2003 Abstracts Presented
Expression of Histone Deacetylase 1 (HDAC 1) in Ovarian Tissue
Dineo Khabele, Fung Yee Chan, Abbie L. Fields, WanCai Yang, Karen Livne, Melissa Lopez-Jones, John M. Mariadason, Gary L. Goldberg, Leonard Augenlicht. Albert Einstein College of Medicine, Albert Einstein Cancer Center & Montefiore Medical Center, New York, NY
Objective: Histone deacetylases (HDACs), together with their counterparts histone acetyltransferases, determine the net acetylation state of DNA bound core histones. By modifying the local chromatin architecture, HDACs can influence the interaction between DNA and transcription-regulatory protein complexes and play a critical role in the regulation of gene expression. Increased HDAC expression has been observed in several cancer types and HDAC inhibitors are known to induce growth arrest in cancer cells in vitro and in vivo. Our research interest is the role of HDACs in ovarian carcinogenesis. The objective of this study was to evaluate gene and protein expression of HDAC1 (a member of Class I HDACs) in malignant versus benign human ovarian tissue and its potential role in ovarian carcinogenesis.
Methods: Histologically confirmed ovarian tissue samples (n=34) were collected from women undergoing surgery (IRB approved CCI #00218). RNA Expression: Total RNA was extracted using the modified Trizol® protocol, reverse transcribed to cDNA, and amplified using quantitative real time RT-PCR (QPCR) with sequence specific primers for HDAC1 and the SYBR® Green PCR Reagents kit. Samples were run in triplicate and gene expression levels were quantified using reference RNA as a standard after normalization to GAPDH. Means and standard errors of the means were obtained. Protein Expression: A tissue microarray was created from paraffin-embedded ovarian tissue specimens that were triple spotted onto a recipient paraffin block. Immunohistochemistry was performed on slides created from the array using rabbit anti-mouse HDAC1 as the primary antibody. Staining was graded on a scale of 0 to 3+, with 0 and 1+ representing negative stain, 2 and 3+ representing positive stain.
Results: RNA Expression: Analysis of the QPCR data demonstrated a statistically significant increase of over 2-fold in HDAC1 gene expression levels in papillary serous ovarian tissue in comparison to benign ovarian tissue (p = 0.036). There was a trend towards HDAC1 overexpression (though not statistically significant) in several other malignant ovarian tumor types in comparison to benign ovarian tissue. Protein expression: Immunohistochemistry demonstrated higher HDAC1 staining in malignant (2-3+) compared to benign ovarian tissues (0-1+).
Conclusions: Based on these data, there appears to be an upregulation in HDAC1 expression at both the transcriptional and translational levels in malignant ovarian tissues compared to benign. Further study is necessary to elucidate the role of HDACs in ovarian tumorigenesis, and to determine whether HDACs are potential targets for treatment in ovarian cancer.
Multidrug Resistance 1 (MDR1) Gene Polymorphism Is Associated with Poor Prognosis of Paclitaxel-Based Chemotherapy in Patients with Ovarian Cancer
Yi-Jen Chen, Ming-Yi Chung,Su-Wen Nei, Ming-Shyen Yen , Peng-Hui Wang , Nae-Fang Twu, Heung-Tat Ng, Chiou-Chung Yuan. Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei, Taiwan
Background: Contributing to multidrug resistance of several types of tumor cells, MDR1 expression was found to be associated with poor prognosis in patients suffering from various types of cancer. Resistance to paclitaxel has been associated with the induction of the MDR1 gene, encoding p-glycoprotein. Regulation of basal expression of MDR1 and mechanisms of induction as a result of exposure to cytotoxic substances are still not completely understood. Sauer et al showed association of non-expressing or weak basal expression in mammary and ovarian carcinoma cell lines with a homozygous TT genotype at the 3435 position in exon 26. In this study, we evaluate whether MDR1 polymorphism is associated with prognosis of paclitaxel-based chemotherapy for patients with ovarian cancer.
Methods: Genotype distributions of 8 SNPs of the MDR1 gene, including T-620C, T-440C, T-129C, T1236C, G2677A/T, C3435T, A4036G, G4485A, were investigated in 94 patients with ovarian cancer. All patients received debulking surgery. Forty-eight Patients underwent six cycles of cisplatin-based chemotherapy 2-3 weeks after primary surgery. Twenty-seven Patients underwent six cycles of paclitaxel-based chemotherapy 2-3 weeks after primary surgery. There was no difference of stage, optimal or suboptimal debulking surgery, and age between two groups. Medians and life tables were computed using the product-limit estimate by the Kaplan-Meier method and the log-rank test was employed to assess statistical significance.
