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PURPOSE: Genomic test results, regardless of laboratory variant classification, require clinical practitioners to judge the applicability of a variant for medical decisions. Teaching and standardizing clinical interpretation of genomic variation calls for a methodology or tool. METHODS: To generate such a tool, we distilled the Clinical Genome Resource framework of causality and the American College of Medical Genetics/Association of Molecular Pathology and Quest Diagnostic Laboratory scoring of variant deleteriousness into the Clinical Variant Analysis Tool (CVAT). Applying this to 289 clinical exome reports, we compared the performance of junior practitioners with that of experienced medical geneticists and assessed the utility of reported variants. RESULTS: CVAT enabled performance comparable to that of experienced medical geneticists. In total, 124 of 289 (42.9%) exome reports and 146 of 382 (38.2%) reported variants supported a diagnosis. Overall, 10.5% (1 pathogenic [P] or likely pathogenic [LP] variant and 39 variants of uncertain significance [VUS]) of variants were reported in genes without established disease association; 20.2% (23 P/LP and 54 VUS) were in genes without sufficient phenotypic concordance; 7.3% (15 P/LP and 13 VUS) conflicted with the known molecular disease mechanism; and 24% (91 VUS) had insufficient evidence for deleteriousness. CONCLUSION: Implementation of CVAT standardized clinical interpretation of genomic variation and emphasized the need for collaborative and transparent reporting of genomic variation.

Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC) is an aggressive clinical entity. Current diagnostic guidelines for premalignant lesions are ambiguous, and their molecular profile and progression events are still unclear. We selected 75 samples, from 40 patients, including 33 VSCC, 8 verrucous carcinomas (VC), 13 differentiated-type vulvar intraepithelial neoplasia (dVIN), 11 suspicious for dVIN (?dVIN), 6 differentiated exophytic vulvar intraepithelial lesions (DE-VIL), 2 vulvar acanthosis with altered differentiation (VAAD), and 2 usual-type vulvar intraepithelial neoplasia (uVIN/HSIL). Invasive and precursor lesions were matched in 29 cases. Clinical information, p16 immunohistochemistry, and mutation analysis were performed on all lesions. All dVIN, ?dVIN, DE-VIL, and VAAD were p16 negative, all uVIN/HSIL were p16 positive. In the HPV-independent group, mutations were identified in 6 genes: TP53 (n = 40), PIK3CA (n = 20), HRAS (n = 12), MET (n = 5), PTEN (n = 4), and BRAF (n = 1). TP53 mutations occurred in 73% (22/30) VSCC, 85% (11/13) dVIN, 70% (7/10) ?dVIN and no VC (0/8), DE-VIL (0/6) nor VAAD (0/2). Basal atypia was the only reliable feature of TP53 mutations. ?dVIN lesions that were non-acanthotic and atypical but obscured by inflammation, all harbored TP53 mutations. In lesions without TP53 mutations, PIK3CA (50% VC, 33% DE-VIL, 100% VAAD, 40% VSCC) and HRAS (63% VC, 33% DE-VIL, 0% VAAD, 20% VSCC) mutations were found. Mutational progression from in situ to invasive was seen (7/26, 27%) and usually involved TP53 (4/26, 15%). Cases with TP53 and PIK3CA co-mutations had the worse clinical outcomes (p

The recent literature has shown that vulvar squamous cell carcinoma (VSCC) can be stratified into two prognostically relevant groups based on human papillomavirus (HPV) status. The prognostic value of p53 for further sub-stratification, particularly in the HPV-independent group, has not been agreed upon. This disagreement is likely due to tremendous variations in p53 immunohistochemical (IHC) interpretation. To address this problem, we sought to compare p53 IHC patterns with TP53 mutation status. We studied 61 VSCC (48 conventional VSCC, 2 VSCC with sarcomatoid features, and 11 verrucous carcinomas) and 42 in situ lesions (30 differentiated vulvar intraepithelial neoplasia [dVIN], 9 differentiated exophytic vulvar intraepithelial lesions [deVIL], and 3 high-grade squamous intraepithelial lesions or usual vulvar intraepithelial neoplasia [HSIL/uVIN]). IHC for p16 and p53, and sequencing of TP53 exons 4-9 were performed. HPV in situ hybridization (ISH) was performed in selected cases. We identified six major p53 IHC patterns, two wild-type patterns: (1) scattered, (2) mid-epithelial expression (with basal sparing), and four mutant patterns: (3) basal overexpression, (4) parabasal/diffuse overexpression, (5) absent, and (6) cytoplasmic expression. These IHC patterns were consistent with TP53 mutation status in 58/61 (95%) VSCC and 39/42 (93%) in situ lesions. Cases that exhibited strong scattered staining and those with a weak basal overexpression pattern could be easily confused. The mid-epithelial pattern was exclusively observed in p16-positive lesions; the basal and parabasal layers that had absent p53 staining, appeared to correlate with the cells that were positive for HPV-ISH. This study describes a pattern-based p53 IHC interpretation framework, which can be utilized as a surrogate marker for TP53 mutational status in both VSCC and vulvar in situ lesions.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.Article SummaryA scalable, non-proprietary, magnetic bead-based automated nucleic acid extraction protocol optimised for minimum cross-well contamination

We studied the whole-genome point mutation and structural variation patterns of 133 tumors (59 high-grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell (GCT)) as a substrate for class discovery in ovarian cancer. Ab initio clustering of integrated point mutation and structural variation signatures identified seven subgroups both between and within histotypes. Prevalence of foldback inversions identified a prognostically significant HGSC group associated with inferior survival. This finding was recapitulated in two independent cohorts (n = 576 cases), transcending BRCA1 and BRCA2 mutation and gene expression features of HGSC. CCOC cancers grouped according to APOBEC deamination (26%) and age-related mutational signatures (40%). ENOCs were divided by cases with microsatellite instability (28%), with a distinct mismatch-repair mutation signature. Taken together, our work establishes the potency of the somatic genome, reflective of diverse DNA repair deficiencies, to stratify ovarian cancers into distinct biological strata within the major histotypes.

