The role of CTNNB1 mutations and matrix metalloproteinases (MMPs) in anti-angiogenesis treatment of endometrial carcinoma.

OBJECTIVE: Treatment options and associated biomarkers for advanced and recurrent disease are limited. Endometrial cancers (ECs) with CTNNB1 exon 3 mutations appear to have preferential response to bevacizumab, an anti-angiogenesis treatment, though the mechanism of action is unknown. We aim to identify mediators of bevacizumab-responsive endometrial cancers. METHODS: We analyzed RNA expression from TCGA and protein expression from CPTAC to identify likely targets for ß-catenin overactivity. We then transiently and stably overexpressed ß-catenin in EC cells to confirm the results suggested by our in silico analysis. We performed corroborative experiments by silencing CTNNB1 in mutated cell lines to demonstrate functional specificity. We implanted transduced cells into xenograft models to study microvessel density. RESULTS: CTNNB1-mutated ECs were associated with increased ß-catenin and MMP7 protein abundance (P < 0.001), but not VEGF-A protein abundance. Overexpressing ß-catenin in EC cells did not increase VEGF-A abundance but did increase expression and secretion of MMP7 (P < 0.03). Silencing CTNNB1 in CTNNB1-mutated cells decreased MMP7 gene expression in EC (P < 0.0001). Microvessel density was not increased. CONCLUSIONS: These data provide a mechanistic understanding for bevacizumab-response in CTNNB1-mutated ECs demonstrated in GOG-86P. We hypothesize that overexpressed and secreted MMP7 potentially digests VEGFR-1, releasing VEGF-A, and increasing its availability. These activities may drive the formation of permeable vessels, which contributes to tumor progression, metastasis, and immune suppression. This mechanism is unique to EC and advocates for further clinical trials evaluating this treatment-related biomarker.

CCNE1 amplification among metastatic sites in patients with gynecologic high-grade serous carcinoma.

OBJECTIVE: We sought to characterize the variability of CCNE1 amplification among metastatic sites of CCNE1 amplified high grade serous carcinoma (HGSC) cases to investigate the feasibility of targeting this alteration for therapeutic purposes. METHODS: Patients with CCNE1 amplified HGSC who underwent surgical cytoreduction with metastatic sites were identified from institutional molecular profiling reports and a population of HGSC cases screened using digital droplet PCR (ddPCR). Cases with normal CCNE1 copy number were included as controls. Slides from metastatic sites were cut from formalin-fixed paraffin-embedded tissue blocks, dissected for tumor of > 50% purity, and underwent DNA extraction. CCNE1 copy number was determined by ddPCR. Tumor purity was confirmed with mutant TP53 allele fraction from targeted massively parallel sequencing. RESULTS: Four of 15 patients from an institutional database screened by ddPCR were found to have CCNE1 amplification. Three additional patients were identified from a query of institutional commercial clinical reports. Among these 7 CCNE1 amplified cases (2 uterine, 5 ovarian), 5 showed preservation of CCNE1 amplification (copy number > 5) among all metastatic sites. The remaining 2 cases had multiple metastatic sites without preserved CCNE1 amplification. Non-amplified cases had predominantly normal CCNE1 copy number across metastatic sites. CONCLUSIONS: CCNE1 amplification is an early genomic event in HGSC and is preserved in most metastatic sites suggesting a uniform response to pathway targeting therapies.

Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer.