Results: The genotype of 8 SNPs of the MDR1 gene was not distributed differently according to age, FIGO stage, histotype, optimal or suboptimal debulking surgery, presence of ascites, or tumor grade. There was no significant difference of clinical outcome based on genotypes of the T1236C polymorphism in patients receiving cisplatin-based chemotherapy while, in the subgroup of patients who received paclitaxel-based chemotherapy, CT heterozygotes and CC homozygotes at the T1236C site showed a longer time to progression (median 18 months compared to 8 months in TT homozygotes; P=0.007) and overall survival (median 29 months compared to 26 months in TT homozygotes; P=0.009). In the multivariate analysis, only T1236C retained an independent role in disease free survival. For the C3435T SNP, there was no significant difference of clinical outcome in patients receiving cisplatin-based chemotherapy while, in the subgroup of patients who received paclitaxel-based chemotherapy, CT heterozygotes and TT homozygotes at C3435T showed a shorter time to progression (median 9 months compared to 25 months in CC homozygotes; P=0.078) and overall survival (median 26 months compared to >50% survival in CC homozygotes; P=0.024). No association between variant genotype and clinical outcome in paclitaxel-based or cisplatin-based chemotherapy was observed with the others SNPs.
Conclusions: The T1236C and C3435T SNPs of the MDR1 gene were related to paclitaxel-based, but not cisplatin-based chemotherapy. The clinical correlation of the C3435T SNP may be due to non-expression or low expression of the MDR1 gene. The association of the T1236C SNP and MDR1 gene expression level is yet to be studied. These results provide potential markers to assist prediction of clinical outcome for paclitaxel-based chemotherapy in ovarian cancer.
On The Development of a Methodology for Comprehensive Analysis of Haptoglobin Post-Translational Modification in Ovarian Cancer
Alex J. Rai, Zombor Zoltani, and Daniel W. Chan. The Johns Hopkins University School of Medicine, Dept. of Pathology, Baltimore, MD
Background: The advancements in proteomic technologies over the last 15 years, coupled with the availability of new antibody reagents, have provided new weapons in the armamentarium against cancer. Using these tools, we have optimized the separation and detection conditions for the analysis of haptoglobin (Hp) isoforms, which we have previously identified as part of a diagnostic panel useful for ovarian cancer detection (Rai et al, Archives of Pathology 126(12), 1518-1526). The haptoglobins comprise a multigene family wherein the members display allelic differences, and are also subject to heavy post-translational modification via glycosylation. As such, there are multiple forms of this protein which need to be resolved in blood samples. A comprehensive analysis of this sort requires a suitable means to visualize and detect these multiple members.
Methods: Haptoglobin gene structure has been well characterized by a number of groups over the last two decades. The protein products have also been studied, albeit to a lesser degree. These previous studies have primarily used anion exchange chromatography to resolve the different protein isoforms, but were conducted under conditions where not all forms of this protein family were resolvable. Our procedure employs 2D gel electrophoresis conducted under narrow range isoelectric focusing (IEF) gradients followed by western blot analysis using a specific, high affinity polyclonal antiserum. Our optimized conditions allow us to better spatially resolve the various members of this protein family and permit the identification of abnormal variants after a quick visual inspection.
Results: Our results demonstrate that there are at least 12 different isoforms detectable using our antiserum. Further, these can be divided into three different protein species, at molecular weights of approximately 44, 20, and 10kD. Our methodology was employed to compare sera (n=48) from normal healthy volunteers with individuals suffering from benign conditions, non-ovarian gynecological cancers, early stage ovarian cancer and late stage ovarian cancer. We find that there are several notable differences among the various groups with regard to the haptoglobin family members. The differences manifest as detectable abnormal isoelectric variants in the ovarian cancer population. They reflect altered post-translational modification of the 10 and 20 kD protein species harboring both higher and lower isoelectric points when compared to non-diseased samples.
Conclusions: Our methodology provides a viable alternative to existing methods and permits a more comprehensive analysis of this protein family, useful in comparison of normal and disease conditions. In addition, characterization of the different glycosylation patterns will provide a handle to identify the enzymes responsible for this modifying capacity. Such proteins are potential candidates as new diagnostic biomarkers for ovarian cancer.
Gemcitabine and Cisplatin Chemotherapy Demonstrates Synergistic Activity in the Treatment of Platinum Resistant Ovarian and Peritoneal Carcinoma
Devansu Tewari, Mark Hunter, Camille Falkner, Bradley J Monk. Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange, CA
Objective: To evaluate the activity of the combination of gemcitabine and cisplatin chemotherapy in women with platinum resistant ovarian and peritoneal carcinoma.
Methods: Patients were treated with the doublet regimen of gemcitabine (450 û 6 00 mg/m2) and cisplatin (30 mg/m2) on days 1 and 8 of a 21 day cycle. Dose adjustments were made based upon patient toxicity profiles. Response to the combination therapy was studied. Therapy was discontinued if either a clinical remission was obtained or progression of disease or intolerable side-effects was encountered.
