Molecular determinants of clinical outcomes of pembrolizumab in recurrent ovarian cancer: Exploratory analysis of KEYNOTE-100.

OBJECTIVE: This prespecified exploratory analysis evaluated the association of gene expression signatures, tumor mutational burden (TMB), and multiplex immunohistochemistry (mIHC) tumor microenvironment-associated cell phenotypes with clinical outcomes of pembrolizumab in advanced recurrent ovarian cancer (ROC) from the phase II KEYNOTE-100 study. METHODS: Pembrolizumab-treated patients with evaluable RNA-sequencing (n = 317), whole exome sequencing (n = 293), or select mIHC (n = 125) data were evaluated. The association between outcomes (objective response rate [ORR], progression-free survival [PFS], and overall survival [OS]) and gene expression signatures (T-cell-inflamed gene expression profile [TcellinfGEP] and 10 non-TcellinfGEP signatures), TMB, and prespecified mIHC cell phenotype densities as continuous variables was evaluated using logistic (ORR) and Cox proportional hazards regression (PFS; OS). One-sided p-values were calculated at prespecified α = 0.05 for TcellinfGEP, TMB, and mIHC cell phenotypes and at α = 0.10 for non-TcellinfGEP signatures; all but TcellinfGEP and TMB were adjusted for multiplicity. RESULTS: No evidence of associations between ORR and key axes of gene expression was observed. Negative associations were observed between outcomes and TcellinfGEP-adjusted glycolysis (PFS, adjusted-p = 0.019; OS, adjusted-p = 0.085) and hypoxia (PFS, adjusted-p = 0.064) signatures. TMB as a continuous variable was not associated with outcomes (p > 0.05). Positive associations were observed between densities of myeloid cell phenotypes CD11c+ and CD11c+/MHCII-/CD163-/CD68- in the tumor compartment and ORR (adjusted-p = 0.025 and 0.013, respectively). CONCLUSIONS: This exploratory analysis in advanced ROC did not find evidence for associations between gene expression signatures and outcomes of pembrolizumab. mIHC analysis suggests CD11c+ and CD11c+/MHCII-/CD163-/CD68- phenotypes representing myeloid cell populations may be associated with improved outcomes with pembrolizumab in advanced ROC. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT02674061.

Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

Small cell cancers of the female genital tract: Molecular and clinical aspects.

OBJECTIVE: Extra-pulmonary small cell carcinomas of the gynecologic tract (EPSCC-GTs) are a rare group of aggressive malignancies associated with poor prognoses and limited treatment options. Here, we review the clinical and molecular aspects of EPSCC-GTs and discuss how understanding their molecular features can assist in their diagnosis and the identification of novel effective treatments. METHODS: We searched PubMed and Scopus for articles using the following keywords: "small cell carcinoma" in combination with "neuroendocrine", "ovary", "vagina", "fallopian tube", "vulva", "endometrium", "uterus", "cervix", or "gynecologic". Articles were limited to those published in English from January 1984 to October 2017. RESULTS: EPSCC-GTs account for 2% of all gynecologic malignancies. The molecular features of EPSCC-GTs are largely understudied and unknown, with the exception of small cell carcinoma (SCC) of the ovary, hypercalcemic type (SCCOHT) and SCC of the cervix (SCCC). In nearly all cases, SCCOHT displays mutation in a single gene, SMARCA4, a member of the SWI/SNF chromatin remodeling complex. The loss of expression of the SWI/SNF protein SMARCA2 is another feature of SCCOHT. Dual negative staining for SMARCA2 and SMARCA4 is specific for SCCOHT and is generally used by gynecologic pathologists for the accurate diagnosis of this malignancy. Mutational analysis of SCCC has shown alterations in PIK3CA, KRAS and TP53, of which the last is the most common, although other actionable mutations have been identified. The molecular features of other EPSCC-GTs are largely unknown. CONCLUSIONS: Due to their rarity, the majority of EPSCC-GTs are understudied and poorly understood. As demonstrated in the case of SCCOHT, unraveling the mutational profiles of these tumors can lead to improved diagnosis and the identification of novel therapeutic targets.

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.

