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PURPOSE Tubo-ovarian cancer (TOC) is a sentinel cancer for BRCA1 and BRCA2 pathogenic variants (PVs). Identification of a PV in the first member of a family at increased genetic risk (the proband) provides opportunities for cancer prevention in other at-risk family members. Although Australian testing rates are now high, PVs in patients with TOC whose diagnosis predated revised testing guidelines might have been missed. We assessed the feasibility of detecting PVs in this population to enable genetic risk reduction in relatives. PATIENTS AND METHODS In this pilot study, deceased probands were ascertained from research cohort studies, identification by a relative, and gynecologic oncology clinics. DNA was extracted from archival tissue or stored blood for panel sequencing of 10 risk-associated genes. Testing of deceased probands ascertained through clinic records was performed with a consent waiver. RESULTS We identified 85 PVs in 84 of 787 (11%) probands. Familial contacts of 39 of 60 (65%) deceased probands with an identified recipient (60 of 84; 71%) have received a written notification of results, with follow-up verbal contact made in 85% (33 of 39). A minority of families (n = 4) were already aware of the PV. For many (29 of 33; 88%), the genetic result provided new information and referral to a genetic service was accepted in most cases (66%; 19 of 29). Those who declined referral (4 of 29) were all male next of kin whose family member had died more than 10 years before. CONCLUSION We overcame ethical and logistic challenges to demonstrate that retrospective genetic testing to identify PVs in previously untested deceased probands with TOC is feasible. Understanding reasons for a family member's decision to accept or decline a referral will be important for guiding future TRACEBACK projects.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

MCC Patient characteristics (S1); Extension cohort of MCCs (S2); List of genes represented on the targeted sequencing panel (S3); Performance metrics for targeted sequencing and low coverage WGS (S4); Validation of mutations by orthogonal genotyping methods (S5); Immunohistochemistry scores for MCCs (S6); SNV and Indels called from targeted sequencing (S7); Mutational burden with respect to copy number changes through low coverage whole genome sequencing (S8); Gene level copy-number from LC-WGS (S9); Copies and viral integration sites for MCV positive MCCs (S10); Drug sensitivity data for 3 MCC cell lines (S11).

Focal copy number alterations proximal to viral integrations in V+MCC.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell–infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities. Cancer Res; 75(24); 5228–34. ©2015 AACR.

Objective Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. Methods We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117–166). Results Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). Conclusions Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pheochromocytomas (PC) and paragangliomas (PGL) are endocrine tumors for which the genetic and clinicopathological features of metastatic progression remain incompletely understood. As a result, the risk of metastasis from a primary tumor cannot be predicted. Early diagnosis of individuals at high risk of developing metastases is clinically important and the identification of new biomarkers that are predictive of metastatic potential is of high value. Activation of TERT has been associated with a number of malignant tumors, including PC/PGL. However, the mechanism of TERT activation in the majority of PC/PGL remains unclear. As TERT promoter mutations occur rarely in PC/PGL, we hypothesized that other mechanisms – such as structural variations – may underlie TERT activation in these tumors. From 35 PC and four PGL, we identified three primary PCs that developed metastases with elevated TERT expression, each of which lacked TERT promoter mutations and promoter DNA methylation. Using whole genome sequencing, we identified somatic structural alterations proximal to the TERT locus in two of these tumors. In both tumors, the genomic rearrangements led to the positioning of super-enhancers proximal to the TERT promoter, that are likely responsible for the activation of the normally tightly repressed TERT expression in chromaffin cells

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector.

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell–infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities. Cancer Res; 75(24); 5228–34. ©2015 AACR.

Background Genome-wide association studies (GWASs) have identified numerous single-nucleotide polymorphisms (SNPs) associated with small increases in breast cancer risk. Studies to date suggest that some SNPs alter the expression of the associated genes, which potentially mediates risk modification. On this basis, we hypothesised that some of these genes may be enriched for rare coding variants associated with a higher breast cancer risk. Methods The coding regions and exon-intron boundaries of 56 genes that have either been proposed by GWASs to be the regulatory targets of the SNPs and/or located

Introduction PALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing. Methods In this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls. Results We identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3–18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism (p = 0.002; OR 1.24 (95 % CI 1.09–1.47). Conclusions Our findings support truncating variants in PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.