Mesonephric carcinoma is a rare malignancy, thought to derive from Wolffian remnants. To date, no targeted molecular therapeutic options have been identified. On the basis of limited case reports, c-KIT immunohistochemical expression has been reported in female adnexal tumors of Wolffian origin, and targeted therapy with Imatinib has been attempted with mixed success. Currently, it is unclear whether c-KIT immunohistochemical expression is seen in mesonephric carcinoma, a tumor that is thought to be related to female adnexal tumors of Wolffian origin, and how this correlates with KIT mutational status. In this study, we assessed the immunohistochemical expression of c-KIT and KIT mutational status, in a series of 13 mesonephric neoplasms (5 cervical [including 2 cervical carcinosarcomas], 3 uterine corpora, 4 ovarian, and 1 vaginal/pelvic). The intensity of staining and proportion of cells showing cytoplasmic/membranous staining for c-KIT were recorded. KIT was sequenced using a next-generation sequencing panel that targeted 120 hotspots and 17 exons in 33 known actionable cancer genes. This panel included KIT exons 9, 11, and 13, and 6 hotspots (T670, D816, D820, N822, Y823, A829). Although c-KIT immunohistochemical expression was observed in the majority of mesonephric carcinomas (10/12 cases; 83%), no KIT mutations were detected. This cautions pathologists against the use of c-KIT immunohistochemistry as a surrogate marker for KIT-activating mutations in this setting. Consistent with previous studies, the majority of mesonephric neoplasms (10/13; 77%) harbored KRAS mutations. Additional mutations were found in CTNNB1 (2/13, 15%), TP53 (2/13, 15%), and PIK3CA (1/13, 8%).

Introduction Activation of the RAS/RAF/MEK/ERK pathway may confer resistance to chemotherapy in non-small cell lung cancer (NSCLC). Selumetinib (AZD6244, ARRY142886), a MEK1/2 inhibitor combined with chemotherapy in patients with NSCLC was evaluated in two schedules to evaluate efficacy and toxicity. Methods IND.219 was a three-arm study of first line pemetrexed/platinum chemotherapy with two schedules of selumetinib (Arm A: intermittent given on days 2–19; Arm B: continuous given on days 1–21) versus chemotherapy alone (Arm C). The primary endpoint was objective response rate (ORR); secondary objectives were tolerability, progression-free survival (PFS), overall survival (OS). The trial was stopped at the planned interim analysis. Results Arms A/B/C enrolled 20/21/21 patients, ORR was 35% (95% CI 15–59% median duration 3.8 months), 62% (95% CI 38–82%; median duration 6.3 months), 24% (95% CI 8–47%; median duration 11.6 months) respectively. The PFS (months Arm A, B, C) was 7.5, 6.7, 4.0 respectively (hazard ratio (HR) PFS Arm A over Arm C: 0.76 [95% CI, 0.38–1.51, 2-sided p = 0.42]; Arm B over Arm C 0.75 [95% CI 0.37–1.54, p = 0.43]. Skin and gastrointestinal adverse events were more common with the addition of selumetinib. A high incidence of venous thromboembolism was seen in all arms. Conclusions Selumetinib combined with chemotherapy was associated with a higher response rate. Continuous selumetinib appeared to be superior to an intermittent schedule. PFS was prolonged with the addition of selumetinib, however this was not statistically significant.

We performed phylogenetic analysis of high-grade serous ovarian cancers (68 samples from seven patients), identifying constituent clones and quantifying their relative abundances at multiple intraperitoneal sites. Through whole-genome and single-nucleus sequencing, we identified evolutionary features including mutation loss, convergence of the structural genome and temporal activation of mutational processes that patterned clonal progression. We then determined the precise clonal mixtures comprising each tumor sample. The majority of sites were clonally pure or composed of clones from a single phylogenetic clade. However, each patient contained at least one site composed of polyphyletic clones. Five patients exhibited monoclonal and unidirectional seeding from the ovary to intraperitoneal sites, and two patients demonstrated polyclonal spread and reseeding. Our findings indicate that at least two distinct modes of intraperitoneal spread operate in clonal dissemination and highlight the distribution of migratory potential over clonal populations comprising high-grade serous ovarian cancers.