Prevalence and prognostic value of PD-L1 expression and tumor mutational burden in persistent, recurrent, or metastatic cervical cancer.

OBJECTIVE: To evaluate the prevalence and prognostic role of programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB) in patients with non-immunotherapy-treated advanced cervical cancer. METHODS: Clinical data were retrospectively collected from medical records between January 1, 2008, and December 31, 2016, at Asan Medical Center (Korea); archived tumor samples were assessed for PD-L1 expression (combined positive score [CPS] ≥1) and TMB (≥175 mutations/exome). Overall survival (OS) was defined as time from advanced diagnosis or initiation of first-line or second-line systemic therapy until death/last follow-up. The association of OS with PD-L1 expression and TMB were analyzed using the log-rank test and Cox proportional hazards model adjusted for covariates. RESULTS: Of 267 patients, 76.0% had squamous cell carcinoma (SCC), 24.0% had adenocarcinoma (AC)/adenosquamous carcinoma (ASC), 64.4% had PD-L1 CPS ≥1, and 32.6% had TMB ≥175 mutations/exome. PD-L1 CPS ≥1 and TMB ≥175 mutations/exome were more prevalent in SCC than in AC/ASC (73.9% and 37.2% vs. 34.4% and 17.7%). There was no association between OS and PD-L1 expression (CPS ≥1 vs. <1: adjusted hazard ratio [HR]=1.14; 95% confidence interval [CI]=0.84-1.53 from advanced diagnosis); OS trended shorter for the subgroup with TMB ≥175 versus <175 mutations/exome (adjusted HR=1.29; 95% CI=0.95-1.75). CONCLUSION: Retrospective analysis of non-immunotherapy-treated patients with advanced cervical cancer demonstrated a higher prevalence of PD-L1 CPS ≥1 and TMB ≥175 mutations/exome in SCC versus AC/ASC. PD-L1 CPS ≥1 was not associated with OS; TMB ≥175 mutations/exome showed a trend toward shorter OS. Additional studies are needed to confirm these findings.

Contribution of tumour and immune cells to PD-L1 expression as a predictive biomarker in metastatic triple-negative breast cancer: exploratory analysis from KEYNOTE-119.

The efficacy of pembrolizumab monotherapy versus chemotherapy increased with increasing programmed death ligand 1 (PD-L1) expression, as quantified by combined positive score (CPS; PD-L1 expression on both tumour cells and immune cells) in patients with previously treated metastatic triple-negative breast cancer (mTNBC) in the phase 3 KEYNOTE-119 study. This exploratory analysis was conducted to determine whether the expression of PD-L1 on tumour cells contributes to the predictive value of PD-L1 CPS in mTNBC. PD-L1 expression in tumour samples was assessed using PD-L1 IHC 22C3 pharmDx and quantified using both CPS and tumour proportion score (TPS; PD-L1 expression on tumour cells alone). Calculated immune cell density (CID) was defined as CPS minus TPS. The ability of each scoring method (CPS, TPS, and CID) to predict clinical outcomes with pembrolizumab was evaluated. With pembrolizumab, the area under the receiver operating characteristic curve was 0.69 (95% CI = 0.58-0.80) for CPS, 0.55 (95% CI = 0.46-0.64) for TPS, and 0.67 (95% CI = 0.56-0.77) for CID. After correction for cutoff prevalence, CPS performed as well as, if not better than, CID with respect to predicting objective response rate, progression-free survival, and overall survival. Data from this exploratory analysis suggest that, although PD-L1 expression on immune cells alone is predictive of response to programmed death 1 blockade in mTNBC, adding tumour PD-L1 expression assessment (i.e. CPS, which combines immune cell and tumour cell PD-L1 expression) may improve prediction. PD-L1 CPS thus remains an effective and broadly applicable uniform scoring system for enriching response to programmed death 1 blockade with pembrolizumab in mTNBC as well as other tumour types.

Evaluation of potential biomarkers for lenvatinib plus pembrolizumab among patients with advanced endometrial cancer: results from Study 111/KEYNOTE-146.

