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.

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.

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.

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.

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.

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.

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.

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.

Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

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.

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.

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.

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.

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.

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.

Prognostic significance of vascular endothelial growth factor serum determination in women with ovarian cancer.

Introduction. We performed a review of the literature to elucidate the potential prognostic significance of serum vascular endothelial growth factor (sVEGF) levels in ovarian cancer. Methods. Eligible studies in English and Italian were identified in MEDLINE/PubMed from VEGF discovery to October 2011. All studies evaluating: (i) sVEGF levels before any surgical and chemotherapeutic treatment; (ii) the association between sVEGF levels and the established prognostic variables; (iii) the value of sVEGF levels in predicting patients' outcomes, were selected for this review. Results. The search resulted in 758 titles. Nine studies met the inclusion criteria. A statistically significant association between the level of sVEGF and FIGO stage, tumour grade, residual tumour size, lymph node involvement, and presence of ascites was found in at least one study. sVEGF, in comparison with the established prognostic factors, appears to be the best prognostic marker for overall survival, since it stands out as an independent prognostic factor in most of the studies considered. Moreover, sVEGF levels were shown to be independent prognostic factors by 2 out of the 3 studies that considered DFS as an end point. Conclusion. High levels of sVEGF identify a subgroup of patients with higher risk of death and/or recurrence. These patients should be eligible for individually tailored therapeutic interventions.

Serum human epididymis protein 4 and risk for ovarian malignancy algorithm as new diagnostic and prognostic tools for epithelial ovarian cancer management.

BACKGROUND: The aim of this work was to analyze the diagnostic and prognostic value of serum human epididymis protein 4 (HE4) and Risk for Ovarian Malignancy Algorithm (ROMA) in epithelial ovarian cancer (EOC). METHODS: Preoperative serum samples of 419 women (140 healthy controls, 131 ovarian benign cysts, 34 endometriosis, and 114 EOC) were tested for CA125 and HE4 using fully automated methods (Abbott ARCHITECT) and validated cutoff values. RESULTS: For the discrimination of benign masses from EOC, in premenopausal women, the sensitivity and specificity were 92.3% and 59.4% for CA125, 84.6% and 94.2% for HE4, and 84.6% and 81.2% for ROMA, whereas in postmenopausal women, the sensitivity and specificity were 94.3% and 82.3% for CA125, 78.2% and 99.0% for HE4, and 93.1% and 84.4% for ROMA. In patients with EOC, elevated CA125, HE4, and ROMA levels were associated with advanced Federation of Gynaecologists and Obstetricians (FIGO) stage, suboptimally debulking, ascites, positive cytology, lymph node involvement, and advanced age (all P ≤ 0.05). Elevated HE4 and ROMA (both P ≤ 0.01), but not CA125 (P = 0.0579), were associated with undifferentiated tumors. In multivariable analysis, elevated HE4 and ROMA (all P ≤ 0.05) were independent prognostic factors for shorter overall, disease-free, and progression-free survival. CONCLUSIONS AND IMPACT: This study underlines the high specificity of HE4 in discriminating endometriosis and ovarian benign cysts from EOC and the high sensitivity of CA125 in detecting EOC. We showed HE4 and ROMA as independent prognostic factors. Multicenter studies are needed to draw firm conclusions about the applicability of HE4 and ROMA in clinical practice.

HE4 and epithelial ovarian cancer: comparison and clinical evaluation of two immunoassays and a combination algorithm.

BACKGROUND: Two commercial immunoassays for HE4 have been compared and the diagnostic accuracy of HE4, CA 125 and the combinatory ROMA algorithm for epithelial ovarian cancer (EOC) has been evaluated. METHODS: HE4 and CA125 were measured on sera obtained from 259 women (73 healthy, 90 with benign ovarian or adnexal diseases, 96 with EOC). The ARCHITECT CMIA HE4 assay was compared with the Fujirebio EIA HE4, and the risk for EOC by the combinatory ROMA algorithm (HE4+CA 125) was assessed with both HE4 assays. RESULTS: The CMIA HE4 assay showed a good linearity (r>0.9998) and precision (interassay and total CVs <4%). The correlation with EIA HE4 was linear (r=0.994), with an average bias of 0.4%. By ROC curve analysis, the sensitivity for EOC at a fixed specificity of 90%, 95% and 99% was 89.6%, 84.4% and 79.2% by CMIA HE4, 84.4%, 83.3% and 79.2% by EIA HE4, 86.5%, 76.0% and 59.4% by CMIA CA125. The accuracy of the ROMA algorithm determined by CMIA or EIA HE4 was very similar (AUC 87.1% vs. 87.6%; p=n.s.) and greater in menopause. CONCLUSIONS: The two HE4 assays showed a good correlation and similar clinical value, with a greater precision for CMIA. HE4 was more specific and accurate than CA125, supporting its use in addition to clinical and imaging criteria for the discrimination of benign from malignant ovarian lesions. The ROMA algorithm showed a good accuracy for discriminating women at high risk for EOC.

Trop-2 overexpression as an independent marker for poor overall survival in ovarian carcinoma patients.

BACKGROUND: Prognostic factors currently available are insufficient to predict the clinical course of epithelial ovarian cancer (EOC). In a previous microarray study we identified the human trophoblast cell surface antigen Trop-2 as one of the top differentially expressed genes in serous papillary EOCs compared to normal human ovarian surface epithelial (HOSE) short-term cultures. The aim of the present investigation was to analyse Trop-2 expression at mRNA and protein level and to assess its prognostic significance in EOC. METHODS: Using quantitative real-time PCR we tested a total of 104 fresh-frozen EOC tissues and 24 HOSE for Trop-2 mRNA expression. Trop-2 protein expression was then examined by immunohistochemistry in matched formalin-fixed paraffin-embedded EOC samples and in 13 normal ovaries. Finally, we correlated Trop-2 expression to EOC conventional clinicopathological features and patient outcomes. RESULTS: We found a significant Trop-2 mRNA and protein upregulation in EOCs compared to normal controls (p<0.001). Trop-2 protein overexpression was significantly associated with the presence of ascites (p=0.04) and lymph node metastases (p=0.04). By univariate survival analysis, Trop-2 protein overexpression was significantly associated with decreased progression-free (p=0.02) and overall survival (p=0.01). Importantly, Trop-2 protein overexpression was an independent prognostic marker for shortened survival time in multivariate Cox regression analysis (p=0.04, HR=2.35, CI(95%)=1.03-5.34). CONCLUSIONS: Our results indicate, for the first time, that Trop-2 protein overexpression correlates with an aggressive malignant phenotype and may constitute a novel prognostic factor for EOC. The targeting of Trop-2 overexpression by immunotherapeutic strategies may represent an attractive and potentially effective approach in patients harbouring EOC.

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.