STUDY QUESTION Does incisional endometriosis (IE) harbor somatic cancer-driver mutations? SUMMARY ANSWER We found that approximately one-quarter of IE cases harbor somatic-cancer mutations, which commonly affect components of the MAPK/RAS or PI3K-Akt-mTor signaling pathways. WHAT IS KNOWN ALREADY Despite the classification of endometriosis as a benign gynecological disease, it shares key features with cancers such as resistance to apoptosis and stimulation of angiogenesis and is well-established as the precursor of clear cell and endometrioid ovarian carcinomas. Our group has recently shown that deep infiltrating endometriosis (DE), a form of endometriosis that rarely undergoes malignant transformation, harbors recurrent somatic mutations. STUDY DESIGN, SIZE, DURATION In a retrospective study comparing iatrogenically induced and endogenously occurring forms of endometriosis unlikely to progress to cancer, we examined endometriosis specimens from 40 women with IE and 36 women with DE. Specimens were collected between 2004 and 2017 from five hospital sites in either Canada, Germany or the Netherlands. IE and DE cohorts were age-matched and all women presented with histologically typical endometriosis without known history of malignancy. PARTICIPANTS/MATERIALS, SETTING, METHODS Archival tissue specimens containing endometriotic lesions were macrodissected and/or laser-capture microdissected to enrich endometriotic stroma and epithelium and a hypersensitive cancer hotspot sequencing panel was used to assess for presence of somatic mutations. Mutations were subsequently validated using droplet digital PCR. PTEN and ARID1A immunohistochemistry (IHC) were performed as surrogates for somatic events resulting in functional loss of respective proteins. MAIN RESULTS AND THE ROLE OF CHANCE Overall, we detected somatic cancer-driver events in 11 of 40 (27.5%) IE cases and 13 of 36 (36.1%) DE cases, including hotspot mutations in KRAS, ERBB2, PIK3CA and CTNNB1. Heterogeneous PTEN loss occurred at similar rates in IE and DE (7/40 vs 5/36, respectively), whereas ARID1A loss only occurred in a single case of DE. While rates of detectable somatic cancer-driver events between IE and DE are not statistically significant (P > 0.05), KRAS activating mutations were more prevalent in DE. LIMITATIONS, REASONS FOR CAUTION Detection of somatic cancer-driver events were limited to hotspots analyzed in our panel-based sequencing assay and loss of protein expression by IHC from archival tissue. Whole genome or exome sequencing, or epigenetic analysis may uncover additional somatic alterations. Moreover, because of the descriptive nature of this study, the functional roles of identified mutations within the context of endometriosis remain unclear and causality cannot be established. WIDER IMPLICATIONS OF THE FINDINGS The alterations we report may be important in driving the growth and survival of endometriosis in ectopic regions of the body. Given the frequency of mutation in surgically displaced endometrium (IE), examination of similar somatic events in eutopic endometrium, as well as clinically annotated cases of other forms of endometriosis, in particular endometriomas that are most commonly linked to malignancy, is warranted. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by a Canadian Cancer Society Impact Grant [701603, PI Huntsman], Canadian Institutes of Health Research Transitional Open Operating Grant [MOP-142273, PI Yong], the Canadian Institutes of Health Research Foundation Grant [FDN-154290, PI Huntsman], the Canadian Institutes of Health Research Project Grant [PJT-156084, PIs Yong and Anglesio], and the Janet D. Cottrelle Foundation through the BC Cancer Foundation [PI Huntsman]. D.G. Huntsman is a co-founder and shareholder of Contextual Genomics Inc., a for profit company that provides clinical reporting to assist in cancer patient treatment. R. Aguirre-Hernandez, J. Khattra and L.M. Prentice have a patent MOLECULAR QUALITY ASSURANCE METHODS FOR USE IN SEQUENCING pending and are current (or former) employees of Contextual Genomics Inc. The remaining authors have no competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable.

Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE) material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG) treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (

Endometriosis is a significant risk factor for clear cell and endometrioid ovarian cancers and is often found contiguous with these cancers. Using whole‐genome shotgun sequencing of seven clear cell ovarian carcinomas (CCC) and targeted sequencing in synchronous endometriosis, we have investigated how this carcinoma may evolve from endometriosis. In every case we observed multiple tumour‐associated somatic mutations in at least one concurrent endometriotic lesion. ARID1A and PIK3CA mutations appeared consistently in concurrent endometriosis when present in the primary CCC. In several cases, one or more endometriotic lesions carried the near‐complete complement of somatic mutations present in the index CCC tumour. Ancestral mutations were detected in both tumour‐adjacent and ‐distant endometriotic lesions, regardless of any cytological atypia. These findings provide objective evidence that multifocal benign endometriotic lesions are clonally related and that CCCs arising in these patients progress from endometriotic lesions that may already carry sufficient cancer‐associated mutations to be considered neoplasms themselves, albeit with low malignant potential. We speculate that genomically distinct classes of endometriosis exist and that ovarian endometriosis with high mutational burden represents one class at high risk for malignant transformation. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational landscapes. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting evolutionary dynamics of copy number alterations (CNA) and loss of heterozygosity (LOH) in whole-genome sequencing data remain underdeveloped. We present a novel probabilistic model, TITAN, to infer CNA and LOH events while accounting for mixtures of cell populations, thereby estimating the proportion of cells harboring each event. We evaluate TITAN on idealized mixtures, simulating clonal populations from whole-genome sequences taken from genomically heterogeneous ovarian tumor sites collected from the same patient. In addition, we show in 23 whole genomes of breast tumors that the inference of CNA and LOH using TITAN critically informs population structure and the nature of the evolving cancer genome. Finally, we experimentally validated subclonal predictions using fluorescence in situ hybridization (FISH) and single-cell sequencing from an ovarian cancer patient sample, thereby recapitulating the key modeling assumptions of TITAN.

Background: Ovarian carcinoma is composed of five major histologic types, which associate with outcome and predict therapeutic response. Our aim was to evaluate histologic type assessments across the centers participating in the Ovarian Tumor Tissue Analysis (OTTA) consortium using an immunohistochemical (IHC) prediction model. Methods: Tissue microarrays (TMA) and clinical data were available for 524 pathologically confirmed ovarian carcinomas. Centralized IHC was conducted for ARID1A, CDKN2A, DKK1, HNF1B, MDM2, PGR, TP53, TFF3, VIM, and WT1, and three histologic type assessments were compared: the original pathologic type, an IHC-based calculated type (termed TB_COSPv2), and a WT1-assisted TMA core review. Results: The concordance between TB_COSPv2 type and original type was 73%. Applying WT1-assisted core review, the remaining 27% discordant cases subdivided into unclassifiable (6%), TB_COSPv2 error (6%), and original type error (15%). The largest discordant subgroup was classified as endometrioid carcinoma by original type and as high-grade serous carcinoma (HGSC) by TB_COSPv2. When TB_COSPv2 classification was used, the difference in overall survival of endometrioid carcinoma compared with HGSC became significant [RR 0.60; 95% confidence interval (CI), 0.37–0.93; P = 0.021], consistent with previous reports. In addition, 71 cases with unclear original type could be histologically classified by TB_COSPv2. Conclusions: Research cohorts, particularly those across different centers within consortia, show significant variability in original histologic type diagnosis. Our IHC-based reclassification produced more homogeneous types with respect to outcome than original type. Impact: Biomarker-based classification of ovarian carcinomas is feasible, improves comparability of results across research studies, and can reclassify cases which lack reliable original pathology. Cancer Epidemiol Biomarkers Prev; 22(10); 1677–86. ©2013 AACR.