BACKGROUND: Lenvatinib plus pembrolizumab demonstrated clinically meaningful benefit in patients with previously treated advanced endometrial carcinoma in Study 111/KEYNOTE-146 (NCT02501096). In these exploratory analyses from this study, we evaluated the associations between clinical outcomes and gene expression signature scores and descriptively summarized response in biomarker subpopulations defined by tumor mutational burden (TMB) and DNA variants for individual genes of interest. METHODS: Patients with histologically confirmed metastatic endometrial carcinoma received oral lenvatinib 20 mg once daily plus intravenous pembrolizumab 200 mg every 3 weeks for 35 cycles. Archived formalin-fixed paraffin-embedded tissue was obtained from all patients. T-cell-inflamed gene expression profile (TcellinfGEP) and 11 other gene signatures were evaluated by RNA sequencing. TMB, hotspot mutations in PIK3CA (oncogene), and deleterious mutations in PTEN and TP53 (tumor suppressor genes) were evaluated by whole-exome sequencing (WES). RESULTS: 93 and 79 patients were included in the RNA-sequencing-evaluable and WES-evaluable populations, respectively. No statistically significant associations were observed between any of the RNA-sequencing signature scores and objective response rate or progression-free survival. Area under the receiver operating characteristic curve values for response ranged from 0.39 to 0.54; all 95% CIs included 0.50. Responses were seen regardless of TMB (≥175 or <175 mutations/exome) and mutation status. There were no correlations between TcellinfGEP and TMB, TcellinfGEP and microvessel density (MVD), or MVD and TMB. CONCLUSIONS: This analysis demonstrated efficacy for lenvatinib plus pembrolizumab regardless of biomarker status. Results from this study do not support clinical utility of the evaluated biomarkers. Further investigation of biomarkers for this regimen is warranted. TRIAL REGISTRATION NUMBER: NCT02501096.

Validated gene targets associated with curatively treated advanced serous ovarian carcinoma.

OBJECTIVES: High-grade serous ovarian cancer (HGSOC) mostly presents at an advanced stage and has a low overall survival rate. However, a subgroup of patients are seemingly cured after standard initial therapy. We hypothesize that the molecular profiles of these patients vary from long-term survivors who recur. METHODS: Patients with advanced HGSOC who underwent primary cytoreductive surgery and platinum-based chemotherapy were identified from The Cancer Genome Atlas (TCGA) and institutional (MSKCC) samples. A curative-intent group was defined by recurrence-free survival of >5years. A long-term recurrent group was composed of patients who recurred but survived >5years. RNA was hybridized to Affymetrix U133A transcription microarrays. The NanoString nCounter gene expression system was used for validation in an independent patient population. RESULTS: In 30 curative and 84 recurrent patients, class comparison identified twice as many differentially expressed probes between the groups than expected by chance alone. TCGA and MSKCC data sets had 19 overlapping genes. Pathway analyses identified over-represented networks that included nuclear factor kappa B (NFkB) transcription and extracellular signal-regulated kinase (ERK) signaling. External validation was performed in an independent population of 28 curative and 38 recurrent patients. Three genes (CYP4B1, CEPT1, CHMP4A) in common between our original data sets remained differentially expressed in the external validation data. CONCLUSIONS: There are distinct transcriptional elements in HGSOC from patients likely to be cured by standard primary therapy. Three genes have withstood rigorous validation and are plausible targets for further study, which may provide insight into molecular features associated with long-term survival and chemotherapy resistance mechanisms.

Association Between Biomarkers and Clinical Outcomes of Pembrolizumab Monotherapy in Patients With Metastatic Triple-Negative Breast Cancer: KEYNOTE-086 Exploratory Analysis.

