Alain G. Zeimet

DNA Hypomethylation and Ovarian Cancer Biology

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5′ gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.

Why did p53 gene therapy fail in ovarian cancer

Promising preclinical and clinical data led to the initiation of an international randomised phase II/III trial of p53 gene-therapy trial for first-line treatment of patients with ovarian cancer. In that trial, replication-deficient adenoviral vectors carrying wild-type p53 were given intraperitoneally in combination with standard chemotherapy to patients with ovarian cancers harbouring p53 mutations. The study was closed after the first interim analysis because an adequate therapeutic benefit was not shown. In this review, we discuss the possible reasons for failure of p53 gene therapy, which include the multiple genetic changes in cancer and epigenetic dysregulations leading to aberrant silencing of genes. These complex interactions lead us to conclude that repair of single genes might not be a suitable strategy for the treatment of cancer. Moreover, dominant negative cross talk between ectopic wild-type p53 and recently identified dominant p53 mutants and splice variants of p63 and p73--which are frequently overexpressed in ovarian cancers--could seriously compromise the effectiveness of p53 gene therapy. Other substantial problems in targeting tumour cells with adenoviral vectors are the heterogeneity or lack of expression of coxsackie-adenovirus receptors and integrin co-receptors in ovarian tumours and the presence of adenovirus-neutralising antibodies in ovarian cancer-related ascites.

Transdominant ΔTAp73 Isoforms Are Frequently Up-regulated in Ovarian Cancer. Evidence for Their Role as Epigenetic p53 Inhibitors in Vivo

Despite strong homology, the roles of TP53 and TP73 in tumorigenesis seem to be fundamentally different. In contrast to TP53, tumor-associated overexpression of TP73 in many different cancers, combined with virtual absence of inactivating mutations and lack of a cancer phenotype in the TP73 null mouse are inconsistent with a suppressor function but instead support an oncogenic function. The discovery of NH2-terminally truncated p73 isoforms, collectively called ΔTAp73, is now the focus of intense interest because they act as potent transdominant inihibitors of wild-type p53 and transactivation-competent TAp73. Therefore, establishing deregulated ΔTAp73 expression in tumors could be the crucial link to decipher which of the two opposing roles of this bipolar gene is the biologically relevant one. This study is the largest to date and encompasses 100 ovarian carcinomas with complete expression profile of all NH2-terminal isoforms, discriminating between TAp73 and ΔTAp73 (ΔNp73, ΔN′p73, Ex2p73, and Ex2/3p73) by isoform-specific real-time reverse transcription-PCR. We find that the set of NH2-terminal p73 isoforms distinguishes ovarian cancer patients from healthy controls and thus is a molecular marker for this diagnosis. Ovarian cancers strongly and almost universally overexpress ΔN′p73 compared with normal tissues (95% of cancers). About one-third of tumors also exhibit concomitant up-regulation of the antagonistic TAp73, whereas only a small subgroup of tumors overexpress ΔNp73. Thus, deregulation of the E2F1-responsive P1 promoter, rather than the alternate P2 promoter, is mainly responsible for the production of transdominant p53/TAp73 antagonists in ovarian cancer. Tumor stage, grade, presence of metastases, p53 status, and residual disease after resection are significant prognostic markers for overall and recurrence-free survival. A trend is found for better overall survival in patients with low expression of ΔN′p73/ΔNp73, compared with patients with high expression. A strong correlation between deregulated ΔTAp73 and p53 status exists. p53 wild-type cancers exhibit significantly higher deregulation of ΔN′p73, ΔNp73, and Ex2/3p73 than p53 mutant cancers. This data strongly supports the hypothesis that overexpression of transdominant p73 isoforms can function as epigenetic inhibitors of p53 in vivo, thereby alleviating selection pressure for p53 mutations in tumors.

Clinical trials in recurrent ovarian cancer.

The 4th Ovarian Cancer Consensus Conference of the Gynecologic Cancer InterGroup was held in Vancouver, Canada, in June 2010. Representatives of 23 cooperative research groups studying gynecologic cancers gathered to establish international consensus on issues critical to the conduct of large randomized trials. Group C, 1 of the 3 discussion groups, examined recurrent ovarian cancer, and we report the consensus reached regarding 4 questions. These included the following: (1) What is the role of cytoreductive surgery for recurrent ovarian cancer? (2) How do we define distinct patient populations in need of specific therapeutic approaches? (3) Should end points for trials with recurrent disease vary from those of first-line trials? (4) Is CA-125 progression alone sufficient for entry/eligibility into clinical trials?