Few biomarkers of ovarian cancer prognosis have been established, partly because subtype-specific associations might be obscured in studies combining all histopathological subtypes. We examined whether tumour expression of the progesterone receptor (PR) and oestrogen receptor (ER) was associated with subtype-specific survival.12 studies participating in the Ovarian Tumor Tissue Analysis consortium contributed tissue microarray sections and clinical data to our study. Participants included in our analysis had been diagnosed with invasive serous, mucinous, endometrioid, or clear-cell carcinomas of the ovary. For a patient to be eligible, tissue microarrays, clinical follow-up data, age at diagnosis, and tumour grade and stage had to be available. Clinical data were obtained from medical records, cancer registries, death certificates, pathology reports, and review of histological slides. PR and ER statuses were assessed by central immunohistochemistry analysis done by masked pathologists. PR and ER staining was defined as negative (1% tumour cell nuclei), weak (1 to50%), or strong (≥50%). Associations with disease-specific survival were assessed.2933 women with invasive epithelial ovarian cancer were included: 1742 with high-grade serous carcinoma, 110 with low-grade serous carcinoma, 207 with mucinous carcinoma, 484 with endometrioid carcinoma, and 390 with clear-cell carcinoma. PR expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p0·0001) and high-grade serous carcinoma (log-rank p=0·0006), and ER expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p0·0001). We recorded no significant associations for mucinous, clear-cell, or low-grade serous carcinoma. Positive hormone-receptor expression (weak or strong staining for PR or ER, or both) was associated with significantly improved disease-specific survival in endometrioid carcinoma compared with negative hormone-receptor expression, independent of study site, age, stage, and grade (hazard ratio 0·33, 95% CI 0·21-0·51; p0·0001). Strong PR expression was independently associated with improved disease-specific survival in high-grade serous carcinoma (0·71, 0·55-0·91; p=0·0080), but weak PR expression was not (1·02, 0·89-1·18; p=0·74).PR and ER are prognostic biomarkers for endometrioid and high-grade serous ovarian cancers. Clinical trials, stratified by subtype and biomarker status, are needed to establish whether hormone-receptor status predicts response to endocrine treatment, and whether it could guide personalised treatment for ovarian cancer.Carraresi Foundation and others.

The classification of endometrial carcinomas is based on pathological assessment of tumour cell type; the different cell types (endometrioid, serous, carcinosarcoma, mixed, undifferentiated, and clear cell) are associated with distinct molecular alterations. This current classification system for high-grade subtypes, in particular the distinction between high-grade endometrioid (EEC-3) and serous carcinomas (ESC), is limited in its reproducibility and prognostic abilities. Therefore, a search for specific molecular classifiers to improve endometrial carcinoma subclassification is warranted. We performed target enrichment sequencing on 393 endometrial carcinomas from two large cohorts, sequencing exons from the following nine genes: ARID1A, PPP2R1A, PTEN, PIK3CA, KRAS, CTNNB1, TP53, BRAF, and PPP2R5C. Based on this gene panel, each endometrial carcinoma subtype shows a distinct mutation profile. EEC-3s have significantly different frequencies of PTEN and TP53 mutations when compared to low-grade endometrioid carcinomas. ESCs and EEC-3s are distinct subtypes with significantly different frequencies of mutations in PTEN, ARID1A, PPP2R1A, TP53, and CTNNB1. From the mutation profiles, we were able to identify subtype outliers, ie cases diagnosed morphologically as one subtype but with a mutation profile suggestive of a different subtype. Careful review of these diagnostically challenging cases suggested that the original morphological classification was incorrect in most instances. The molecular profile of carcinosarcomas suggests two distinct mutation profiles for these tumours: endometrioid-type (PTEN, PIK3CA, ARID1A, KRAS mutations) and serous-type (TP53 and PPP2R1A mutations). While this nine-gene panel does not allow for a purely molecularly based classification of endometrial carcinoma, it may prove useful as an adjunct to morphological classification and serve as an aid in the classification of problematic cases. If used in practice, it may lead to improved diagnostic reproducibility and may also serve to stratify patients for targeted therapeutics. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

We characterized BRCA1 and BRCA2 status (mutation/methylation) in a consecutive series of cases of ovarian carcinoma in order to identify differences in clinicopathological features, molecular characteristics, and outcome between the pelvic high-grade serous cancers with (i) germline or somatic mutations in BRCA1 or BRCA2, (ii) methylation of BRCA1, and (iii) normal BRCA1 or BRCA2. In all, 131 women were identified prospectively, who were undergoing surgical staging and agreed to germline testing for BRCA1 and BRCA2 mutations. Histopathology, germline and somatic BRCA1 or BRCA2 mutations, BRCA1 methylation, and BRCA1 and BRCA2 mRNA expression levels distinguished four subgroups. In all, 103 cases were high-grade serous carcinoma and of these 31 (30%) had germline or somatic BRCA1 or BRCA2 mutations (20% BRCA1 and 10% BRCA2) (group 1), 21 (20%) had methylation of BRCA1 (group 2), and in 51 (50%) there was no BRCA loss (group 3). Group 4 consisted of 28 cases of non-high-grade serous, none of which had BRCA loss. BRCA1 and BRCA2 mRNA expression levels correlated with designated group (P=0.0008). Among high-grade serous carcinomas, there were no differences between groups 1–3 with respect to stage, ascites, CA125 level, platinum sensitivity, cytoreduction rate, neoadjuvant chemotherapy, or survival. Tumors with BRCA1 or BRCA2 mutations had increased immune infiltrates (CD20 and TIA-1) compared with high-grade serous without mutations (P=0.034, 0.027). TP53 expression differed between groups (P

Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described.We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas.ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions.These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

Recent advances in next generation sequencing have made it possible to precisely characterize all somatic coding mutations that occur during the development and progression of individual cancers. Here we used these approaches to sequence the genomes (>43-fold coverage) and transcriptomes of an oestrogen-receptor-alpha-positive metastatic lobular breast cancer at depth. We found 32 somatic non-synonymous coding mutations present in the metastasis, and measured the frequency of these somatic mutations in DNA from the primary tumour of the same patient, which arose 9 years earlier. Five of the 32 mutations (in ABCB11, HAUS3, SLC24A4, SNX4 and PALB2) were prevalent in the DNA of the primary tumour removed at diagnosis 9 years earlier, six (in KIF1C, USP28, MYH8, MORC1, KIAA1468 and RNASEH2A) were present at lower frequencies (1-13%), 19 were not detected in the primary tumour, and two were undetermined. The combined analysis of genome and transcriptome data revealed two new RNA-editing events that recode the amino acid sequence of SRP9 and COG3. Taken together, our data show that single nucleotide mutational heterogeneity can be a property of low or intermediate grade primary breast cancers and that significant evolution can occur with disease progression.