The paucity of genetically informed, immunocompetent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high-grade serous tubo-ovarian cancer (HGSC) models exhibiting mutational combinations seen in patients with HGSC. Detailed analysis of homologous recombination (HR)-proficient (Trp53-/-;Ccne1OE;Akt2OE;KrasOE ), HR-deficient (Trp53-/-;Brca1-/-;MycOE ), and unclassified (Trp53-/-;Pten-/-;Nf1-/- ) organoids revealed differences in in vitro properties (proliferation, differentiation, and "secretome"), copy-number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, and evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T cell-dependent responses in Trp53-/-;Ccne1OE;Akt2OE;Kras HGSC; in contrast, Trp53-/-;Pten-/-;Nf1-/- tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics. SIGNIFICANCE: The lack of genetically informed, diverse, immunocompetent models poses a major barrier to therapeutic development for many malignancies. Using engineered fallopian tube organoids to study the cell-autonomous and cell-nonautonomous effects of specific combinations of mutations found in HGSC, we suggest an effective combination treatment for the currently intractable CCNE1-amplified subgroup.This article is highlighted in the In This Issue feature, p. 211.

Massively parallel sequencing analysis of mucinous ovarian carcinomas: genomic profiling and differential diagnoses.

OBJECTIVE: Mucinous ovarian cancer (MOC) is a rare type of epithelial ovarian cancer resistant to standard chemotherapy regimens. We sought to characterize the repertoire of somatic mutations in MOCs and to define the contribution of massively parallel sequencing to the classification of tumors diagnosed as primary MOCs. METHODS: Following gynecologic pathology and chart review, DNA samples obtained from primary MOCs and matched normal tissues/blood were subjected to whole-exome (n = 9) or massively parallel sequencing targeting 341 cancer genes (n = 15). Immunohistochemical analysis of estrogen receptor, progesterone receptor, PTEN, ARID1A/BAF250a, and the DNA mismatch (MMR) proteins MSH6 and PMS2 was performed for all cases. Mutational frequencies of MOCs were compared to those of high-grade serous ovarian cancers (HGSOCs) and mucinous tumors from other sites. RESULTS: MOCs were heterogeneous at the genetic level, frequently harboring TP53 (75%) mutations, KRAS (71%) mutations and/or CDKN2A/B homozygous deletions/mutations (33%). Although established criteria for diagnosis were employed, four cases harbored mutational and immunohistochemical profiles similar to those of endometrioid carcinomas, and one case for colorectal or endometrioid carcinoma. Significant differences in the frequencies of KRAS, TP53, CDKN2A, FBXW7, PIK3CA and/or APC mutations between the confirmed primary MOCs (n = 19) and HGSOCs, mucinous gastric and/or mucinous colorectal carcinomas were found, whereas no differences in the 341 genes studied between MOCs and mucinous pancreatic carcinomas were identified. CONCLUSIONS: Our findings suggest that the assessment of mutations affecting TP53, KRAS, PIK3CA, ARID1A and POLE, and DNA MMR protein expression may be used to further aid the diagnosis and treatment decision-making of primary MOC.

A pair of datasets for microRNA expression profiling to examine the use of careful study design for assigning arrays to samples.

We set out to demonstrate the logistic feasibility of careful experimental design for microarray studies and its level of scientific benefits for improving the accuracy and reproducibility of data inference. Towards this end, we conducted a study of microRNA expression using endometrioid endometrial tumours (n=96) and serous ovarian tumours (n=96) that were primary, untreated, and collected from 2000 to 2012 at Memorial Sloan Kettering Cancer Center. The same set of tumour tissue samples were profiled twice using the Agilent microRNA microarrays: once under an ideal experimental condition with balanced array-to-sample allocation and uniform handling; a second time by mimicking typical practice, with arrays assigned in the order of sample collection and processed by two technicians in multiple batches. This paper provides a detailed description of the generation and validation of this unique dataset pair so that the research community can re-use it to investigate other statistical questions regarding microarray study design and data analysis, and to address biological questions on the relevance of microRNA expression in gynaecologic cancer.

Noninvasive ovarian cancer biomarker detection via an optical nanosensor implant.