Nonplatinum Topotecan Combinations Versus Topotecan Alone for Recurrent Ovarian Cancer: Results of a Phase III Study of the North-Eastern German Society of Gynecological Oncology Ovarian Cancer Study Group

PURPOSE The management of recurrent ovarian cancer remains controversial. Single-agent topotecan is an established treatment option, and preliminary evidence suggests improved tumor control by combining topotecan with etoposide or gemcitabine. PATIENTS AND METHODS Women with relapsed ovarian cancer after primary surgery and platinum-based chemotherapy were randomly assigned to topotecan monotherapy 1.25 mg/m(2)/d, topotecan 1.0 mg/m(2) plus oral etoposide 50 mg/d, or topotecan 0.5 mg/m(2)/d plus gemcitabine 800 mg/m(2) on day 1 and 600 mg/m(2) on day 8 every 3 weeks. Patients were stratified for platinum-refractory and platinum-sensitive disease according to a recurrence-free interval of less or more than 12 months, respectively. The primary end point was overall survival. Secondary end points included progression-free survival, objective response rates, toxicity, and quality of life (as measured by the European Organisation for Research and Treatment of Cancer [EORTC] 30-item Quality-of-Life Questionnaire). RESULTS The trial enrolled 502 patients with a mean age of 60.5 years (+/- 10.2 years), 208 of whom were platinum resistant. Median overall survival was 17.2 months (95% CI, 13.5 to 21.9 months) with topotecan, 17.8 months (95% CI, 13.7 to 20.0 months) with topotecan plus etoposide (log-rank P = .7647), and 15.2 months (95% CI, 11.3 to 20.9 months) with topotecan plus gemcitabine (log-rank P = .2344). Platinum-sensitive patients lived significantly longer than platinum-refractory patients (21.9 v 10.6 months). The median progression-free survival was 7.0, 7.8, and 6.3 months, respectively. Objective response rates were 27.8%, 36.1%, and 31.6%, respectively. Patients under combined treatment were at higher risk of severe thrombocytopenia. CONCLUSION Nonplatinum topotecan combinations do not provide a survival advantage over topotecan alone in women with relapsed ovarian cancer.

Clinical Relevance of Dominant-Negative p73 Isoforms for Responsiveness to Chemotherapy and Survival in Ovarian Cancer: Evidence for a Crucial p53-p73 Cross-talk In vivo

Purpose: We aimed to determine the clinical role of the p53 family members p53 and p73 in the responsiveness to platinum-based chemotherapy and survival in ovarian cancer, considering their cross-talk and the p53 polymorphism at codon 72. Experimental Design: A detailed analysis of p53 and p73 in a series of 122 ovarian cancers was done. We used a functional yeast-based assay to determine the p53 mutational status. Red yeast colonies, indicating mutant p53, were subsequently sequenced to determine the specific p53 alteration. p53 mutations were divided into two groups according to their previous characterization in the literature: those that efficiently inhibit transcriptionally active TAp73 function and those that do not. A p53 polymorphism at codon 72 was determined in corresponding normal tissue or blood of ovarian cancer patients. Isoform-specific p73 expression analysis using real-time reverse transcription-PCR has previously been done in the majority of ovarian cancers included in this study. In a retrospective chart review, responsiveness to chemotherapy was assessed, and survival data with long follow-up times were collected. Results: Eighty of 122 (65.6%) of ovarian cancers harbored p53 mutations. p53 mutational status was an important determinant of responsiveness to platinum-based chemotherapy in all patients with a residual tumor of <2 cm in diameter after initial surgery (wild-type versus mutant, P = 0.029). In addition, p53 mutational status was a strong prognosticator for recurrence-free and overall survival (P < 0.0001 and P = 0.003, respectively) in univariate analyses. High expression levels of dominant-negative p73 isoforms (ΔNp73 and ΔN′p73) significantly correlated with chemotherapeutic failure (P = 0.048) and with worse recurrence-free and overall survival in patients with p53 mutant cancers (P = 0.048 and P = 0.005, respectively). Eight p53 mutations, present in 19 cases, were found that efficiently inhibit TAp73 (i.e., 175H, 220C, 245S, 245D, 248W, 248Q, 266E, and 273H). Patients with p53 mutations that efficiently inhibit TAp73 function had a significantly shorter overall survival than patients with p53 mutations of unknown effect on TAp73 (P = 0.044). The p53 polymorphism at codon 72 had no influence on responsiveness to chemotherapy or survival. Conclusion: We provide the first clinical evidence that dominant-negative p73 isoforms contribute to drug resistance in vivo, underscoring the importance of a p53-p73 cross-talk. NH2-terminally truncated p73 isoforms were of significant clinical effect by providing an additional unfavorable factor for response to platinum-based chemotherapy and survival in p53 mutant ovarian cancers.

