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Differential microRNA expression in cis-platinum-resistant versus -sensitive ovarian cancer cell lines
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ABSTRACTS / Gynecologic Oncology 120 (2011) S2–S133
Conclusions: Differential microarray gene expression between cancer and control groups of at-risk obese women indicate significant genetic or molecular factors potentially involved in endometrial pathogenesis that could become primary prevention targets. doi:10.1016/j.ygyno.2010.12.115
109 Differential microRNA expression in cis-platinum-resistant versus -sensitive ovarian cancer cell lines A. Sherman1, L. Hu1, C. Melton1, D. Kapp2, J. Chan1 1 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, 2Stanford University Medical Center, Stanford, CA Objective: The objective of this study was to determine the association of differential microRNA expression and cis-platinum-resistant versus -sensitive ovarian cancer. MTT assays were used to determine drug resistance in cisplatinum-resistant (A2780-CP) versus -sensitive (A2780) ovarian cancer cell lines. MicroRNA microarray was performed to identify potential targets for reversing drug resistance. Microarray results were further validated via quantitative real-time PCR (qRT-PCR). To confirm our in vitro findings, we employed The Cancer Genomic Atlas (TCGA) data portal. Using the Agilent microRNA array platform, we evaluated data from 443 patients with corresponding genomic, chemotherapy, recurrence, and outcome data. We divided patients into two groups, those who were platinum sensitive (>24-month progression-free survival [PFS]) and those who were platinum resistant (<6-month PFS), and identified differential microRNA expression. Student's t test, Kaplan–Meier survival estimates, and Cox proportional hazards models were employed for statistical analyses. Targetscan and Pictar genomic sequence analyses were used to identify gene targets of microRNAs. Results: The A2780-CP ovarian cancer cells had a 3.2-fold higher resistance to cis-platinum (IC50 5.74 vs 1.80 μM, P = 0.049). MicroRNA microarray analysis identified differentially expressed (> 2-fold) microRNAs including mir-181a, mir-10b, mir-27b, and mir-126. Specifically, mir-181a had a 3.4-fold overexpression in resistant compared with sensitive cancer cells, confirmed with qRT-PCR. Of 443 tumor specimens from TCGA data, a 1.25-fold higher expression of miR-181a was associated with a median recurrence-free survival of 23 months versus 59 months in those with lower miR-181a expression (P = 0.013). Additionally, a greater proportion of patients with high miR-181a expression recurred within 24 months (28.7% vs 18.8%, P = 0.062). Furthermore, miRanda algorithms for complementarity identified target genes associated with miR-181a and BCL2L11, a known facilitator of apoptosis. Conclusions: Our data suggest that miR-181a may be implicated in platinum-resistant serous ovarian cancer. Targeting microRNA expression may have significant promise in the treatment of drugresistant ovarian cancer. doi:10.1016/j.ygyno.2010.12.116
110 DNA methylation markers associated with serous ovarian cancer subtypes U. Chandavarkar1, M. Campan1, S. Houshdaran1, C. Pearce1, H. Shen1, M. Widschwendter2, A. Berchuck3, L. Roman4, P. Laird1 1 University of Southern California, Los Angeles, CA, 2University College, London, UK, 3Duke University Medical Center, Durham, NC, 4USC/LAC Medical Center Women and Children's Hospital, Los Angeles, CA
Objective: Ovarian cancer is the most deadly of the gynecologic malignancies. Several clinicopathologic factors have been used to prognosticate clinical outcome and select therapy, but none has provided sufficient insight into the biology of the disease. Even within a single histology, there may be variability in outcome and exposure history. DNA methylation is an epigenetic modification that consists of the addition of a methyl group to a cytosine followed by a guanine (mCpG). DNA methylation is important for gene transcription and in cancer cells, DNA methylation patterns are altered and this may lead to transcriptional changes. The purpose of this study was to identify DNA methylation-based subtypes of a single histology (serous ovarian carcinoma) and to design sensitive and specific DNA methylation markers to be tested in paraffin-embedded tissues in order to correlate the subtypes with known clinical characteristics. High-throughput quantitative DNA methylation analysis was performed using the Illumina Infinium platform for 389 serous ovarian cancer samples and 27,578 CpG dinucleotides spanning 14,495 genes. Statistical analyses identified the most biologically variant CpG sites. DNA methylation values from these selected probes and all samples were