Background: Ciliated muconodular papillary tumors (CMPTs) are newly recognized rare peripheral lung nodules that are histologically characterized by ciliated columnar, goblet, and basal cells. Although recent studies have shown that CMPTs constitute a neoplastic disease, the complete histogenesis of CMPTs is not fully understood and molecular data are limited. Methods: We reviewed four cases of CMPT and performed immunohistochemical and genomic analyses to establish CMPT profiles. Results: All cases were positive for hepatocyte nuclear factor-4α and mucin 5B and negative for programmed death ligand 1 expression, as determined by immunohistochemistry. The genetic analysis revealed three pathogenic mutations (BRAF V600E, AKT1 E17K, and KRAS G12D), with the KRAS mutation reported here for the first time. Conclusion: Histological and genetic profiles indicate that CMPTs are likely neoplastic and exhibit features similar to mucinous adenocarcinoma. This suggests that some CMPTs may be a precursor lesion of mucinous adenocarcinoma., Diagnostic Pathology, 12, 62; 2017

Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 75% (9/12) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.

Ovarian endometrioid carcinomas and endometrial endometrioid carcinomas share many histological and molecular alterations. These similarities are likely due to a common endometrial epithelial precursor cell of origin, with most ovarian endometrioid carcinomas arising from endometriosis. To directly compare the mutation profiles of two morphologically similar tumor types, endometrial endometrioid carcinomas (n=307) and ovarian endometrioid carcinomas (n=33), we performed select exon capture sequencing on a panel of genes: ARID1A, PTEN, PIK3CA, KRAS, CTNNB1, PPP2R1A, TP53. We found that PTEN mutations are more frequent in low-grade endometrial endometrioid carcinomas (67%) compared with low-grade ovarian endometrioid carcinomas (17%) (P

High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug-resistance mechanisms.

Background Ovarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies. Methods We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis. Results Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements. Conclusions: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.

Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.

Germline truncating mutations in DICER1, an endoribonuclease in the RNase III family that is essential for processing microRNAs, have been observed in families with the pleuropulmonary blastoma-family tumor and dysplasia syndrome. Mutation carriers are at risk for nonepithelial ovarian tumors, notably sex cord-stromal tumors.We sequenced the whole transcriptomes or exomes of 14 nonepithelial ovarian tumors and noted closely clustered mutations in the region of DICER1 encoding the RNase IIIb domain of DICER1 in four samples. We then sequenced this region of DICER1 in additional ovarian tumors and in certain other tumors and queried the effect of the mutations on the enzymatic activity of DICER1 using in vitro RNA cleavage assays.DICER1 mutations in the RNase IIIb domain were found in 30 of 102 nonepithelial ovarian tumors (29%), predominantly in Sertoli-Leydig cell tumors (26 of 43, or 60%), including 4 tumors with additional germline DICER1 mutations. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for microRNA interaction and cleavage, and were somatic in all 16 samples in which germline DNA was available for testing. We also detected mutations in 1 of 14 nonseminomatous testicular germ-cell tumors, in 2 of 5 embryonal rhabdomyosarcomas, and in 1 of 266 epithelial ovarian and endometrial carcinomas. The mutant DICER1 proteins had reduced RNase IIIb activity but retained RNase IIIa activity.Somatic missense mutations affecting the RNase IIIb domain of DICER1 are common in nonepithelial ovarian tumors. These mutations do not obliterate DICER1 function but alter it in specific cell types, a novel mechanism through which perturbation of microRNA processing may be oncogenic. (Funded by the Terry Fox Research Institute and others.).

14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t (10;17) genomic rearrangement, leading to fusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE–FAM22 fusion oncoproteins was demonstrated by immunoblot in t (10;17)-bearing frozen tumor and cell line samples. YWHAE–FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t (10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE–FAM22 maintains a structurally and functionally intact 14-3-3ε (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE–FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE–FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions.

Next generation sequencing has now enabled a cost-effective enumeration of the full mutational complement of a tumor genome-in particular single nucleotide variants (SNVs). Most current computational and statistical models for analyzing next generation sequencing data, however, do not account for cancer-specific biological properties, including somatic segmental copy number alterations (CNAs)-which require special treatment of the data. Here we present CoNAn-SNV (Copy Number Annotated SNV): a novel algorithm for the inference of single nucleotide variants (SNVs) that overlap copy number alterations. The method is based on modelling the notion that genomic regions of segmental duplication and amplification induce an extended genotype space where a subset of genotypes will exhibit heavily skewed allelic distributions in SNVs (and therefore render them undetectable by methods that assume diploidy). We introduce the concept of modelling allelic counts from sequencing data using a panel of Binomial mixture models where the number of mixtures for a given locus in the genome is informed by a discrete copy number state given as input. We applied CoNAn-SNV to a previously published whole genome shotgun data set obtained from a lobular breast cancer and show that it is able to discover 21 experimentally revalidated somatic non-synonymous mutations in a lobular breast cancer genome that were not detected using copy number insensitive SNV detection algorithms. Importantly, ROC analysis shows that the increased sensitivity of CoNAn-SNV does not result in disproportionate loss of specificity. This was also supported by analysis of a recently published lymphoma genome with a relatively quiescent karyotype, where CoNAn-SNV showed similar results to other callers except in regions of copy number gain where increased sensitivity was conferred. Our results indicate that in genomically unstable tumors, copy number annotation for SNV detection will be critical to fully characterize the mutational landscape of cancer genomes.