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.

Significance of thrombocytosis in patients with epithelial ovarian cancer

Abstract OBJECTIVES: Our objective was to determine the relevance of a preoperative platelet count in ovarian cancer. Special interest was directed to a possible prognostic significance of thrombocytosis in this disease. STUDY DESIGN: Charts of 130 patients with epithelial ovarian cancer were reviewed retrospectively. Survival analyses were performed with the Kaplan-Meier method. Differences in survival were examined according to Mantel and Breslow. Platelet counts were furthermore correlated to other clinical parameters. For this purpose the Spearman rank test and a stepwise logistic regression model were used. Data comparison was accomplished with the Mann-Whitney U test and the χ 2 test. RESULTS: Prevalence of thrombocytosis was 38%. No differences in survival between patients with or without thrombocytosis was found. Volume of ascities and hemoglobin concentrations were independently associated with thrombocytosis (p = 0.002 and p = 0.004, respectively). CONCLUSIONS: Thrombocytosis is not a useful prognostic factor in ovarian cancer. Elevated platelets are predominately associated with the presence of ascites and marked anemia. (A M J O BSTET G YNECOL 1994;170:549-54.)

Up‐regulation of L1CAM is linked to loss of hormone receptors and E‐cadherin in aggressive subtypes of endometrial carcinomas

Endometrial carcinomas (ECs) are classified into type 1 (less aggressive) and type 2 (aggressive) tumours that differ in genetic alterations. So far, reliable immunohistochemical markers that can identify patients with high risk for recurrence are rare. We have defined the expression of L1 cell adhesion molecule (L1CAM), a biomarker previously identified for EC, and compared its expression to oestrogen receptor (ER)/progesterone receptor (PR) and E‐cadherin. We found that L1CAM was absent in normal endometrium and the vast majority of endometrioid ECs (type 1) but was strongly expressed in serous and clear‐cell ECs, considered as type 2. 78/272 cases were identified as L1CAM‐positive endometrioid ECs that were correlated with a poor prognosis. Strikingly, we observed an inverse relationship between L1CAM and ER/PR/E‐cadherin expression in all ECs. In mixed ECs, composed of endometrioid (L1CAM− ER/PR+ E‐cadherin+) and clear‐cell/serous (L1CAM+ ER/PR− E‐cadherin−), both phenotypes were co‐expressed. In some of these cases L1CAM was up‐regulated at the leading edge of the tumour, where ER/PR and E‐cadherin expression were selectively lost. In EC cell lines treated with the epithelial–mesenchymal transition (EMT) inducer TGFβ1, L1CAM and vimentin were strongly up‐regulated, while E‐cadherin expression was reduced. The treatment also resulted in an increased expression of the EMT transcription factor Slug and an enhanced cell invasion. Depletion of Slug by siRNA knockdown prevented both L1CAM up‐regulation and enhanced cell invasion. According to our analysis, we suggest that L1CAM is a novel marker for EMT in ECs and that L1CAM‐typing could identify endometrioid ECs that have type 2‐like features and are at high risk for recurrence. Copyright

Epigenetic downregulation of the retinoic acid receptor-beta2 gene in breast cancer.

A growing body of evidence supports the hypothesis that the retinoic acid receptor β2 (RAR-β2) gene is a tumor suppressor gene which induces apoptosis and that the chemopreventive and therapeutic effects of retinoids are due to induction of RAR-β2. During breast cancer progression, RAR-β2 is reduced or even lost. It is known from studies of other tumor-suppressor genes that methylation of the 5′-region is the cause of loss of expression. Several groups demonstrated that this is also true for the RAR-β2 in breast cancer by treating breast cancer cell lines with a demethylating agent and examining expression of the RAR-β2 gene in response to a challenge with retinoic acid. Studies using sodium bisulfite genomic sequencing as well as methylation specific PCR showed that a number of breast cancer cell lines as well as breast cancer tissue showed signs of methylation. The RAR-β2 gene was unmethylated in non-neoplastic breast tissue as well as in other normal tissues. A combination of retinoic acid with demethylating agents as well as with histone deacetylase inhibitors acts synergistically to inhibit growth. This review presents data that suggest that treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality. Both the time of commencement of chemoprevention and the choice of substances that are able either to prevent de novo methylation or to reverse methylation-caused gene silencing may be important considerations.