Patients with high-grade serous ovarian carcinoma (HGSC) exhibit poor 5-year survival rates, which may be significantly improved by early-stage detection. The U.S. Food and Drug Administration-approved biomarkers for HGSC-CA-125 (cancer antigen 125) and HE4 (human epididymis protein 4)-do not generally appear at detectable levels in the serum until advanced stages of the disease. An implantable device placed proximal to disease sites, such as in or near the fallopian tube, ovary, uterine cavity, or peritoneal cavity, may constitute a feasible strategy to improve detection of HGSC. We engineered a prototype optical sensor composed of an antibody-functionalized carbon nanotube complex, which responds quantitatively to HE4 via modulation of the nanotube optical bandgap. The complexes measured HE4 with nanomolar sensitivity to differentiate disease from benign patient biofluids. The sensors were implanted into four models of ovarian cancer, within a semipermeable membrane, enabling the optical detection of HE4 within the live animals. We present the first in vivo optical nanosensor capable of noninvasive cancer biomarker detection in orthotopic models of disease.

Immune-Active Microenvironment in Small Cell Carcinoma of the Ovary, Hypercalcemic Type: Rationale for Immune Checkpoint Blockade.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a highly aggressive monogenic cancer driven by SMARCA4 mutations. Here, we report responses to anti-PD1 immunotherapy in four patients and characterize the immune landscape of SCCOHT tumors using quantitative immunofluorescence and gene expression profiling. Unexpectedly for a low mutation burden cancer, the majority of the tumors (eight of 11 cases) demonstrated PD-L1 expression with strong associated T-cell infiltration (R2 = 0.60-0.95). PD-L1 expression was detected in both tumor and stromal cells, with macrophages being the most abundant PD-L1-positive cells in some tumors (three of 11 cases). Transcriptional profiling revealed increased expression of genes related to Th1 and cytotoxic cell function in PD-L1-high tumors, suggesting that PD-L1 acts as a pathway of adaptive immune resistance in SCCOHT. These findings suggest that although SCCOHT are low-mutational burden tumors, their immunogenic microenvironment resembles the landscape of tumors that respond well to treatment with PD-1/PD-L1 blockade.

Genetic predisposition to bevacizumab-induced hypertension.

OBJECTIVE: Bevacizumab, a monoclonal antibody to VEGF, has shown efficacy in ovarian, cervical and endometrial cancer in addition to several other solid tumors. Serious side effects include hypertension, proteinuria, bowel perforation, and thrombosis. We tested the hypothesis that genetic variation in hypertension-associated genes is associated with bevacizumab-induced hypertension (BIH). METHODS: Patients with solid tumors treated with bevacizumab in combination with other therapy were identified from six clinical trials. Haplotype-tagging (ht) SNPs for 10 candidate genes associated with hypertension were identified through the International Hapmap Project. Germline DNA was genotyped for 103 htSNPs using mass spectrometry. Bevacizumab toxicities were identified from clinical trial reports. Haplotypes were reconstructed from diploid genotyping data and frequencies were compared using standard two-sided statistical tests. RESULTS: The study included 114 patients with breast, lung, ovarian, or other cancers, of whom 38 developed BIH. WNK1, KLKB1, and GRK4 were found to contain single loci associated with BIH. Haplotype analysis of WNK1, KLKB1, and GRK4 identified risk haplotypes in each gene associated with grade 3/4 BIH. A composite risk model was created based on these haplotypes. Patients with the highest risk score were the most likely to develop grade 3/4 BIH (OR=6.45; P=0.005; 95%CI, 1.86-22.39). CONCLUSIONS: We concluded that genetic variation in WNK1, KLKB1, and GRK4 may be associated with BIH. These genes are biologically plausible mediators due to their role in blood pressure control, regulating sodium homeostasis and vascular tone. This preliminary risk model performed better than population-based risk models and when further validated may help risk-stratify patients for BIH prior to initiating therapy.

Molecular analysis of high-grade serous ovarian carcinoma with and without associated serous tubal intra-epithelial carcinoma.

