Expression and promotor hypermethylation of miR-34a in the various histological subtypes of ovarian cancer.

BACKGROUND: An increasing body of evidence shows that miR-34 family has tumor suppressive properties mediating apoptosis, cell cycle arrest and senescence. In ovarian cancer, miR34 family members were found to be under expressed. Particularly miR-34a has been revealed to be a direct transcriptional target of p53 which is frequently mutated in epithelial ovarian carcinomas especially in high grade serous cancer. Moreover, methylation of miR-34a CpG Islands was found to down-regulate miR-34a expression. The aim of this study was to investigate the clinical relevance of mir34a as well as its promoter methylation in a subset of 133 ovarian cancers with a special focus on the p53 mutation status, the dualistic type I and type II ovarian cancer model and the different histotypes. METHODS: One hundred thirty-three epithelial ovarian cancers and 8 samples of healthy ovarian surface epithelium were retrospectively analysed for miR-34a expression with quantitative real-time reverse transcription PCR (qRT-PCR). Gene-specific DNA methylation was evaluated with MethyLight technique. RESULTS: Significantly lower miR-34a expression was found in ovarian cancers than in healthy ovarian epithelium (p = 0.002). The expression of miR-34a was found lower in type II than in type I cancers (p = 0.037), in p53 mutated as compared to p53 wild type cancers (p = 0.003) and in high grade compared to in low grade cancers (p = 0.028). In multivariate COX regression model low expressing miR-34a cancers exhibited a reduced PFS (p = 0.039) and OS (p = 0.018). In serous cancers low miR-34a levels showed a worse OS confirmed also in multivariate analysis (p = 0.022). miR-34a promoter methylation was found higher in type II cancers than in type I (p = 0.006). mir34a expression and promoter methylation showed an inverse correlation in cancer samples (p = 0.05). CONCLUSION: We demonstrated a clinical independent role of miR-34a in epithelial ovarian cancers. Moreover, we corroborated the correlation between miR-34a expression and its promoter methylation in a large set of ovarian cancers. The inverse association between miR-34a expression and grading, p53 mutation status and dualistic tumor type classification, together with its prognostic relevance may underline the tumor-suppressive character of miR-34a in ovarian cancer.

Intratumoral interferon regulatory factor (IRF)-1 but not IRF-2 is of relevance in predicting patient outcome in ovarian cancer.

IRF-1 and IRF-2 expression was determined by real-time PCR in 138 ovarian cancer samples and 30 healthy ovarian biopsies and was correlated with the expression of other relevant immunologic parameters and common clinicopathologic variables. Regulation of IRF-1 and IRF-2 was evaluated by cytokine treatment of various ovarian cancer cell lines, human peritoneal mesothelial cells and ovarian surface epithelium. IRF-1 but not IRF-2 was constitutively over-expressed in 5 of 7 ovarian cancer cell lines. Both IRFs were inducible with IFN-gamma and to a lesser extent with IL-1 or TNF-alpha, but not with IL-6. Epidermal growth factor (EGF) treatment down-regulated both IRFs. In ovarian cancer samples only IRF-1, but not IRF-2 mRNA, was up-regulated when compared with healthy ovarian tissue. IRF-1 but not IRF-2 expression was significantly associated with interferon (IFN)-gamma and forkhead box P3 (FoxP3). In univariate survival analysis, strong expression of IRF-1 and IRF-2 predicted improved disease-free survival (DFS) and overall survival (OS). In Cox regression analyses, IRF-1 retained independent prognostic significance for DFS and OS and IFN-gamma for OS. In contrast to other solid tumors, IRF-2 expression cannot be regarded as a classic oncoprotein associated with poor prognosis in ovarian cancer. Of the immunologic parameters investigated, intratumoral IRF-1 expression is the most powerful independent predictor of a favorable clinical outcome.

Expression of coxsackie-adenovirus receptor is related to estrogen sensitivity in breast cancer.