used to perform consensus k-means clustering, and cluster labels were assigned. We designed MethyLight reactions for six of the discriminant markers and tested them in 30 serous ovarian cancer samples. Results: We uncovered three novel DNA methylation-based clusters within serous ovarian cancer best categorized by a panel of biologically variant CpG probes. MethyLight reactions for five of these markers identified DNA methylation differences in 30 serous ovarian cancers. Conclusions: We have identified three unique serous ovarian cancer subtypes best distinguished by variant DNA methylation features. Further investigations will be aimed at testing the five designed MethyLight markers in tissues linked to clinical information. It is hoped that this work will provide a better understanding of the pathogenesis of serous ovarian cancer and will lead to a more individualized approach to care. doi:10.1016/j.ygyno.2010.12.117
111 Epithelial ovarian cancer tumor microenvironment is a favorable biomarker resource E. Hoskins1, B. Hood2, M. Sun2, T. Krivak1, T. Conrads3, R. Edwards1 1 Magee–Womens Hospital of UPMC, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA, 3Women's Health Integrated Research Center at Inova Health System, Annadale, VA Objective: Serum proteomics for biomarker detection has limitations: the presence of highly abundant and nonspecific proteins, which preclude detection of specific ovarian cancer biomarkers. Ascites and tissue interstitial fluid (TIF) obtained from patients affected with epithelial ovarian cancer (EOC) are an alternative source of biomarkers for ovarian cancer screening test development. We hypothesized that TIF is a superior protein-rich medium that contains ovarian cancerspecific proteins for biomarker discovery compared with ascitic fluid. EOC tumor and ascites were collected within 30 minutes of removal at the time of debulking surgery. Samples used were from four chemotherapy-naïve patients with pathologically proven papillary serous EOC. For TIF collection, we implemented a standardized sampling strategy, involving incubation of tumor tissue in saline for 1 hour at 37 °C immediately following resection. In parallel, TIF and ascitic fluid were clarified of cellular debris at 1200 rpm for 20 minutes. The supernantants of both fluids were placed at –80 °C until analysis. TIF and ascites underwent a rigorous work flow including application of the Multiple Affinity Removal System, protein quantification, one-dimensional gel
ABSTRACTS / Gynecologic Oncology 120 (2011) S2–S133
Conclusions: Differential microarray gene expression between cancer and control groups of at-risk obese women indicate significant genetic or molecular factors potentially involved in endometrial pathogenesis that could become primary prevention targets. doi:10.1016/j.ygyno.2010.12.115
109 Differential microRNA expression in cis-platinum-resistant versus -sensitive ovarian cancer cell lines A. Sherman1, L. Hu1, C. Melton1, D. Kapp2, J. Chan1 1 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, 2Stanford University Medical Center, Stanford, CA Objective: The objective of this study was to determine the association of differential microRNA expression and cis-platinum-resistant versus -sensitive ovarian cancer. MTT assays were used to determine drug resistance in cisplatinum-resistant (A2780-CP) versus -sensitive (A2780) ovarian cancer cell lines. MicroRNA microarray was performed to identify potential targets for reversing drug resistance. Microarray results were further validated via quantitative real-time PCR (qRT-PCR). To confirm our in vitro findings, we employed The Cancer Genomic Atlas (TCGA) data portal. Using the Agilent microRNA array platform, we evaluated data from 443 patients with corresponding genomic, chemotherapy, recurrence, and outcome data. We divided patients into two groups, those who were platinum sensitive (>24-month progression-free survival [PFS]) and those who were platinum resistant (<6-month PFS), and identified differential microRNA expression. Student's t test, Kaplan–Meier survival estimates, and Cox proportional hazards models were employed for statistical analyses. Targetscan and Pictar genomic sequence analyses were used to identify gene targets of microRNAs. Results: The A2780-CP ovarian cancer cells had a 3.2-fold higher resistance to cis-platinum (IC50 5.74 vs 1.80 μM, P = 0.049). MicroRNA microarray analysis identified differentially expressed (> 2-fold) microRNAs including mir-181a, mir-10b, mir-27b, and mir-126. Specifically, mir-181a had a 3.4-fold overexpression in resistant compared with sensitive cancer cells, confirmed with qRT-PCR. Of 443 tumor specimens from TCGA data, a 1.25-fold higher expression of miR-181a was associated with a median recurrence-free survival of 23 months versus 59 months in those with lower miR-181a expression (P = 0.013). Additionally, a greater proportion of patients with high miR-181a expression recurred within 24 months (28.7% vs 18.8%, P = 0.062). Furthermore, miRanda algorithms for complementarity identified target genes associated with miR-181a and BCL2L11, a known facilitator of apoptosis. Conclusions: Our data suggest that miR-181a may be implicated in platinum-resistant serous ovarian cancer. Targeting microRNA expression may have significant promise in the treatment of drugresistant ovarian cancer. doi:10.1016/j.ygyno.2010.12.116