Many high-grade serous carcinomas (HGSCs) of the pelvis are thought to originate in the distal portion of the fallopian tube. Serous tubal intra-epithelial carcinoma (STIC) lesions are the putative precursor to HGSC and identifiable in ~ 50% of advanced stage cases. To better understand the molecular etiology of HGSCs, we report a multi-center integrated genomic analysis of advanced stage tumors with and without STIC lesions and normal tissues. The most significant focal DNA SCNAs were shared between cases with and without STIC lesions. The RNA sequence and the miRNA data did not identify any clear separation between cases with and without STIC lesions. HGSCs had molecular profiles more similar to normal fallopian tube epithelium than ovarian surface epithelium or peritoneum. The data suggest that the molecular features of HGSCs with and without associated STIC lesions are mostly shared, indicating a common biologic origin, likely to be the distal fallopian tube among all cases.High-grade serous carcinomas (HGSCs) are associated with precursor lesions (STICs) in the fallopian epithelium in only half of the cases. Here the authors report the molecular analysis of HGSCs with and without associated STICs and show similar profiles supporting a common origin for all HGSCs.

miR-200c-driven Mesenchymal-To-Epithelial Transition is a Therapeutic Target in Uterine Carcinosarcomas.

Uterine carcinosarcomas (UCSs) are highly aggressive malignancies associated with poor prognoses and limited treatment options. These tumors are hypothesized to develop from the endometrial adenocarcinoma (EAC) through epithelial-mesenchymal transition (EMT). We test this long-standing hypothesis by depleting miR-200, a family of microRNAs critical for EMT, in EAC cell lines. Our data suggest that UCSs do not develop from EACs via EMT. Clinically more relevant, we show that miR-200 expression in UCS cells induces a robust mesenchymal-epithelial transition (MET). Using in vitro and murine xenograft models, we demonstrate decreased growth and aggressiveness of miR-200-overexpressing UCS cell lines. Whole transcriptome analysis confirmed changes consistent with an MET and also revealed changes in angiogenic genes expression. Finally, by treatment of UCS-xenografted mice with miR-200c incorporated in DOPC nanoliposomes, we demonstrate anti-tumor activities. These findings suggest that ectopic miR-200 expression using advanced microRNA therapeutics may be a potential treatment approach for patients with UCS.

Antibodies Against Specific MUC16 Glycosylation Sites Inhibit Ovarian Cancer Growth.

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.

Uterine Cancer After Risk-Reducing Salpingo-oophorectomy Without Hysterectomy in Women With BRCA Mutations.

IMPORTANCE: The link between BRCA mutations and uterine cancer is unclear. Therefore, although risk-reducing salpingo-oophorectomy (RRSO) is standard treatment among women with BRCA mutations (BRCA+ women), the role of concomitant hysterectomy is controversial. OBJECTIVE: To determine the risk for uterine cancer and distribution of specific histologic subtypes in BRCA+ women after RRSO without hysterectomy. DESIGN, SETTING, AND PARTICIPANTS: This multicenter prospective cohort study included 1083 women with a deleterious BRCA1 or BRCA2 mutation identified from January 1, 1995, to December 31, 2011, at 9 academic medical centers in the United States and the United Kingdom who underwent RRSO without a prior or concomitant hysterectomy. Of these, 627 participants were BRCA1+; 453, BRCA2+; and 3, both. Participants were prospectively followed up for a median 5.1 (interquartile range [IQR], 3.0-8.4) years after ascertainment, BRCA testing, or RRSO (whichever occurred last). Follow up data available through October 14, 2014, were included in the analyses. Censoring occurred at uterine cancer diagnosis, hysterectomy, last follow-up, or death. New cancers were categorized by histologic subtype, and available tumors were analyzed for loss of the wild-type BRCA gene and/or protein expression. MAIN OUTCOMES AND MEASURES: Incidence of uterine corpus cancer in BRCA+ women who underwent RRSO without hysterectomy compared with rates expected from the Surveillance, Epidemiology, and End Results database. RESULTS: Among the 1083 women women who underwent RRSO without hysterectomy at a median age 45.6 (IQR: 40.9 - 52.5), 8 incident uterine cancers were observed (4.3 expected; observed to expected [O:E] ratio, 1.9; 95% CI, 0.8-3.7; P = .09). No increased risk for endometrioid endometrial carcinoma or sarcoma was found after stratifying by subtype. Five serous and/or serous-like (serous/serous-like) endometrial carcinomas were observed (4 BRCA1+ and 1 BRCA2+) 7.2 to 12.9 years after RRSO (BRCA1: 0.18 expected [O:E ratio, 22.2; 95% CI, 6.1-56.9; P < .001]; BRCA2: 0.16 expected [O:E ratio, 6.4; 95% CI, 0.2-35.5; P = .15]). Tumor analyses confirmed loss of the wild-type BRCA1 gene and/or protein expression in all 3 available serous/serous-like BRCA1+ tumors. CONCLUSIONS AND RELEVANCE: Although the overall risk for uterine cancer after RRSO was not increased, the risk for serous/serous-like endometrial carcinoma was increased in BRCA1+ women. This risk should be considered when discussing the advantages and risks of hysterectomy at the time of RRSO in BRCA1+ women.

Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type.

Small cell carcinoma of the ovary, hypercalcemic type is an aggressive tumor generally affecting young women with limited treatment options. Mutations in SMARCA4, a catalytic subunit of the SWI/SNF chromatin remodeling complex, have recently been identified in nearly all small cell carcinoma of the ovary, hypercalcemic type cases and represent a signature molecular feature for this disease. Additional biological dependencies associated with small cell carcinoma of the ovary, hypercalcemic type have not been identified. SMARCA2, another catalytic subunit of the SWI/SNF complex mutually exclusive with SMARCA4, is thought to be post-translationally silenced in various cancer types. We analyzed 10 archival small cell carcinoma of the ovary, hypercalcemic type cases for SMARCA2 protein expression by immunohistochemistry and found that SMARCA2 expression was lost in all but one case. None of the 50 other tumors that primarily or secondarily involved the ovary demonstrated concomitant loss of SMARCA2 and SMARCA4. Deep sequencing revealed that this loss of SMARCA2 expression is not the result of mutational inactivation. In addition, we established a small cell carcinoma of the ovary, hypercalcemic type patient-derived xenograft and confirmed the loss of SMARCA2 in this in vitro model. This patient-derived xenograft model, established from a recurrent tumor, also had unexpected mutational features for this disease, including functional mutations in TP53 and POLE. Taken together, our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of small cell carcinoma of the ovary, hypercalcemic type-a finding that offers new opportunities for therapeutic interventions.

Loss of SMARCA4 Expression Is Both Sensitive and Specific for the Diagnosis of Small Cell Carcinoma of Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare ovarian neoplasm that occurs in young women and has a poor prognosis. The histologic diagnosis of SCCOHT can be challenging due to its rarity and relatively nonspecific histologic features, which range from the classic, first-described small cell morphology to a pattern in which there are large cells with abundant eosinophilic cytoplasm. Many entities can be in the differential diagnosis and to date, immunohistochemical stains have shown no distinctive profile and have been of limited aid. SMARCA4 (also known as BRG1) mutations have recently been reported at high frequency in these tumors. SMARCA4 is an important component of the SWI/SNF complex that regulates gene expression through alteration of nucleosome conformation. Studies to date have suggested that immunohistochemical loss of expression of SMARCA4 is associated with the presence of a SMARCA4 mutation in most cases. In this study, the sensitivity and specificity of the immunohistochemical loss of SMARCA4 expression for the diagnosis of SCCOHT is examined in the context of the differential diagnosis with other primary or metastatic ovarian tumors. All but one of the SCCOHT showed loss of SMARCA4 expression (16/17; 94%), while of 279 other tumors tested, only two tumors (one clear cell carcinoma and one ovarian melanoma) showed loss of SMARCA4 expression. We conclude that SMARCA4 immunohistochemistry is highly sensitive and specific for a diagnosis of SCCOHT and is of clinical utility in the differential diagnosis of poorly differentiated ovarian tumors.