This study analyzes the relationship between coxsackie-adenovirus receptor (CAR) expression (transmembrane and soluble isoforms) and hormone sensitivity in 95 breast cancers. Furthermore, prognostic significance of the expression of the various CAR isoforms was investigated. In addition, inducibility of CAR expression by estradiol and tamoxifen was assessed in various breast cancer cell lines. Expression of transmembrane CAR (hCAR) highly correlated with estrogen receptivity, but was independent of the expression of progesterone receptor (PR). Furthermore, hCAR expression was significantly higher in tumors with low-grade malignancy. However, no relationship between hCAR expression and tumor size, lymph node status, or survival was revealed. In the hormone receptor-positive breast cancer cell line T47-D expression of hCAR and its soluble isoforms was increased by treatment with estradiol and tamoxifen. In contrast, no induction of either CAR isoform was achieved in receptor-negative cell lines. Furthermore, enhancement of hCAR expression was significantly greater when cells were treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) than when treated with estradiol or tamoxifen. Moreover, sensitivity to TSA induction of hCAR was considerably greater in receptor-positive than in receptor-negative cell lines. No additive effect on CAR expression was found when TSA was combined with either estradiol or tamoxifen. In conclusion, the so far undescribed association between estrogen receptivity and the expression of hCAR in breast cancer seems to not only reflect a phenotype of low malignancy, but expression of hCAR may also be directly influenced by ER-specific ligands.

Clinical relevance of E2F family members in ovarian cancer--an evaluation in a training set of 77 patients.

PURPOSE: The major obstacle in treating ovarian cancer is the rapid development of platinum resistance during therapy. Deregulation of members of the E2F family of transcription factors is crucially involved in carcinogenesis and probably in mechanisms underlying platinum resistance. We therefore investigated the relevance of the whole set of E2F family members in predicting clinical outcome and their significance in predicting platinum resistance. EXPERIMENTAL DESIGN: Real-time PCR of all E2F family members was done from 77 ovarian carcinomas, defined as our training set, and 8 healthy control samples. The correlation with clinicopathologic characteristics, platinum resistance, and survival was investigated. Furthermore, the cross-talk of E2F family members was assessed for its value in predicting survival and platinum resistance. RESULTS: The proliferation-promoting E2F1 and E2F2 were associated with grade 3 tumors and residual disease >2 cm in diameter after initial surgery. Survival analyses showed low expression of E2F1 or E2F2 to be significantly associated with favorable disease-free and overall survival (E2F1, P = 0.039 and 0.047, respectively; E2F2, P = 0.009 and 0.006, respectively). In contrast, high expression of inhibiting E2F4 or E2F7 predicted favorable disease-free and overall survival (E2F4, P = 0.047 and 0.042, respectively; E2F7, P = 0.048 and 0.042, respectively). A high E2F2 to E2F4 ratio was the most valuable prognostic variable for disease-free survival in multivariate analysis (hazard ratio, 6.494; P = 0.002). Tumors considered platinum resistant were associated with lower E2F4 and E2F7 expression (P = 0.012 and 0.009, respectively) compared with platinum-sensitive tumors. Again, ratios of E2F1 or E2F2 to E2F7 were the most favorable variables in predicting platinum resistance. CONCLUSIONS: We here show that deregulation of both proliferation-promoting and proliferation-inhibiting E2F transcription factors and their cross-talk is crucially involved in the tumor biology of ovarian cancer and influences clinical outcome. Furthermore, down-regulation of E2F7 may contribute to mechanisms underlying platinum resistance, and calculation of ratios of proliferation-promoting E2F1 to E2F7 could serve as a putative predictor of platinum resistance.

Evaluation of Vav3.1 as prognostic marker in endometrial cancer.