Objective A family history of prostate cancer (PC) is a well-recognized high-risk factor for the development of clinically significant PC. To date, traditional linkage and association studies have identified only a limited number of genes and specific gene variants that account for only a small proportion of PC risk. To identify novel PC predisposition genes we performed whole-exome sequencing of PC-affected men from families with a significant history of PC. Methods and materials Exome sequencing was performed on 5 PC-affected men from 3 families where there were multiple cases of PCs and where diagnostic testing returned a negative result for BRCA1 and BRCA2 mutations. Genotyping was performed for all potentially predisposing variants detected within each family on the affected and unaffected male participants. Results Essential splice site, missense, and stop-lost variants were filtered against a recently published candidate gene list. A total of 19 truncating variants and 17 missense variants were identified for genotyping in all prostate-affected and unaffected male participants. In all, 3 missense variants, PCTP , MCRS1 , and ATRIP , demonstrated complete segregation and 1 missense variant, PARP2 , demonstrated partial segregation with PC. In addition, 3 truncating variants, CYP3A43 , DOK3 , and PLEKHH3 , demonstrated complete segregation and 3 truncation mutations, HEATR5B , GPR124 , and HKR1 , demonstrated partial segregation with PC. No segregating variants between the 3 families were shared. Conclusions In all, 10 truncating or missense variants showed either complete or partial segregation with PC in the relevant families. CYP3A43 and PARP2 variants have been shown to occur in other familial PCs and our findings add to the contribution that these variants potentially have in the risk and development of PC in BRCAX cases.

Motivation: In light of the increasing adoption of targeted resequencing (TR) as a cost-effective strategy to identify disease-causing variants, a robust method for copy number variation (CNV) analysis is needed to maximize the value of this promising technology. Results: We present a method for CNV detection for TR data, including whole-exome capture data. Our method calls copy number gains and losses for each target region based on normalized depth of coverage. Our key strategies include the use of base-level log-ratios to remove GC-content bias, correction for an imbalanced library size effect on log-ratios, and the estimation of log-ratio variations via binning and interpolation. Our methods are made available via CONTRA (COpy Number Targeted Resequencing Analysis), a software package that takes standard alignment formats (BAM/SAM) and outputs in variant call format (VCF4.0), for easy integration with other next-generation sequencing analysis packages. We assessed our methods using samples from seven different target enrichment assays, and evaluated our results using simulated data and real germline data with known CNV genotypes. Availability and implementation: Source code and sample data are freely available under GNU license (GPLv3) at http://contra-cnv.sourceforge.net/ Contact: Jason.Li@petermac.org Supplementary information: Supplementary data are available at Bioinformatics online.

Background/Aims: Merkel cell carcinoma (MCC) is rare but aggressive cutaneous high-grade neuroendocrine cancer. Viral infection and sun-exposure are known risk factors for development of MCC, the latter being important in Australia, which has the highest reported incidence of MCC worldwide. There is growing evidence to suggest that MCC associated with the Merkel cell polyoma virus (MCPyV) is clinically, biologically and genetically distinct to those tumors without viral infection. We aimed to assess mutations arising in MCC in the Australian population by targeted massively-parallel sequencing (MPS) to gain new biological insight and identify novel therapeutic opportunities in this disease. Methods: MCPyV was detected in tumours by PCR amplification of large-T antigen coding DNA. The tumor cohort was comprised of seven MCPyV-positive and 16 MCPyV-negative tumors reflecting the reported prevalence of these subtypes in the Australian population. We also sequenced two viral negative MCC cell lines. Hybridization-based DNA capture was used for enrichment of 625 cancer genes for high-depth MPS of tumor and the matching germline DNA, where available. Single nucleotide variants and small insertions and deletions were detected using GATK and muTect variant calling tools and annotated using ENSEMBL variant effect predictor. Somatic variants were enriched in the data by filtering out any variants found in matching germline samples or population-based polymorphism databases Results: High mutation burden was found exclusively in the MCPyV-negative cases with the hallmark signature of UV induced DNA damage. Somatic mutations were identified in known cancer genes specific to the MCPyV-negative tumors. Deleterious RB1 and TP53 mutations were found in 17 and 16 of the MCPyV-negative cases, respectively. Canonical mutations were identified in PIK3CA, HRAS as well as truncating NF1 mutations, indicating that activation of the PI3K and RAS-MAPK pathways are important for pathogenesis of MCPyV-negative tumors. Treatment of two cell lines harboring either PIK3CA or HRAS mutations, respectively, showed in vitro sensitivity to the dual PI3K/mTOR inhibitor PF-04691502. Conclusions: The identification of a UV-induced DNA damage signature and mutations in PI3K and MAPK pathways specific to viral negative tumors confirms the distinct routes to pathogenesis in this disease subtype. This study provides new insight into the biology of viral negative MCC and potential opportunities for the deployment of targeted therapies in this patient group. Citation Format: Stephen Q. Wong, Kelly Waldeck, Ismael A. Vergara, Jason Li, Richard Lupat, Timothy Semple, Carleen Cullinane, Gisela Mir Arnau, Meredith Johnston, Annette Hogg, Anthony T. Papenfuss, Stephen Fox, Grant McArthur, Anthony Gill, Rodney J. Hicks, Richard W. Tothill. Merkel cell carcinomas in Australia have distinct mutation profiles reflecting viral etiology and UV-related DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2015-3875