Molecular subtypes of uterine leiomyosarcoma and correlation with clinical outcome.

The molecular etiology of uterine leiomyosarcoma (ULMS) is poorly understood, which accounts for the wide disparity in outcomes among women with this disease. We examined and compared the molecular profiles of ULMS and normal myometrium (NL) to identify clinically relevant molecular subtypes. Discovery cases included 29 NL and 23 ULMS specimens. RNA was hybridized to Affymetrix U133A 2.0 transcription microarrays. Differentially expressed genes and pathways were identified using standard methods. Fourteen NL and 44 ULMS independent archival samples were used for external validation. Molecular subgroups were correlated with clinical outcome. Pathway analyses of differentially expressed genes between ULMS and NL samples identified overrepresentation of cell cycle regulation, DNA repair, and genomic integrity. External validation confirmed differential expression in 31 genes (P < 4.4 × 10(-4), Bonferroni corrected), with 84% of the overexpressed genes, including CDC7, CDC20, GTSE1, CCNA2, CCNB1, and CCNB2, participating in cell cycle regulation. Unsupervised clustering of ULMS identified two clades that were reproducibly associated with progression-free (median, 4.0 vs 26.0 months; P = .02; HR, 0.33) and overall (median, 18.2 vs 77.2 months; P = .04; HR, 0.33) survival. Cell cycle genes play a key role in ULMS sarcomagenesis, providing opportunities for therapeutic targeting. Reproducible molecular subtypes associated with clinical outcome may permit individualized adjuvant treatment after clinical trial validation.

Gene expression profiling of lobular carcinoma in situ reveals candidate precursor genes for invasion.

PURPOSE: Lobular carcinoma in situ (LCIS) is both a risk indicator and non-obligate precursor of invasive lobular carcinoma (ILC). We sought to characterize the transcriptomic features of LCIS and ILC, with a focus on the identification of intrinsic molecular subtypes of LCIS and the changes involved in the progression from normal breast epithelium to LCIS and ILC. METHODS: Fresh-frozen classic LCIS, classic ILC, and normal breast epithelium (N) from women undergoing prophylactic or therapeutic mastectomy were prospectively collected, laser-capture microdissected, and subjected to gene expression profiling using Affymetrix HG-U133A 2.0 microarrays. RESULTS: Unsupervised hierarchical clustering of 40 LCIS samples identified 2 clusters of LCIS distinguished by 6431 probe sets (p < 0.001). Genes identifying the clusters included proliferation genes and other genes related to cancer canonical pathways such as TGF beta signaling, p53 signaling, actin cytoskeleton, apoptosis and Wnt-Signaling pathway. A supervised analysis to identify differentially expressed genes (p < 0.001) between normal epithelium, LCIS, and ILC, using 23 patient-matched triplets of N, LCIS, and ILC, identified 169 candidate precursor genes, which likely play a role in LCIS progression, including PIK3R1, GOLM1, and GPR137B. These potential precursor genes map significantly more frequently to 1q and 16q, regions frequently targeted by gene copy number alterations in LCIS and ILC. CONCLUSION: Here we demonstrate that classic LCIS is a heterogeneous disease at the transcriptomic level and identify potential precursor genes in lobular carcinogenesis. Understanding the molecular heterogeneity of LCIS and the potential role of these potential precursor genes may help personalize the therapy of patients with LCIS.

Blocking and randomization to improve molecular biomarker discovery.