PPP2R1A mutations have recently been described in 3/42 (7%) of clear cell carcinomas of the ovary. PPP2R1A encodes the α-isoform of the scaffolding subunit of the serine/threonine protein phosphatase 2A (PP2A) holoenzyme. This putative tumour suppressor complex is involved in growth and survival pathways. Through targeted sequencing of PPP2R1A, we identified somatic missense mutations in 40.8% (20/49) of high-grade serous endometrial tumours, and 5.0% (3/60) of endometrial endometrioid carcinomas. Mutations were also identified in ovarian tumours at lower frequencies: 12.2% (5/41) of endometrioid and 4.1% (2/49) of clear cell carcinomas. No mutations were found in 50 high-grade and 12 low-grade serous carcinomas. Amino acid residues affected by these mutations are highly conserved across species and are involved in direct interactions with regulatory B-subunits of the PP2A holoenzyme. PPP2R1A mutations in endometrial high-grade serous carcinomas are a frequent and potentially targetable feature of this disease. The finding of frequent PPP2R1A mutations in high-grade serous carcinoma of the endometrium but not in high-grade serous carcinoma of the ovary provides clear genetic evidence that these are distinct diseases.

Background Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis. Methods and Findings In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%–2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%–2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%–0.8%). Conclusions The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma., David Huntsman and colleagues describe the associations between biomarker expression patterns and survival in different ovarian cancer subtypes. They suggest that the management of ovarian cancer should reflect differences between these subtypes., Editors' Summary Background. Every year, about 200,000 women develop ovarian cancer and more than 100,000 die from the disease. Ovarian epithelial cancer (carcinoma) occurs when epithelial cells from the ovary or fallopian tube acquire mutations or equivalent changes that allow them to grow uncontrollably within one of the ovaries (two small organs in the pelvis that produce eggs) and acquire the potential to spread around the body (metastasize). While the cancer is confined to the ovaries, cancer specialists call this stage I disease; 70%–80% of women diagnosed with stage I ovarian cancer survive for at least 5 y. However, only a fifth of ovarian cancers are diagnosed at this stage; in the majority of patients the cancer has spread into the pelvis (stage II disease), into the peritoneal cavity (the space around the gut, stomach, and liver; stage III disease), or metastasized to distant organs such as brain (stage IV disease). This peritoneal spread might be associated with often only vague abdominal pain and mild digestive disturbances. Patients with advanced-stage ovarian carcinoma are treated with a combination of surgery and chemotherapy but, despite recent advances in treatment, only 15% of women diagnosed with stage IV disease survive for 5 y. Why Was This Study Done? Although it is usually regarded as a single disease, there are actually several distinct subtypes of ovarian carcinoma. These are classified according to their microscopic appearance as high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous ovarian carcinomas. These subtypes develop differently and respond differently to chemotherapy. Yet scientists studying ovarian carcinoma usually regard this cancer as a single entity, and current treatment protocols for the disease are not subtype specific. Might better progress be made toward understanding ovarian carcinoma and toward improving its treatment if each subtype were treated as a separate disease? Why are some tumors confined to the ovary, whereas the majority spread beyond the ovary at time of diagnosis? In this study, the researchers address these questions by asking whether correlations between the expression of “biomarkers” (molecules made by cancer cells that can be used to detect tumors and to monitor treatment effectiveness) and the stage at diagnosis or length of survival can be explained by differential biomarker expression between different subtypes of ovarian carcinoma. They also address the question of whether early stage and late stage ovarian carcinomas are fundamentally different. What Did the Researchers Do and Find? The researchers measured the expression of 21 candidate protein biomarkers in 500 ovarian carcinoma samples collected in British Columbia, Canada, between 1984 and 2000. For 20 of the biomarkers, the fraction of tumors expressing the biomarker varied significantly between ovarian carcinoma subtypes. Considering all the tumors together, ten biomarkers had different expression levels in early and late stage tumors. However, when each subtype was considered separately, the expression of none of the biomarkers varied with stage. When the researchers asked whether the expression of any of the biomarkers correlated with survival times, they found that nine biomarkers were unfavorable indicators of outcome when all the tumors were considered together. That is, women whose tumors expressed any of these biomarkers had a higher risk of dying from ovarian cancer than women whose tumors did not express these biomarkers. However, only three biomarkers were unfavorable indicators for high-grade serous carcinomas considered alone and the expression of a biomarker called WT1 in this subtype of ovarian carcinoma is associated with a lower risk of dying. Similarly, expression of the biomarker Ki-67 was an unfavorable prognostic indicator when all the tumors were considered, but was not a prognostic indicator for any individual subtype. What Do These Findings Mean? These and other findings indicate that biomarker expression is more strongly associated with ovarian carcinoma subtype than with stage. In other words, biomarker expression is constant from early to late stage, but only within a given subtype. Second, the association of biomarker expression with survival varies between subtypes, hence lumping all subtypes together can yield misleading results. Although these findings need confirming in more tumor samples, they support the view that ovarian carcinoma subtypes are different diseases. In practical terms, therefore, these findings suggest that better ways to detect and treat ovarian cancer are more likely to be found if future biomarker studies and clinical research studies investigate each subtype of ovarian carcinoma separately rather than grouping them all together. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050232. The US National Cancer Institute provides a brief description of what cancer is and how it develops and information on all aspects of ovarian cancer for patients and professionals. It also provides a fact sheet on tumor markers (in English and Spanish) The UK charity Cancerbackup provides general information about cancer and more specific information about ovarian cancer, including tumor staging

