Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial-mesenchymal transition.

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.

MAL gene overexpression as a marker of high-grade serous ovarian carcinoma stem-like cells that predicts chemoresistance and poor prognosis.

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.

Identification of stably expressed reference small non-coding RNAs for microRNA quantification in high-grade serous ovarian carcinoma tissues.

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.

HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) as diagnostic tools for ovarian cancer in patients with a pelvic mass: An Italian multicenter study.

OBJECTIVE: This multicenter study aims to evaluate HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) performance in the differential diagnosis of epithelial ovarian cancer (EOC). METHODS: A total of 405 patients referred to gynecological oncologist with suspicious pelvic mass requiring a surgery for identification of EOC were consecutively enrolled; 387 patients satisfied inclusion criteria: 290 benign diseases; 15 borderline neoplasia and 82 tumors (73 EOC). RESULTS: Good diagnostic performance in discriminating benign from EOC patients was obtained for CA125, HE4 and ROMA when calculating optimal cut-off values: premenopause, specificity (SP) >86.6, sensitivity (SN) >82.6, area under the curves (AUC)≥0.894; postmenopause, SP>93.2, SN>82, AUC≥0.928. Fixing SP at 98%, performance indicators obtained for benign vs EOC patients were: premenopause, SN:65.2%, positive predictive value (+PV): 75%, positive likelihood ratio (+LR): 26.4 for CA125; SN:69.6%, +PV:76.2%, +LR:28.1 for HE4; SN:69.6%, +PV: 80%; +LR:35.1 for ROMA; postmenopause, SN:88%, +PV: 95.7%, +LR:38.7 for CA125; SN:78%, +PV:95.1%, +LR:34.3 for HE4; SN:88%, +PV:97.8%, +LR:77.4 for ROMA. When using routine cut-off thresholds, ROMA showed better well-balanced values of both SP and SN (premenopause, SN:87%, SP:86.1%; postmenopause, SN:90%; SP:94.3%). CONCLUSIONS: Overall, ROMA showed well balanced diagnostic performance to differentiate EOC from benign diseases. Meaningful differences of +PVs and +LRs between HE4 and CA125 suggest that the two markers may play at least in part different roles in EOC diagnosis, with HE4 seeming to be more efficient than CA125 in ruling in EOC patients in the disease group, also in early stages tumors, both in pre and postmenopause.

Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma.

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 NuRD-chromatin-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.

Functional characterization of epithelial ovarian cancer histotypes by drug target based protein signaling activation mapping: implications for personalized cancer therapy.

Epithelial ovarian carcinoma (EOC) is a deadly disease, with a 5-year survival of 30%. The aim of the study was to perform broad-scale protein signaling activation mapping to evaluate if EOC can be redefined based on activated protein signaling network architecture rather than histology. Tumor cells were isolated using laser capture microdissection (LCM) from 72 EOCs. Tumors were classified as serous (n = 38), endometrioid (n = 13), mixed (n = 8), clear cell (CCC; n = 7), and others (n = 6). LCM tumor cells were lysed and subjected to reverse-phase protein microarray to measure the expression/activation level of 117 protein drug targets. Unsupervised hierarchical clustering analysis was utilized to explore the overall signaling network. ANOVA was used to detect significant differences among the groups (p < 0.05). Regardless of histology, unsupervised analysis revealed five pathway-driven clusters. When the EOC histotypes were compared by ANOVA, only CCC showed a distinct signaling network, with activation of EGFR, Syk, HER2/ErbB2, and SHP2 (p = 0.0007, p = 0.0021, p < 0.0001, and p = 0.0410, respectively). The histological classification of EOC fails to adequately describe the underpinning protein signaling network. Nevertheless, CCC presents unique signaling characteristics compared to the other histotypes. EOC may need to be characterized by functional signaling activation mapping rather than pure histology.

Secretoglobin expression in ovarian carcinoma: lipophilin B gene upregulation as an independent marker of better prognosis.

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 (rs =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.

Cancer antigen 125, human epididymis 4, kallikrein 6, osteopontin and soluble mesothelin-related peptide immunocomplexed with immunoglobulin M in epithelial ovarian cancer diagnosis.