Randomization and blocking have the potential to prevent the negative impacts of nonbiologic effects on molecular biomarker discovery. Their use in practice, however, has been scarce. To demonstrate the logistic feasibility and scientific benefits of randomization and blocking, we conducted a microRNA study of endometrial tumors (n = 96) and ovarian tumors (n = 96) using a blocked randomization design to control for nonbiologic effects; we profiled the same set of tumors for a second time using no blocking or randomization. We assessed empirical evidence of differential expression in the two studies. We performed simulations through virtual rehybridizations to further evaluate the effects of blocking and randomization. There was moderate and asymmetric differential expression (351/3,523, 10%) between endometrial and ovarian tumors in the randomized dataset. Nonbiologic effects were observed in the nonrandomized dataset, and 1,934 markers (55%) were called differentially expressed. Among them, 185 were deemed differentially expressed (185/351, 53%) and 1,749 not differentially expressed (1,749/3,172, 55%) in the randomized dataset. In simulations, when randomization was applied to all samples at once or within batches of samples balanced in tumor groups, blocking improved the true-positive rate from 0.95 to 0.97 and the false-positive rate from 0.02 to 0.002; when sample batches were unbalanced, randomization was associated with the true-positive rate (0.92) and the false-positive rate (0.10) regardless of blocking. Normalization improved the detection of true-positive markers but still retained sizeable false-positive markers. Randomization and blocking should be used in practice to more fully reap the benefits of genomics technologies.

Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis.

Recurrent SMARCA4 mutations in small cell carcinoma of the ovary.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, highly aggressive form of ovarian cancer primarily diagnosed in young women. We identified inactivating biallelic SMARCA4 mutations in 100% of the 12 SCCOHT tumors examined. Protein studies confirmed loss of SMARCA4 expression, suggesting a key role for the SWI/SNF chromatin-remodeling complex in SCCOHT.

BRCA1 immunohistochemistry in a molecularly characterized cohort of ovarian high-grade serous carcinomas.

BRCA1 and BRCA2 dysfunction, frequently seen in high-grade serous ovarian carcinomas, often results from germline mutations, somatic mutations, and promoter methylation. Identification of tumors with BRCA defects has therapeutic and prognostic implications. Identifying germline BRCA mutations is also important given the increased risk for hereditary breast and ovarian carcinoma. Our goal was to assess whether immunohistochemical analysis (IHC) for BRCA1 is an effective method for the detection of BRCA1 dysfunction in molecularly characterized high-grade ovarian serous carcinoma. We identified 43 high-grade ovarian serous carcinomas with known events in BRCA1 and BRCA2 included in The Cancer Genome Atlas Project. BRCA1 stain was first assessed without knowledge of the BRCA status, and a semiquantitative assessment for intensity and amount of staining was performed. The stains were reevaluated and divided into 3 categories (retained, loss, and equivocal) on the basis of correlation with genotyping data. Presence of retained BRCA staining was considered normal, whereas the other patterns, including equivocal staining or loss of staining, were considered abnormal. Two pathologists, blinded to the BRCA status, then scored 2 sets of validation cases selected on the basis of available molecular data-1 with only germline mutation status available (n=31) and 1 with comprehensive genomic data (n=39). The pathologists agreed 88% of the time in the training set and 91% in the validation sets. In the training set, abnormal BRCA staining was seen in 24 cases, of which 21 (87%) showed BRCA1 genetic abnormalities, 1 showed BRCA2 mutations, and 2 showed no BRCA abnormalities. Abnormal BRCA1 staining was noted in all 5 cases with BRCA1 germline mutations, in 3 (60%) of 5 with BRCA1 somatic mutations, and in 13 (93%) of 14 with BRCA1 promoter methylation. The 2 validation sets included 70 additional patients, and all cases with germline BRCA1 mutations (n=11) showed abnormal BRCA1 staining. Tumors with BRCA1 promoter methylation also showed abnormal staining in 6 (86%) of 7 cases. In the entire study, no cases with BRCA1 germline mutation showed intact immunostaining (negative predictive value=100%). This study shows that BRCA1 IHC is well correlated with molecular events in ovarian carcinoma. Considering the high negative predictive value for germline mutations, BRCA1 IHC appears to be an effective approach to stratify patients for germline genetic testing and to detect other mechanisms of BRCA1 dysfunction in high-grade serous ovarian carcinomas.