heavy-chain biosynthesis, which is vital to the adaptive immune response. Programmed V-(D)-J somatic rearrangement and the complex duplicated nature of the locus have impeded attempts to reconcile its genomic organization based on traditional B-lymphocyte derived genetic material. As a result, sequence descriptions of germline variation within IGHV are lacking, haplotype inference using traditional linkage disequilibrium methods has been difficult, and the human genome reference assembly is missing several expressed IGHV genes. By using a hydatidiform mole BAC clone resource, we present the most complete haplotype of IGHV, IGHD, and IGHJ gene regions derived from a single chromosome, representing an alternate assembly of ∼1 Mbp of high-quality finished sequence. From this we add 101 kbp of previously uncharacterized sequence, including functional IGHV genes, and characterize four large germline copy-number variants (CNVs). In addition to this germline reference, we identify and characterize eight CNV-containing haplotypes from a panel of nine diploid genomes of diverse ethnic origin, discovering previously unmapped IGHV genes and an additional 121 kbp of insertion sequence. We genotype four of these CNVs by using PCR in 425 individuals from nine human populations. We find that all four are highly polymorphic and show considerable evidence of stratification (Fst = 0.3-0.5), with the greatest differences observed between African and Asian populations. These CNVs exhibit weak linkage disequilibrium with SNPs from two commercial arrays in most of the populations tested.

Analysis of FOXO1 mutations in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines. FOXO1 mutations were found in 8.6% (24/279) of DLBCL cases: 92.3% (24/26) of mutations were in the first exon, 46.2% (12/26) were recurrent mutations affecting the N-terminal region, and another 38.5% (10/26) affected the Forkhead DNA binding domain. Recurrent mutations in the N-terminal region resulted in diminished T24 phosphorylation, loss of interaction with 14-3-3, and nuclear retention. FOXO1 mutation was associated with decreased overall survival in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (P = .037), independent of cell of origin (COO) and the revised International Prognostic Index (R-IPI). This association was particularly evident (P = .003) in patients in the low-risk R-IPI categories. The independent relationship of mutations in FOXO1 to survival, transcending the prognostic influence of the R-IPI and COO, indicates that FOXO1 mutation is a novel prognostic factor that plays an important role in DLBCL pathogenesis.

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Acquired TNFRSF14 mutations in follicular lymphoma are associated with worse prognosis.

Clinical correlative studies have linked 1p36 deletions with worse prognosis in follicular lymphoma (FL). In this study, we sought to identify the critical gene(s) in this region that is responsible for conferring inferior prognosis. BAC array technology applied to 141 FL specimens detected a minimum region of deletion (MRD) of ∼97 kb within 1p36.32 in 20% of these cases. Frequent single-nucleotide polymorphism-detected copy-neutral loss of heterozygosity was also found in this region. Analysis of promoter CpGs in the MRD did not reveal differential patterns of DNA methylation in samples that differed in 1p36 status. Exon sequencing of MRD genes identified somatic alterations in the TNFRSF14 gene in 3 of 11 selected cases with matching normal DNA. An expanded cohort consisting of 251 specimens identified 46 cases (18.3%) with nonsynonymous mutations affecting TNFRSF14. Overall survival (OS) and disease-specific survival (DSS) were associated with the presence of TNFRSF14 mutation in patients whose overall treatment included rituximab. We further showed that inferior OS and DSS were most pronounced in patients whose lymphomas contained both TNFRSF14 mutations and 1p36 deletions after adjustment for the International Prognostic Index [hazard ratios of 3.65 (95% confidence interval, 1.35-9.878, P=0.011) and 3.19 (95% confidence interval, 1.06-9.57, P=0.039), respectively]. Our findings identify TNFRSF14 as a candidate gene associated with a subset of FL, based on frequent occurrence of acquired mutations and their correlation with inferior clinical outcomes.

Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin.

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

Circos: an information aesthetic for comparative genomics.

We created a visualization tool called Circos to facilitate the identification and analysis of similarities and differences arising from comparisons of genomes. Our tool is effective in displaying variation in genome structure and, generally, any other kind of positional relationships between genomic intervals. Such data are routinely produced by sequence alignments, hybridization arrays, genome mapping, and genotyping studies. Circos uses a circular ideogram layout to facilitate the display of relationships between pairs of positions by the use of ribbons, which encode the position, size, and orientation of related genomic elements. Circos is capable of displaying data as scatter, line, and histogram plots, heat maps, tiles, connectors, and text. Bitmap or vector images can be created from GFF-style data inputs and hierarchical configuration files, which can be easily generated by automated tools, making Circos suitable for rapid deployment in data analysis and reporting pipelines.

De novo transcriptome assembly with ABySS.

MOTIVATION: Whole transcriptome shotgun sequencing data from non-normalized samples offer unique opportunities to study the metabolic states of organisms. One can deduce gene expression levels using sequence coverage as a surrogate, identify coding changes or discover novel isoforms or transcripts. Especially for discovery of novel events, de novo assembly of transcriptomes is desirable. RESULTS: Transcriptome from tumor tissue of a patient with follicular lymphoma was sequenced with 36 base pair (bp) single- and paired-end reads on the Illumina Genome Analyzer II platform. We assembled approximately 194 million reads using ABySS into 66 921 contigs 100 bp or longer, with a maximum contig length of 10 951 bp, representing over 30 million base pairs of unique transcriptome sequence, or roughly 1% of the genome. AVAILABILITY AND IMPLEMENTATION: Source code and binaries of ABySS are freely available for download at http://www.bcgsc.ca/platform/bioinfo/software/abyss. Assembler tool is implemented in C++. The parallel version uses Open MPI. ABySS-Explorer tool is implemented in Java using the Java universal network/graph framework. CONTACT: ibirol@bcgsc.ca.

Novel expressed sequences identified in a model of androgen independent prostate cancer.

BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in American men, and few effective treatment options are available to patients who develop hormone-refractory prostate cancer. The molecular changes that occur to allow prostate cells to proliferate in the absence of androgens are not fully understood. RESULTS: Subtractive hybridization experiments performed with samples from an in vivo model of hormonal progression identified 25 expressed sequences representing novel human transcripts. Intriguingly, these 25 sequences have small open-reading frames and are not highly conserved through evolution, suggesting many of these novel expressed sequences may be derived from untranslated regions of novel transcripts or from non-coding transcripts. Examination of a large metalibrary of human Serial Analysis of Gene Expression (SAGE) tags demonstrated that only three of these novel sequences had been previously detected. RT-PCR experiments confirmed that the 6 sequences tested were expressed in specific human tissues, as well as in clinical samples of prostate cancer. Further RT-PCR experiments for five of these fragments indicated they originated from large untranslated regions of unannotated transcripts. CONCLUSION: This study underlines the value of using complementary techniques in the annotation of the human genome. The tissue-specific expression of 4 of the 6 clones tested indicates the expression of these novel transcripts is tightly regulated, and future work will determine the possible role(s) these novel transcripts may play in the progression of prostate cancer.