Background Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter might suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas. Methods Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p value equal to or less than 0.05 was considered significant. Results Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p = 0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54 is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102). Conclusion Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

Introduction: Clear Cell ovarian carcinomas (CCC) represent 10% of ovarian carcinomas, with outcomes for high-stage significantly worse than high-grade serous form. The complete mutational landscape, the molecular basis of the transformation of endometriosis, the putative precursor, and patterns of clonal evolution in CCC are not well understood. Methods: Whole genome sequencing and gene expression profiling was done to uncover somatic alterations and measure effects on transcriptional networks. Targeted deep sequencing of primary tumors, metastases and endometriosis was also performed and statistical modeling approaches were used to validate mutations, quantify clonal diversity, and trace patterns of selection. Results: Mutations in ARID1A (11/19) and PIK3CA (8/19) were by far the most frequent aberrations seen. Three other SWI/SNF components also showed somatic alteration: two in non-ARID1A mutant cases and a truncating mutation of ARID1B in an ARID1A-null case. Amongst 24 significant “candidate drivers” impacting expression, five “cancer genes” have been previously described: PIK3CA, ARID1A, CTNNB1, TP53, and PPP2R1A. We observed no association between PIK3CA or ARID1A status with disease stage, genomic instability, or mutation load. Deep sequencing data suggested multiple clones in every case. In cases with matching precursor lesions, we observed multiple mutations in at least one such lesion. In precursor lesions where tumor-matched somatic mutations were observed, ARID1A and PIK3CA mutations were also always present, if observed in the matched tumor. Conclusions: ARID1A and PIK3CA mutations appear as early and histo-type defining events in CCC. Pattern of endometriosis transformation can be associated with somatic mutations in all cases, including histologically “Atypical” and non-atypical endometriosis. Finally, patterns of mutational conservation across the series of precursor lesions may present an opportunity for early screening of endometriosis tissues as an indicator of transformation potential. Citation Format: Michael S Anglesio, Ali Bashashati, Yikan Wang, Gavin Ha, Janine Senz, Winnie Yang, Steve E Kalloger, Leah M Prentice, Satoshi Yanagida, Clara Salamanca, Galina Soukhatcheva, Anthony Kazernis, Hector Chang, Anne-Marie Mes-Mason, Aikou Okamoto, Marco A Marra, Blake Gilks, Sohrab P Shah, David G Huntsman. The somatic mutational landscape of endometriosis associated ovarian cancers and precursor lesions [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS18.

Introduction: Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is arguably the most aggressive ovarian cancer. Most patients are diagnosed at an advanced stage, do not respond to chemotherapy, and die of disease within 1-2 years. It affects children and young women and is reported to occur in families. The cause of the disease is poorly understood. Therefore, we used next generation sequencing technology to identify the genetic basis of the disease. Method: We performed whole genome, whole exome sequencing or targeted sequencing on tumours and germline samples from 17 SCCOHT patients and on the SCCOHT cell line BIN-67 and SCCOHT-1. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumors from 23 patients and on a tissue microarray of 485 primary ovarian tumours of other subtypes. BIN-67 cells harbouring biallelic inactivation of SMARCA4 were transduced with a lentivirus expressing wild type SMARCA4. Result: We identified inactivating germline and somatic mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (11/14) of SCCOHT patients, 2 of whom bore germline mutations, and in both cell lines. SMARCA4 protein was lost in 87% (20/23) of SCCOHT tumours but in only 0.4% (2/485) of other primary ovarian tumours. Reintroduction of wild-type SMARCA4 into BIN-67 cells resulted in altered cell morphology and growth arrest. Conclusions: The mutation spectrum, IHC profile and cell culture phenotype implicate SMARCA4 as a critical tumour suppressor in SCCOHT pathogenesis. Citation Format: Pilar Ramos, Anthony N. Karnezis, David W. Craig, Aleksandar Sekulic, Megan L. Russell, William P.D. Hendricks, Jason J. Corneveaux, Michael T. Barrett, Karey Shumansky, Yidong Yang, Sohrab P. Shah, Leah M. Prentice, Marco A. Marra, Jeffrey Kiefer, Victoria L. Zismann, Troy A. McEachron, Bodour Salhia, Jaime Prat, Blaise A. Clarke, Joseph G. Pressey, John H. Farley, Stephen P. Anthony, Richard B.S. Roden, Heather E. Cunliffe, David G. Huntsman, Jeffrey M. Trent. Small cell carcinoma of the ovary, hypercalcemic type displays frequent inactivating germline and somatic mutations in SMARCA4 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1327.

Rearrangements of the neuregulin (NRG1) gene have been implicated in breast carcinoma oncogenesis. To determine the frequency and clinical significance of NRG1 aberrations in clinical breast tumors, a breast cancer tissue microarray was screened for NRG1 aberrations by fluorescent in situ hybridization (FISH) using a two-color split-apart probe combination flanking the NRG1 gene. Rearrangements of NRG1 were identified in 17/382 cases by FISH, and bacterial artificial chromosome array comparative genomic hybridization was applied to five of these cases to further map the chromosome 8p abnormalities. In all five cases, there was a novel amplicon centromeric to NRG1 with a minimum common region of amplification encompassing two genes, SPFH2 and FLJ14299. Subsequent FISH analysis for the novel amplicon revealed that it was present in 63/262 cases. Abnormalities of NRG1 did not correlate with patient outcome, but the novel amplicon was associated with poor prognosis in univariate analysis, and in multivariate analysis was of prognostic significance independent of nodal status, tumor grade, estrogen receptor status, and human epidermal growth factor receptor (HER)2 overexpression. Of the two genes in the novel amplicon, expression of SPFH2 correlated most significantly with amplification. This amplicon may emerge as a result of breakpoints and chromosomal rearrangements within the NRG1 locus.