Results: Twenty-three platinum resistant patients treated with the combination of gemcitabine and cisplatin were identified. Twenty-two patients with a median of four prior chemotherapy regimens including two previous platinum-based regimens were able to be evaluated for response to therapy. This included two patients who were treated with the combination therapy on three and two separate occasions, respectively, constituting twenty-five evaluable responses. The overall response rate was 68% (95% C.I. 48 - 88%) with eight (32%) complete and nine (36%) partial responses documented. The median progression-free interval was 5.9 months for responding patients with an overall progression-free interval of 3.8 months. Median survival for patients with positive responses was 15.8 months compared to 8.8 months for patients with no response to therapy. Overall survival of both groups was 11.4 months. Grade III or IV toxicity was encountered in 60% of the treatments: neutropenia Grade III (36%) and Grade IV (28%); thrombocytopenia Grade III (28%) and Grade IV (16%); anemia Grade III (12%); and peripheral neuropathy Grade III (4%). No deaths were associated with treatment although two patients with partial responses had to discontinue the regimen secondary to drug toxicity.
Conclusions: The use of gemcitabine and cisplatin chemotherapy is an active and tolerable combination in platinum resistant ovarian and peritoneal carcinoma.
This regimen warrants consideration in the overall management of patients with recurrent disease.
Effects of Intraperitoneal Hyperthermic Chemotherapy in Ovarian Cancer
Joon Mo Lee, Soo Young Hur, Jae-Hoon Kim. Department of Obstetrics and Gynecology, Kang Nam Saint Mary’s Hospital, The Catholic University of Korea, Seoul, Korea
Objectives: To describe the clinical effect of intraperitoneal hyperthermic chemotherapy (IPHC) in ovarian cancer patients.
Methods: We retrospectively reviewed 117 Stage IC-III ovarian cancer patients diagnosed at the gynecologic department of Kangnam St. Mary’s Hospital from January 1994 to January 2000. Among patients, 57 patients underwent conventional treatment (cytoreductive of tumor mass and chemotherapy) and IPHC and 60 patients (control group) underwent conventional treatment only. IPHC was performed by the protocol administering the mixture of 350 mg/m2 of carboplatin and 5,000,000 IU/m2 of interferon alpha; and maintaining the intraperitoneal temperature at 43-44°C; during operation.
Results: The 5-year survival rate in all patients was 58.6%, with 63.4% in the IPHC group and 52.8% in the control group, showing a significantly higher level in the IPHC group (P=0.0078). Especially, confined to only stage III ovarian cancer patients (n=74), it was 53.8% in IPHC group (n=35) and 33.3% in control group (n=39), showing a significantly higher rate in IPHC group (P=0.0015). Among stage III ovarian cancer patients whose tumor size reduced to less than 1 cm during secondary surgery (n=53), the 5-year survival rate was 65.6% in patients who underwent IPHC (n=26) and was 40.7% in control patients (n=27) (P=0.0046). IPHC was shown to be an independent prognostic factor not affected by surgical staging, tumor size noted after second surgery, and patient age according to the result of multivariate analysis (Hazard ratio=0.496, P=0.0176).
Conclusions: Our study suggests that IPHC would be one of novel promising treatment modalities in ovarian cancer.
Clinical Outcomes and Fertility Results after Conservative Treatment of Epithelial Ovarian Cancer: Analysis of 37 Followed-Up Patients
Philippe Morice, D Wicart-Poque, A Rey, S Camatte, P Pautier, C Pomel, C Lhomme, P Duvillard, D Castaigne. Institut Gustave Roussy, Villejuif, France
Objectives: The aim of this study is to assess and evaluate the clinical outcome and fertility in patients treated conservatively for epithelial ovarian cancer (EOC).
Methods: Data from 42 patients treated conservatively for EOC were reviewed. Thirty-seven followed-up patients with complete data were analyzed. Optimal surgical staging was performed in 2 cases during the initial surgery and in 33 patients during a reassessment surgery. Six patients underwent hysterectomy during this restaging surgery.
Results: Among 31 patients treated conservatively following the restaging surgery, the FIGO staging distribution was: 24 stage IA (grade 1 n=10; grade 2 n= 12; grade 3 n=2); 2 stage IC; 2 stage II and 3 patients with initial stage unknown. Ten patients had recurrence (8 on the remaining ovary). The disease free survival at 5 years for patients with stage IA grade 1 and 2 tumors was respectively 89% and 66%. All patients with stage > IA recurred. Only five pregnancies (4 spontaneous and 1 following IVF procedure) were obtained.
Conclusions: Conservative surgery for patients with EOC could be considered in young patient with stage IA grade 1 disease adequately staged and desiring to preserve fertility potential but should not performed in patients with FIGO stage > IA.