PURPOSE: In the two-cohort phase II KEYNOTE-086 study (ClinicalTrials.gov identifier: NCT02447003), first-line and second-line or later pembrolizumab monotherapy demonstrated antitumor activity in metastatic triple-negative breast cancer (mTNBC; N = 254). This exploratory analysis evaluates the association between prespecified molecular biomarkers and clinical outcomes. METHODS: Cohort A enrolled patients with disease progression after one or more systemic therapies for metastatic disease irrespective of PD-L1 status; Cohort B enrolled patients with previously untreated PD-L1-positive (combined positive score [CPS] ≥ 1) metastatic disease. The association between the following biomarkers as continuous variables and clinical outcomes (objective response rate [ORR], progression-free survival [PFS], and overall survival [OS]) was evaluated: PD-L1 CPS (immunohistochemistry), cluster of differentiation 8 (CD8; immunohistochemistry), stromal tumor-infiltrating lymphocyte (sTIL; hematoxylin and eosin staining), tumor mutational burden (TMB; whole-exome sequencing [WES]), homologous recombination deficiency-loss of heterozygosity, mutational signature 3 (WES), mutational signature 2 (apolipoprotein B mRNA editing catalytic polypeptide-like; WES), T-cell-inflamed gene expression profile (TcellinfGEP; RNA sequencing), and 10 non-TcellinfGEP signatures (RNA sequencing); Wald test P values were calculated, and significance was prespecified at α = 0.05. RESULTS: In the combined cohorts (A and B), PD-L1 (P = .040), CD8 (P < .001), sTILs (P = .012), TMB (P = .007), and TcellinfGEP (P = .011) were significantly associated with ORR; CD8 (P < .001), TMB (P = .034), Signature 3 (P = .009), and TcellinfGEP (P = .002) with PFS; and CD8 (P < .001), sTILs (P = .004), TMB (P = .025), and TcellinfGEP (P = .001) with OS. None of the non-TcellinfGEP signatures were associated with outcomes of pembrolizumab after adjusting for the TcellinfGEP. CONCLUSION: In this exploratory biomarker analysis from KEYNOTE-086, baseline tumor PD-L1, CD8, sTILs, TMB, and TcellinfGEP were associated with improved clinical outcomes of pembrolizumab and may help identify patients with mTNBC who are most likely to respond to pembrolizumab monotherapy.

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation.

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation-DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.

Chromatin-informed inference of transcriptional programs in gynecologic and basal breast cancers.

Chromatin accessibility data can elucidate the developmental origin of cancer cells and reveal the enhancer landscape of key oncogenic transcriptional regulators. We develop a computational strategy called PSIONIC (patient-specific inference of networks informed by chromatin) to combine chromatin accessibility data with large tumor expression data and model the effect of enhancers on transcriptional programs in multiple cancers. We generate a new ATAC-seq data profiling chromatin accessibility in gynecologic and basal breast cancer cell lines and apply PSIONIC to 723 patient and 96 cell line RNA-seq profiles from ovarian, uterine, and basal breast cancers. Our computational framework enables us to share information across tumors to learn patient-specific TF activities, revealing regulatory differences between and within tumor types. PSIONIC-predicted activity for MTF1 in cell line models correlates with sensitivity to MTF1 inhibition, showing the potential of our approach for personalized therapy. Many identified TFs are significantly associated with survival outcome. To validate PSIONIC-derived prognostic TFs, we perform immunohistochemical analyses in 31 uterine serous tumors for ETV6 and 45 basal breast tumors for MITF and confirm that the corresponding protein expression patterns are also significantly associated with prognosis.

CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary.

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

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.

The EMSY threonine 207 phospho-site is required for EMSYdriven suppression of DNA damage repair.

BRCA1 and BRCA2 are essential for the repair of double-strand DNA breaks, and alterations in these genes are a hallmark of breast and ovarian carcinomas. Other functionally related genes may also play important roles in carcinogenesis. Amplification of EMSY, a putative BRCAness gene, has been suggested to impair DNA damage repair by suppressing BRCA2 function. We employed direct repeat GFP (DR-GFP) and RAD51 foci formation assays to show that EMSY overexpression impairs the repair of damaged DNA, suggesting that EMSY belongs to the family of BRCAness proteins. We also identified a novel phospho-site at threonine 207 (T207) and demonstrated its role in EMSY-driven suppression of DNA damage repair. In vitro kinase assays established that protein kinase A (PKA) directly phosphorylates the T207 phospho-site. Immunoprecipitation experiments suggest that EMSY-driven suppression of DNA damage repair is a BRCA2-independent process. The data also suggest that EMSY amplification is a BRCAness feature, and may help to expand the population of patients who could benefit from targeted therapies that are also effective in BRCA1/2-mutant cancers.