BACKGROUND: Human epididymis protein 4 (HE4), kallikrein 6 (KLK6), osteopontin (OPN) and soluble mesothelin-related peptide (SMRP) are new promising biomarkers that could integrate CA125 in epithelial ovarian cancer (EOC) diagnosis. The autoantibody response to tumor antigens is a potential tool for improving the diagnostic performances of biomarkers. The aim of this study was to assess the diagnostic potential of these biomarkers in the form of free markers and immunocomplexed with immunoglobulin M (IgM). Moreover, we analyzed the association between these markers and clinico-pathological characteristics of EOC patients. METHODS: Serum and plasma samples of 60 healthy controls, 60 ovarian benign cysts, 60 endometriosis and 60 EOCs, collected before any treatment, were tested for CICs and free antigens by immunoassays. RESULTS: Immunocomplexes were characterized by poor sensitivity and specificity, since they allowed the detection only of a small number of EOC patients and were increased in patients with benign gynecological pathologies. However, the markers in the form of free antigens showed good diagnostic performances. Of note, CA125 and HE4 showed high sensitivity in the detection of the malignancy and HE4 emerged as a useful biomarker in differential diagnosis between EOC and endometriosis. Finally, elevated KLK6 and OPN, were associated with advanced FIGO stage, high grade disease, suboptimally debulked tumor and ascites. CONCLUSIONS: This study confirms the diagnostic role of CA125, HE4, KLK6, OPN and SMRP, and for the first time showed that CA125, HE4, KLK6, OPN and SMRP immunocomplexed with IgM are not a potential tool for EOC diagnosis.

Human epididymis protein 4 as a serum marker for diagnosis of endometrial carcinoma and prediction of clinical outcome.

BACKGROUND: The purpose of this study was to assess the diagnostic and prognostic impact of preoperative serum determination of human epididymis protein 4 (sHE4), and to investigate its potential correlation with clinicopathological features and survival endpoints in endometrial cancer patients. METHODS: Preoperative serum samples from 193 endometrial cancer patients and 125 women with normal endometrium were measured for sHE4 and serum CA125 (sCA125) concentrations by quantitative chemiluminescent microparticle immunoassays on the automated Architect instrument. RESULTS: sHE4 concentrations were significantly higher in endometrial cancer patients regardless of tumour stage and grade compared with normal controls. Setting the specificity at 95 % , the sensitivities in detecting endometrial cancer patients were 66 % for HE4, 33 % for CA125 and 64 % for the combination of the two markers. High concentrations of both HE4 and CA125 significantly correlated with all clinicopathological features characterising a more aggressive tumour phenotype.In multivariate analysis, only high preoperative sHE4 concentrations, but not sCA125, were independent prognostic factors for shorter Overall Survival, Disease-Free Survival and Progression-Free Survival. CONCLUSIONS: HE4 is more sensitive and specifi c than CA125in distinguishing endometrial cancer patients from women with normal endometrium, regardless of tumour stage and grade. sHE4 appears to be associated with a more aggressive tumour variant and it could be clinically useful, in identifying high-risk endometrial cancer patients, for a tailored surgical and postoperative therapy. HE4 significant correlation with decreased Overall Survival, Disease Free Survival and Progression Free Survival suggests its potential role as a novel prognostic marker for endometrial cancer.

Trop-2 protein overexpression is an independent marker for predicting disease recurrence in endometrioid endometrial carcinoma.

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.

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence.

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.

Development and characterization of a human single-chain antibody fragment against claudin-3: a novel therapeutic target in ovarian and uterine carcinomas.

OBJECTIVE: The purpose of this study was to develop and characterize a human antibody in a single-chain antibody fragment format (scFv) that is directed specifically against claudin-3 (CLDN3). STUDY DESIGN: The synthetic ETH-2 Gold human antibody phage display library was used to select scFv specific against CLDN3. scFv binding properties were analyzed by surface plasmon resonance; specificity was confirmed with enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry on a panel of ovarian and uterine serous carcinoma cell lines. RESULTS: Surface plasmon resonance studies indicated scFv H6 to be the clone with the highest affinity against CLDN3 (K(D) of 23.60 nmol/L). scFv H6 efficiently stained CLDN3-expressing cells and recognized its epitope in enzyme-linked immunosorbent assay that was performed with uterine serous papillary carcinoma native protein extract, which suggested that a conformational epitope is recognized by this antibody. Cell surface immunofluorescence with laser scanning confocal microscopy confirmed the specific binding to the native membrane CLDN3. CONCLUSION: scFv H6 may represent a novel antitumor agent against chemotherapy-resistant ovarian and serous papillary carcinomas and other human malignancies that overexpress CLDN3.

Human kallikrein 5: an interesting novel biomarker in ovarian cancer patients that elicits humoral response.