Podocalyxin is a CD34-related cell surface molecule with anti-adhesive qualities. We probed a tissue microarray (n = 272) linked to long-term outcome data and found that podocalyxin was highly overexpressed in a distinct subset of invasive breast carcinomas (n = 15; 6%). Univariate disease-specific (P < 0.01) and multivariate regression (P < 0.0005) analyses indicated that this overexpression is an independent indicator of poor outcome. Forced podocalyxin expression perturbed cell junctions between MCF-7 breast carcinoma cells, and it caused cell shedding from confluent monolayers. Therefore, podocalyxin overexpression is a novel predictor of breast cancer progression that may contribute to the process by perturbing tumor cell adhesion.

Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive malignancy that affects children and young women at a mean age of 24 (range 14 months - 58 years). SCCOHT is refractory to standard of care therapy for ovarian cancer, with ∼75% mortality within 18 months of diagnosis. The early age of onset of SCCOHT and reports of familial occurrence in some cases, strongly suggest an underlying hereditary etiology. To understand the molecular pathogenesis of SCCOHT, we performed next-generation genomic sequencing on a series of tumor and germline samples from SCCOHT patients. This analysis revealed germline and somatic inactivating mutations in SMARCA4, a subunit of the SWI/SNF chromatin-remodeling complex, in 75% (9/12) of SCCOHT patients. Moreover, immunohistochemical (IHC) analysis of 15 tumors revealed that 87% (13/15) of tumors lacked SMARCA4 protein. The high prevalence of SMARCA4 mutations in SCCOHT has not been previously reported in other, more common ovarian carcinomas. We therefore examined the expression of SMARCA4 protein in 300 ovarian carcinomas of different histologies by IHC and found SMARCA4 protein loss in only 6 tumors. In addition, the BIN-67 SCCOHT cell line, which harbors 2 splice site mutations in SMARCA4, showed complete absence of SMARCA4 protein by Western blot while representative cell lines from 4 other ovarian carcinoma subtypes as well as immortalized granulosa cells (SVOG) and adult granulosa tumor cells (KGN) all maintained SMARCA4 expression. The prevalence of germline and sporadic SMARCA4 mutations as well as frequent SMARCA4 protein loss in SCCOHTs implicates this gene as a tumor suppressor in this cancer and more broadly suggests a role for the SWI/SNF complex in its pathogenesis. In addition to providing evidence to the pathogenesis of SCCOHT, this finding provides the opportunity to develop treatment approaches for SCCOHT based on targeting vulnerabilities of SMARCA4-deficient cells. Citation Format: Pilar Ramos, Anthony Karnezis, David Craig, Aleksandar Sekulic, Megan Russell, William Hendricks, Michael Barrett, Karey Shumansky, Yidong Yang, Sohrab Shah, Leah Prentice, Marco Marra, Jeffrey Kiefer, Victoria Zismann, Troy McEachron, Bodour Salhia, Joseph Pressey, John Farley, Stephen Anthony, Richard Roden, Heather Cunliffe, David Huntsman, Jeffrey Trent. The rare, highly malignant small cell carcinoma of the ovary displays common inactivating germline and somatic mutations in the tumor suppressor SMARCA4. [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 LB-202. doi:10.1158/1538-7445.AM2014-LB-202

Clear Cell ovarian carcinomas (CCC) represent ∼10% of ovarian carcinomas, with outcomes for high-stage cases significantly worse than the more common high-grade serous form. Response to standard chemotherapies are poor and efforts to improve treatment strategies are confounded by studies grouping ovarian histologies together, as well as a general lack of molecular background data on CCC. CCC frequently occurs in a background of endometriosis. The complete mutational landscape, the molecular basis of the transformation of endometriosis and patterns of clonal evolution in CCC are not understood. We performed whole genome sequencing and gene expression profiling on 19 CCC to uncover candidate somatic alterations (mutations and, copy number aberrations) and measure their effect on transcriptional networks as candidates for driver mutations. We then performed targeted deep sequencing on the primary tumor samples, metastases and, from a subset of cases, adjacent or distant typical and atypical endometriosis. We used statistical modeling approaches to validate mutations, quantify the degree of clonal diversity and trace patterns of selection through oncogenic transformation. Mutations in ARID1A and PIK3CA were by far the most frequent aberrations seen in the cohort (ARID1A: 10/19 cases; PIK3CA: 8/19 cases). The majority of ARID1A mutant cases exhibited bi-allelic loss of function. Two non-ARID1A mutant cases showed alterations in other SWI/SNF complex components. Amongst the 24 most significant candidate drivers impacting expression, five genes (PIK3CA, CTNNB1, TP53, PPP2R1A and KRAS) were known drivers. No association between PIK3CA or ARID1A status with disease stage, genomic instability, or mutation load was observed. Analysis of deep sequencing data suggested the presence of multiple clones in every case. For each case with matching precursor lesions, we observed multiple mutations in at least one such lesion. Cases with ARID1A and PIK3CA mutations always showed evidence of these mutations in their precursor lesions. The proportion of mutations from the primary tumor that were also present in precursor lesions varied widely across the cohort from approximately 10% to nearly 100%. Our data support both ARID1A and PIK3CA mutations as early events in CCC. The pattern of endometriosis transformation could be associated with somatic mutations in all cases. This suggests that candidate tumor-initiating mutations and global- or individually- targetable features should be a focus to improve management of this disease. Finally, we suggest that patterns of mutational conservation across the series of precursor lesions presents an opportunity for early screening of endometriosis tissues as an indicator of transformation potential. Citation Format: Ali Bashashati, Michael Anglesio, Yikan Wang, Gavin Ha, Janine Senz, Winnie Yang, Steve Kalloger, Leah Prentice, Satoshi Yanagida, Clara Salamanca, Galina Soukhatcheva, Anthony Karnezis, Hector Chang, Martin Hirst, Anne-Marie Mes-Mason, Aikou Okamoto, Marco Marra, Blake Gilks, Sohrab Shah, David Huntsman. The somatic mutational landscape of ovarian clear cell carcinoma and its precursor lesions. [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 LB-312. doi:10.1158/1538-7445.AM2014-LB-312

Background Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. Results We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. Conclusions Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.