PURPOSE: Vav3 is a guanine nucleotide exchange factor that regulates the activity of Rho/Rac family GTPases. In a study on ovarian cancer, we recently demonstrated pronounced prognostic and predictive value of Vav3.1, a specific truncation variant of the parental Vav3 gene. Here, we sought to investigate the role of Vav3.1 in the most prevalent gynecological tumor entity, endometrial cancer. METHODS: Vav3.1 transcript levels were determined in a large cohort of endometrial cancer patients using variant-specific PCR (n = 239), and non-malignant endometrial tissue served as control (n = 26). Expression levels of Vav3.1 were stratified according to established clinicopathological characteristics and correlated to long-term patient survival (average follow-up of > 7.5 years). Type 1 and type 2 cancers were separately investigated. RESULTS: While Vav3.1 was markedly overexpressed in endometrial cancer tissue, we could not detect associations with clinical parameters related to prognosis, such as FIGO stage and tumor grade. Kaplan-Meier estimators of different measures of survival failed to show prognostic significance of Vav3.1 in endometrial cancer. Lack of prognostic value was observed for both type 1 and type 2 cancers. CONCLUSIONS: Our study shows that Vav3.1 is not suited as a marker of cancer progression and/or treatment response in endometrial cancer. Feasibility and potential benefit of targeting Vav3.1 in endometrial cancer needs to be evaluated in future studies, proceeding from its clear, roughly ten-fold, induction in the malignant endometrium.

Heterogeneous cross-talk of E2F family members is crucially involved in growth modulatory effects of interferon-gamma and EGF.

There is growing evidence that deregulation of E2F transcription factors is causatively involved in the patho-physiology of various tumors. However, no data on the role of E2F family members in tumor biology of ovarian cancer are available. We here describe an expression study of all known E2F transcription factors and their coactivators DP-1 and DP-2 in various human ovarian cancer cell lines and the breast cancer cell line T47D and their involvement in pathways affected by interferon-gamma and EGF. A significant overexpression of the proliferation-promoting E2F1 and especially E2F2 points to a pivotal role in modulating the uncontrolled proliferation in ovarian cancer cells. Of special note is the fact that interferon-gamma treatment did not only caused a reduction of the proliferation-promoting transcription factors E2F1 and E2F2, but also increased the inhibiting transcription factors E2F4 and E2F5, thus underlining the importance of an E2F cross-talk in the anti-proliferative function of interferon-gamma. Moreover, an increase in DP-1 and E2F3 is probably involved in the proliferation-enhancing effect of EGF. Our study provides a new insight in the crucial role of E2F cross-talk, especially the role of the inhibiting transcription factors E2F4 and E2F5, in the tumor biology of cancer and its possible usefulness as targets in anti-cancer therapy.

Expression of the E2F family of transcription factors and its clinical relevance in ovarian cancer.

The E2F family of transcription factors plays a pivotal role in the regulation of cellular proliferation. On the basis of sequence homology and function, eight distinct members of E2F transcription factors (E2F-1 to E2F-8) have been distinguished to date. The regulation of E2F transcription factors is closely associated with the function of the retinoblastoma family of tumor suppressors (RB pathway). In the last decade various alterations of distinct components of the RB-E2F pathway were found to be associated with tumor progression. However, no data on the role of E2F family members are available in tumor biology of ovarian cancer. Here we describe an expression study of E2F transcription factors in various human ovarian cancer cell lines; its clinical relevance was examined in a training set of 77 ovarian cancer patients. Expression levels of E2F-1, E2F-2, and E2F-8 were elevated in all the ovarian cancer cell lines studied when compared with human peritoneal mesothelial cells (HPMCs). Interestingly, EGF treatment showed a time-dependent upregulation of the activating transcription factor E2F-3 and a simultaneous increase of DP-1, the heterodimeric partner of E2F-3. High expression of E2F-1, E2F-2, and E2F-8 was found to be associated with histopathologic grade 3 tumors and residual tumor over 2 cm in diameter after primary debulking surgery in ovarian cancer patients. Taken together, these data suggest that the proliferation-promoting E2F transcription factors E2F-1 and especially E2F-2 play a pivotal role in tumor biology of ovarian cancer and may be candidates for specific therapeutic targets.

DNA methylation in serum and tumors of cervical cancer patients.

PURPOSE: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease. EXPERIMENTAL DESIGN: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique. RESULTS: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1. CONCLUSIONS: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Analysis of aberrant DNA methylation and human papillomavirus DNA in cervicovaginal specimens to detect invasive cervical cancer and its precursors.

PURPOSE: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status. EXPERIMENTAL DESIGN: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCR-based methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions. RESULTS: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs. CONCLUSIONS: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.

Human papillomavirus DNA in sera of cervical cancer patients as tumor marker.