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.

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.

New insights into PARP inhibitors' effect on cell cycle and homology-directed DNA damage repair.

In preclinical and clinical studies, olaparib and veliparib are the most represented PARP inhibitors (PARPi), which mainly target homologous DNA damage repair pathway-deficient cancer cells. Their off-target effects are not fully understood, especially with regard to cell cycle and homology-directed DNA damage repair. Our objective was to comparatively evaluate olaparib and veliparib in this context and correlate our findings with their therapeutic potential. We used a well-established direct repeat GFP (DR-GFP) reporter assay in U2OS(DR-GFP) and H1299(DR-GFP) cells and measured DNA damage repair activity upon drug treatment. Olaparib-treated U2OS(DR-GFP) cells showed a dramatic decrease in DNA damage repair versus veliparib irrespective of inhibitory potency. We demonstrate that this effect was a result of olaparib's strong effect on the cell cycle. Unlike in veliparib-treated U2OS(DR-GFP) cells, in olaparib-treated cells S-phase decreased and G(2)-phase increased sharply, indicating a G(2)-phase arrest-like state and replicative stress. This was further confirmed by upregulation of p53 and p21 and accumulation of cyclin A. Lack of the same effect in p53-null H1299(DR-GFP) cells suggested that olaparib's effect is p53 related, which was confirmed in p53-depleted U2OS(DR-GFP) and p53-null HCT116 cells. Importantly, we also demonstrate that olaparib, but not veliparib, induced a robust phosphorylation of Chk1, a crucial component of the replicative stress response pathway. Our data show olaparib and veliparib differ in their off-target effects; olaparib, unlike veliparib, mitigates DNA damage repair activity via G(2) cell-cycle arrest-like effect in a p53-dependent manner. These off-target effects may add to PARPis' anticancer properties.

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.

A novel mammalian complex containing Sin3B mitigates histone acetylation and RNA polymerase II progression within transcribed loci.

Transcription requires the progression of RNA polymerase II (RNAP II) through a permissive chromatin structure. Recent studies of Saccharomyces cerevisiae have demonstrated that the yeast Sin3 protein contributes to the restoration of the repressed chromatin structure at actively transcribed loci. Yet, the mechanisms underlying the restoration of the repressive chromatin structure at transcribed loci and its significance in gene expression have not been investigated in mammals. We report here the identification of a mammalian complex containing the corepressor Sin3B, the histone deacetylase HDAC1, Mrg15, and the PHD finger-containing Pf1 and show that this complex plays important roles in regulation of transcription. We demonstrate that this complex localizes at discrete loci approximately 1 kb downstream of the transcription start site of transcribed genes, and this localization requires both Pf1's and Mrg15's interaction with chromatin. Inactivation of this mammalian complex promotes increased RNAP II progression within transcribed regions and subsequent increased transcription. Our results define a novel mammalian complex that contributes to the regulation of transcription and point to divergent uses of the Sin3 protein homologues throughout evolution in the modulation of transcription.

Sin3B expression is required for cellular senescence and is up-regulated upon oncogenic stress.

Serial passage of primary mammalian cells or strong mitogenic signals induce a permanent exit from the cell cycle called senescence. A characteristic of senescent cells is the heterochromatinization of loci encoding pro-proliferative genes, leading to their transcriptional silencing. Senescence is thought to represent a defense mechanism against uncontrolled proliferation and cancer. Consequently, genetic alterations that allow senescence bypass are associated with susceptibility to oncogenic transformation. We show that fibroblasts genetically inactivated for the chromatin-associated Sin3B protein are refractory to replicative and oncogene-induced senescence. Conversely, overexpression of Sin3B triggers senescence and the formation of senescence-associated heterochromatic foci. Although Sin3B is strongly up-regulated upon oncogenic stress, decrease in expression of Sin3B is associated with tumor progression in vivo, suggesting that expression of Sin3B may represent a barrier against transformation. Together, these results underscore the contribution of senescence in tumor suppression and suggest that expression of chromatin modifiers is modulated at specific stages of cellular transformation. Consequently, these findings suggest that modulation of Sin3B-associated activities may represent new therapeutic opportunities for treatment of cancers.