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Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRDchromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.

MicroRNAs (miRNAs) belong to a family of small non‐coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high‐grade serous ovarian carcinoma (HGS‐OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS‐OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR‐92a‐3p, let‐7a‐5p, SNORD61, SNORD72, SNORD68, miR‐103a‐3p, miR‐423‐3p, miR‐191‐5p, miR‐16‐5p) were analysed on a total of 75 HGS‐OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS‐OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR‐191‐5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS‐OvCa and normal controls, including the first time both the normal tissues supposed to be HGS‐OvCa progenitors. In addition, we recommend miR‐191‐5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS‐OvCa tissues.

Background Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. Methods Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. Results A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology ( p  = 0.001) and advanced FIGO stage ( p  = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. Conclusions FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.

Background The existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance. Methods We isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR. Results Spheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival. Conclusions This investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC. Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial–mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

Background Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients. Methods MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome. Results MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08–0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17–0.99, p = 0.048) had an independent prognostic value for disease-free survival. Conclusion This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.

Background Endometrial cancer is the most common gynecologic malignancy in developed countries. Trop-2 is a glycoprotein involved in cellular signal transduction and is differentially overexpressed relative to normal tissue in a variety of human adenocarcinomas, including endometrioid endometrial carcinomas (EEC). Trop-2 overexpression has been proposed as a marker for biologically aggressive tumor phenotypes. Methods Trop-2 protein expression was quantified using tissue microarrays consisting of formalin-fixed paraffin-embedded specimens from 118 patients who underwent surgical staging from 2001–9 by laparotomy for EEC. Clinicopathologic characteristics including age, stage, grade, lymphovascular space invasion, and medical comorbidities were correlated with immunostaining score. Univariate and multivariate analyses were performed for overall survival, disease-free survival, and progression-free survival in relation to clinical parameters and Trop-2 protein expression. Results Clinical outcome data were available for 103 patients. Strong Trop-2 immunostaining was significantly associated with higher tumor grade (p=0.02) and cervical involvement (p<0.01). Univariate analyses showed a significant association with reduced disease-free survival (DFS) (p=0.01), and a trend towards significance for overall and progression-free survival (p=0.06 and p=0.05, respectively). Multivariate analyses revealed Trop-2 overexpression and advanced FIGO stage to be independent prognostic factors for poor DFS (p=0.04 and p <0.001, respectively). Conclusions Trop-2 protein overexpression is significantly associated with higher tumor grade and serves as an independent prognostic factor for DFS in endometrioid endometrial cancer.

Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.

Background The aim of the present study was to investigate within ovarian carcinoma and normal ovarian biopsies the gene expression of multiple secretoglobin family members relative to mammaglobin B, which we previously reported as a promising novel ovarian carcinoma prognostic marker. Methods Using quantitative real-time Reverse Transcription PCR we tested 53 ovarian carcinoma and 30 normal ovaries for the expression of 8 genes belonging to the secretoglobin family: mammaglobin A, lipophilin A, lipophilin B, uteroglobin, HIN-1, UGRP-1, RYD5 and IIS. Next, we decided to expand the LipB gene expression analysis to a further 48 ovarian carcinoma samples, for a total of 101 tumor tissues of various histologies and to study its protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tumors and normal ovaries. Finally, we correlated lipophilin B gene and protein expression to conventional patient clinico-pathological features and outcome. Results We found significant mammaglobin A, lipophilin A, lipophilin B and RYD5 gene overexpression in ovarian carcinomas compared to normal ovaries. Lipophilin B mRNA showed a higher presence in tumors (75.4%) compared to normal ovaries (16.6%) and the most significant correlation with mammaglobin B mRNA (r s =0.77, p < 0.001). By immunohistochemical analysis, we showed higher lipophilin B expression in the cytoplasm of tumor cells compared to normal ovaries (p < 0.001). Moreover, lipophilin B gene overexpression was significantly associated with serous histology (serous vs clear cell p = 0.027; serous vs undifferentiated p = 0.007) and lower tumor grade (p = 0.02). Lower LipB mRNA levels (low versus high tertiles) were associated to a shorter progression-free (p = 0.03, HR = 2.2) and disease-free survival (p = 0.02, HR = 2.5) by univariate survival analysis and, importantly, they remain an independent prognostic marker for decreased disease-free (p = 0.001, HR = 3.9) and progression-free survival (p = 0.004, HR = 2.8) in multivariate Cox regression analysis. Conclusions The present study represents the first quantitative evaluation of secretoglobin gene expression in normal and neoplastic ovarian tissues. Our results demonstrate lipophilin B gene and protein upregulation in ovarian carcinoma compared to normal ovary. Moreover, lipophilin B gene overexpression correlates with a less aggressive tumor phenotype and represents a novel ovarian carcinoma prognostic factor.