Persistent infection with human papillomavirus (HPV) has been widely recognized to induce carcinoma of the uterine cervix. Viral DNA has been documented to occur as tumor DNA in the circulation of patients with primary tumors caused by viral infection. The aim of this study was to evaluate the utility of serum HPV DNA as tumor marker in cervical cancer patients. Sera taken from 94 cervical cancer patients at the date of diagnosis and follow-up serum samples from 24 patients were examined for HPV DNA using PCR enzyme immunoassay. Serum samples taken at the date of diagnosis were HPV DNA positive in 45% of all investigated samples. Sera which were HPV DNA positive at the time of diagnosis became and also remained negative after primary treatment in patients without recurrence or persistent disease. HPV DNA was positive up to 423 days before the time of clinical diagnosis of recurrence in 10 out of 13 cases (median 72 days, range 0-423 days). Serum HPV DNA seems to reflect biological activity of the tumor. Our results indicate that serum HPV DNA might be a useful additional marker for early detection of recurrence in cervical cancer patients.

A DNA methylation pattern similar to normal tissue is associated with better prognosis in human cervical cancer.

Promoter hypermethylation has been recognized to play an important role in carcinogenesis. We analyzed the methylation status of 25 genes in 14 normal cervical tissue specimens and in 65 tissue specimens from cervical cancer patients using the MethyLight technique. Most of the analyzed genes have been shown to be methylated in various cancers. RB1 was never methylated in any analyzed cervical tissue. DNA methylation status of the remaining 24 genes in every tissue sample was subsequently analyzed using unsupervised agglomerative hierarchical cluster analysis to group specimens and CpG regions. We observed four clusters. All normal cervical tissue specimens were grouped together in one cluster. Neither grade, nor histological type nor age demonstrated any significant association with clusters formed. Interestingly, statistically significantly less patients whose tumor DNA methylation pattern clustered together with normal cervical tissue died within our observation period, as compared to those patients out of the three remaining clusters (P < 0.03) Cervical cancer patients, whose DNA methylation pattern clustered together with normal cervical tissue revealed a strong trend to better survival (P = 0.066) compared to patients grouped in the remaining cluster. This study shows for the first time that working solely with DNA methylation pattern a subgroup of cervical cancer patients can be defined that demonstrated strong similarity to non-neoplastic probands and had a better prognosis.

Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history.

BACKGROUND: Recent studies have revealed a possible role for the human papillomavirus (HPV) in the pathogenesis of breast cancer. In this study, patients having both a history of invasive cervical cancer and breast cancer as second primary cancer were selected for enrolment in a study of breast carcinomas for the presence of HPV. METHODS: Paraffin-embedded tissue from cervical cancer, pelvic lymph nodes, breast cancer and axillary lymph nodes of eleven patients were examined for the presence of HPV DNA using a polymerase chain reaction - enzyme immuno assay. DNA extraction was performed with the "QIAamp Tissue Kit" according to the manufacturer's instructions. Additionally, serum samples taken between diagnosis of cervical and breast cancer, were analyzed for the presence of HPV DNA to examine a possible haematogenous spread of oncogenic HPV DNA. RESULTS: All cervical carcinomas were HPV-positive. HPV DNA was detected in seven out of eleven cases in breast cancer and/or axillary lymph node tissue. Six patients had the same HPV type (HPV-16) in cervical cancer and in the corresponding breast cancer/lymph node tissue. In one case, the same HPV DNA type (HPV 16) was detected in cervical cancer, breast cancer and serum sample. CONCLUSION: These results suggest that HPV DNA might be transported from the original site of infection to the breast tissue by the bloodstream, and that it is possibly involved in the carcinogenesis of breast neoplasia in some patients.

Soluble isoforms but not the transmembrane form of coxsackie-adenovirus receptor are of clinical relevance in epithelial ovarian cancer.