INTRODUCTION: Kallikrein-related peptidases are secreted serine proteases that exert stimulatory or inhibitory effects on tumor progression. A recent study demonstrated that kallikrein-related peptidase 5 (KLK5) concentration is elevated in serum of patients with ovarian carcinoma. At the moment, the presence of KLK5 in other ovarian pathological lesions is not clearly determined. Moreover, the possibility of a spontaneous humoral immune response to KLK5 has not been studied yet. METHODS: In this study, we examined KLK5 levels and antibody (IgG and IgM) response to KLK5 in the serum of 50 healthy women, 50 patients with benign pelvic masses, 17 patients with ovarian borderline tumors, and 50 patients with ovarian carcinomas, using 3 enzyme-linked immunosorbent assay tests available in-house. RESULTS: At 95% specificity on healthy controls, 52% of patients with ovarian carcinoma showed high serum KLK5 (sKLK5) levels, whereas patients with benign pathological lesions or borderline tumors showed almost undetectable sKLK5 levels. Moreover, sKLK5 levels were positively associated to International Federation of Gynaecologists and Obstetricians stage suggesting a possible role of sKLK5 in ovarian cancer progression. Our results about humoral response showed elevated levels of KLK5-specific antibodies in 20% of patients with benign masses, 26% of patients with borderline tumors, and 36% of patients with ovarian carcinomas when compared with healthy controls. Interestingly, KLK5 antibodies were also found in patients with undetectable sKLK5 levels. CONCLUSIONS: In conclusion, our results showed that KLK5 is a potential new biomarker to be used in combination with other biomarkers for ovarian cancer detection. Moreover, the existence of KLK5 antibodies suggests that KLK5 might represent a possible target for immune-based therapies.

Overexpression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in uterine carcinosarcomas.

PURPOSE: To evaluate the expression levels of claudin-3 and claudin-4, the low- and high-affinity receptors, respectively, for the cytotoxic Clostridium perfringens enterotoxin (CPE) in uterine carcinosarcomas and explore the potential for targeting these receptors in the treatment of this aggressive uterine tumor. EXPERIMENTAL DESIGN: We analyzed claudin-3 and claudin-4 receptor expression at mRNA and protein levels in flash frozen and formalin-fixed, paraffin-embedded carcinosarcoma specimens. Recombinant CPE was used as a novel therapy against chemotherapy-resistant carcinosarcoma cell lines in vitro. The therapeutic effect of sublethal doses of CPE was studied in severe combined immunodeficient mouse xenografts harboring large s.c. carcinosarcomas. RESULTS: All flash-frozen carcinosarcoma biopsies (12 of 12) and short-term carcinosarcoma cell lines evaluated overexpressed claudin-3 and claudin-4 by quantitative reverse transcription-PCR. Membranous immunoreactivity for claudin-4 protein expression was documented in 80% (20 of 25) of primary tumors and 100% (6 of 6) of the metastatic carcinosarcomas, whereas negligible staining was found in normal endometrial cells. Regardless of their resistance to chemotherapeutic agents, all short-term carcinosarcoma cell lines tested died within 1 h of exposure to 3.3 microg/mL of CPE in vitro. Intratumoral injections of well-tolerated doses of CPE in large s.c. carcinosarcoma xenografts led to large areas of tumor cell necrosis and tumor disappearance in all treated animals. CONCLUSIONS: Claudin-3 and claudin-4 receptors are highly overexpressed in carcinosarcoma. These proteins may offer promising targets for the use of CPE as a novel type-specific therapy against this biologically aggressive variant of endometrial cancer.

Gene expression profile of ovarian serous papillary carcinomas: identification of metastasis-associated genes.

OBJECTIVE: The purpose of this study was to identify genes that are highly differentially expressed in metastatic serous papillary ovarian tumors (MET) when compared with primary ovarian serous carcinomas (OSPC). STUDY DESIGN: An oligonucleotide microarray with probe sets complementary to >14,500 human genes was used to determine whether patterns of gene expression may differentiate OSPC from MET in 31 snap-frozen serous papillary ovarian carcinomas (ie, 14 primary OSPC and 17 omental metastasis [MET]). RESULTS: Hierarchic cluster analysis of gene expression in OSPC and MET identified 156 genes that exhibited > 2-fold differences (P < .05) and that distinguished OSPC from MET. A number of invasion and metastasis predictive genes (including plasminogen activator; matrix metalloproteinase; matrix structural constituent genes encoding products with collagen, heparin, and hyaluronic acid binding activity; genes encoding receptors for insulin-like growth factors; vascular endothelial growth factor; endothelin type A; fibroblast growth factor; thrombospondin 1 and 2; type A and B integrins, and chemokines [stromal cell-derived factor 1 (CXCL12)]) were found among the 120 genes that were highly differentially overexpressed in MET, when compared with OSPC. Down-regulated genes in MET compared with OSPC included hepsin and testisin. Overexpression of CXCL12, matrix metalloproteinase, plasminogen activator, type A and B integrins, and hepsin genes was validated by quantitative real-time polymerase chain reaction in all samples. Finally, overexpression of CXCL12 in MET, when compared with OSPC, was validated at the protein level by immunohistochemistry. CONCLUSION: Gene expression profiling may differentiate metastatic ovarian cancer from primary OSPC. The identification of metastasis-associated genes may provide a foundation for the development of new type-specific diagnostic strategies and treatment for metastatic ovarian cancer.