110 DNA methylation markers associated with serous ovarian cancer subtypes U. Chandavarkar1, M. Campan1, S. Houshdaran1, C. Pearce1, H. Shen1, M. Widschwendter2, A. Berchuck3, L. Roman4, P. Laird1 1 University of Southern California, Los Angeles, CA, 2University College, London, UK, 3Duke University Medical Center, Durham, NC, 4USC/LAC Medical Center Women and Children's Hospital, Los Angeles, CA
Objective: Ovarian cancer is the most deadly of the gynecologic malignancies. Several clinicopathologic factors have been used to prognosticate clinical outcome and select therapy, but none has provided sufficient insight into the biology of the disease. Even within a single histology, there may be variability in outcome and exposure history. DNA methylation is an epigenetic modification that consists of the addition of a methyl group to a cytosine followed by a guanine (mCpG). DNA methylation is important for gene transcription and in cancer cells, DNA methylation patterns are altered and this may lead to transcriptional changes. The purpose of this study was to identify DNA methylation-based subtypes of a single histology (serous ovarian carcinoma) and to design sensitive and specific DNA methylation markers to be tested in paraffin-embedded tissues in order to correlate the subtypes with known clinical characteristics. High-throughput quantitative DNA methylation analysis was performed using the Illumina Infinium platform for 389 serous ovarian cancer samples and 27,578 CpG dinucleotides spanning 14,495 genes. Statistical analyses identified the most biologically variant CpG sites. DNA methylation values from these selected probes and all samples were used to perform consensus k-means clustering, and cluster labels were assigned. We designed MethyLight reactions for six of the discriminant markers and tested them in 30 serous ovarian cancer samples. Results: We uncovered three novel DNA methylation-based clusters within serous ovarian cancer best categorized by a panel of biologically variant CpG probes. MethyLight reactions for five of these markers identified DNA methylation differences in 30 serous ovarian cancers. Conclusions: We have identified three unique serous ovarian cancer subtypes best distinguished by variant DNA methylation features. Further investigations will be aimed at testing the five designed MethyLight markers in tissues linked to clinical information. It is hoped that this work will provide a better understanding of the pathogenesis of serous ovarian cancer and will lead to a more individualized approach to care. doi:10.1016/j.ygyno.2010.12.117
111 Epithelial ovarian cancer tumor microenvironment is a favorable biomarker resource E. Hoskins1, B. Hood2, M. Sun2, T. Krivak1, T. Conrads3, R. Edwards1 1 Magee–Womens Hospital of UPMC, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA, 3Women's Health Integrated Research Center at Inova Health System, Annadale, VA Objective: Serum proteomics for biomarker detection has limitations: the presence of highly abundant and nonspecific proteins, which preclude detection of specific ovarian cancer biomarkers. Ascites and tissue interstitial fluid (TIF) obtained from patients affected with epithelial ovarian cancer (EOC) are an alternative source of biomarkers for ovarian cancer screening test development. We hypothesized that TIF is a superior protein-rich medium that contains ovarian cancerspecific proteins for biomarker discovery compared with ascitic fluid. EOC tumor and ascites were collected within 30 minutes of removal at the time of debulking surgery. Samples used were from four chemotherapy-naïve patients with pathologically proven papillary serous EOC. For TIF collection, we implemented a standardized sampling strategy, involving incubation of tumor tissue in saline for 1 hour at 37 °C immediately following resection. In parallel, TIF and ascitic fluid were clarified of cellular debris at 1200 rpm for 20 minutes. The supernantants of both fluids were placed at –80 °C until analysis. TIF and ascites underwent a rigorous work flow including application of the Multiple Affinity Removal System, protein quantification, one-dimensional gel