The coxsackie-adenovirus receptor (hCAR) has been extensively studied in context of adenoviral-based gene therapy for cancer. However, there is strong evidence that besides its decisive role in coxsackie and adenovirus cell-entry, hCAR is a component of epithelial tight junctions and involved in cell-cell adhesions in normal and cancer cells. Furthermore, this adhesion molecule behaves like a cell surface receptor endowed with tumor suppressive properties via signal transduction. Moreover, 3 truncated soluble isoforms of hCAR were recently identified. We investigated the quantitative expression of all known CAR isoforms in a training set of 140 ovarian cancer samples and 21 controls by RT-PCR. The expression levels of the various isoforms were compared with clinicopathologic parameters and their prognostic significance was assessed. Expression levels of all CAR isoforms were elevated in ovarian carcinomas as compared with those of non-malignant controls. mRNA-expression correlated with protein levels. Moreover, expression of the soluble isoforms CAR 3/7 and CAR 4/7 but not that of hCAR was significantly increased in advanced ovarian cancer as revealed by a highly significant correlation with FIGO stage and residual disease > 2 cm in diameter after debulking surgery. High expression of CAR 3/7 and 4/7 was shown to be of independent prognostic relevance for progression-free (CAR 4/7) and overall survival (CAR 3/7 and CAR 4/7). In conclusion, soluble CAR isoforms 3/7 and 4/7 may play a pivotal role in ovarian cancer biology, possibly by counteracting migration- and growth-inhibitory properties of the membranous hCAR and thus favoring cancer cell dissemination throughout the peritoneal cavity.

CDH1 and CDH13 methylation in serum is an independent prognostic marker in cervical cancer patients.

Cervical cancer is the principal cause of death due to cancer in women. Five-year survival rate ranges from 15-80%, depending on the extent of the disease. New predictive markers for relapse may increase survival rates by improving treatment of patients at high risk for relapse. The gene products of CDH1 and CDH13, namely E-cadherin and H-cadherin, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system, caused by aberrant methylation, is a common finding in human cancers. To test the hypothesis that CDH1/CDH13 methylation is a prognostic marker in cervical cancer we determined the methylation status of CDH1/CDH13 in serum samples from 93 cervical cancer patients. Methylation analysis was carried out using MethyLight. Aberrant methylation of the 5'-region of CDH1 or CDH13 was observed in 43% (40 of 93) of the patients. Cervical cancer patients with unmethylated CDH1/CDH13 in serum samples showed significantly better disease-free survival in univariate and multivariate analysis. Median disease-free survival for CDH1/CDH13 methylation negative and positive patients was 4.3 years and 1.2 years, respectively. Our results suggest that detection of aberrant methylation of CDH1/CDH13 may be of potential use as a marker for selecting cervical cancer patients at high risk for relapse who could benefit from additional systemic therapy.

Methylation status and expression of human telomerase reverse transcriptase in ovarian and cervical cancer.

OBJECTIVES: Activation of telomerase, the enzyme that synthesizes the telomere ends of linear chromosomes, has been implicated in human cell immortalization and cancer cell pathogenesis. The expression pattern of human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is correlated with telomerase activity. The promotor region of the hTERT gene has been located in a CpG island and may therefore be regulated, at least in part, by DNA methylation. The potential for methylation-mediated regulation of hTERT gene expression in ovarian and cervical cancer tissue has not been investigated up to now. The aim of this study was to investigate the expression and methylation pattern of hTERT in ovarian and cervical cancer tissue and their correlation with clinicopathological features and outcome of the disease. METHODS: A total of 223 tissues were analyzed for hTERT methylation using MethyLight: 65 patients with cervical cancer and 124 with ovarian cancer were studied. The control group consisted of 20 normal ovarian tissues and 14 normal cervical tissues. Quantitative hTERT expression analysis was carried out in a subgroup of patients using real time PCR. RESULTS: hTERT expression was statistically significantly higher in ovarian and cervical cancer tissue in comparison to normal tissue. While methylation of hTERT in cervical cancer was significantly more frequent in comparison to normal cervical tissue, the difference between ovarian cancer and normal ovarian tissue was not significant. No correlation was detected between hypermethylation of hTERT and hTERT mRNA expression. Both ovarian cancer and normal ovary showed an increase in hTERT methylation with increasing age. hTERT expression was not correlated with prognosis, whereas cervical and ovarian cancer patients with unmethylated hTERT had significantly better overall survival. CONCLUSION: At least in some tumor entities, hTERT methylation is a function of age and is associated with a poorer outcome, irrespective of hTERT expression.