Epidermal growth factor receptor detection in serum and saliva as a diagnostic and prognostic tool in oral cancer.

OBJECTIVES/HYPOTHESIS: Epidermal growth factor receptor (EGFR) is a type I transmembrane glycoprotein that is overexpressed in a wide variety of malignancies, including oral squamous cell carcinoma (OSCC). Our objective was to assess the EGFR diagnostic and prognostic value in OSCC by investigating its expression in serum and saliva of patients in comparison with healthy subjects. STUDY DESIGN: Prospective case-control study. METHODS: Serum and saliva samples were collected from a cohort of 63 treatment-naïve OSCC patients before surgery and a matched group of 60 healthy subjects. EGFR concentrations in serum and saliva were quantified by an enzyme-linked immunosorbent assay. RESULTS: OSCC patients showed lower values of serum EGFR compared with controls (P = .0002). Conversely, saliva EGFR concentrations were higher in OSCC patients than in controls (P = .0014). Saliva EGFR levels were also increased in patients with higher T category (pT4 vs. pT <4, median 6.0 vs. 3.8 ng/mL, P = .02). Considering 9.2 ng/mL (fourth quartile) as the cutoff value, patients with higher levels of saliva EGFR had worse prognosis in terms of overall survival (P = .04). Conversely, no association was found between serum EGFR and clinical outcomes in OSCC patients. CONCLUSIONS: Saliva EGFR can be considered as a potential tumor marker for OSCC with both diagnostic and prognostic values. Serum EGFR levels, on the other hand, were lower in OSCC patients, but did not show any prognostic impact. Saliva EGFR levels are worthy of further investigation as a potential diagnostic and prognostic biomarker for OSCC. LEVEL OF EVIDENCE: 3b. Laryngoscope, 127:E408-E414, 2017.

FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients.

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.

Overexpression of kallikrein 10 (hK10) in uterine serous papillary carcinomas.

OBJECTIVE: Kallikrein 10 is a secreted serine protease recently implicated in the growth and invasion of several human tumors. The goal of this study was to investigate the expression and secretion levels in vitro and in vivo of kallikrein 10 in uterine serous papillary carcinoma, a highly aggressive variant of endometrial tumor. STUDY DESIGN: Human kallikrein 10 gene expression levels were evaluated in 11 snap-frozen uterine serous papillary carcinoma biopsies and 6 normal endometrial cell biopsies by real-time polymerase chain reaction. Secretion of kallikrein 10 protein by 10 primary tumor cultures including 3 uterine serous papillary carcinomas, 2 endometrioid carcinomas, and 5 ovarian serous papillary tumors was measured using a sensitive ELISA. Finally, kallikrein 10 concentration in 75 serum and plasma samples from 22 healthy women, 20 women with benign diseases, 21 women with endometrioid carcinomas, and 12 uterine serous papillary carcinoma patients was studied. RESULTS: Kallikrein 10 gene expression levels were significantly higher in uterine serous papillary carcinoma when compared with normal endometrial cell biopsies (mean copy number by real time polymerase chain reaction = 743 versus 1.4; uterine serous papillary carcinoma versus endometrioid carcinoma: P < .02). In vitro kallikrein 10 secretion was detected in all primary uterine serous papillary carcinoma cell lines tested (mean = 2.7 microg/L), and the secretion levels were not significantly different to those found in primary ovarian serous papillary tumor cultures (mean 4.2 microg/L). In contrast, no kallikrein 10 secretion was detectable in primary endometrioid carcinomas. Kallikrein 10 serum and plasma concentrations (microg/L; mean +/- SEM) among normal healthy females (0.6 +/- 0.04), patients with benign diseases (0.6 +/- 0.06), and patients with endometrioid carcinomas (0.7 +/- 0.06) were not significantly different. In contrast, serum and plasma kallikrein 10 values in uterine serous papillary carcinoma patients (1.2 +/- 0.1) were significantly higher than those in the non-cancer group (P = .002), benign group (P = .002), and endometrioid carcinoma patients (P = .005). CONCLUSION: Kallikrein 10 is highly expressed in uterine serous papillary carcinoma, and it is released in the plasma and serum of uterine serous papillary carcinoma patients. Kallikrein 10 may represent a novel biomarker for uterine serous papillary carcinoma.

The HIV-protease inhibitor saquinavir reduces proliferation, invasion and clonogenicity in cervical cancer cell lines.

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.

Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery.

Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients.