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Expression patterns of the Wnt pathway inhibitor dickKOPF-3 (Dkk3) and secreted frizzled-related proteins (sFRP) 1 and 4 in endometrial endometrioid adenocarcinoma: A gynecologic oncology group study
Abstracts / Gynecologic Oncology 127 (2012) S1–S34
complex hyperplasia without atypia, 3 were simple hyperplasia with atypia, 5 were complex hyperplasia with atypia, and 11 were endometrial adenocarcinoma. STMN and pSTMN expression levels had a statistically significant difference between groups (p = 0.0156 and 0.0456 respectively). When evaluating for PTEN expression, there was no difference in PTEN copy number between groups when analyzing in comparison to STMN and pSTMN expressions (p N 0.05). However, when excluding complex hyperplasia without atypia and simple hyperplasia with atypia due to low sample size in these groups, there was a statistical difference between the remaining three groups (p = 0.046). Conclusion: Stathmin (STMN) expression could identify aggressive endometrial hyperplasia and be used as a marker for physicians to identify those patients needing increased surveillance. Further studies are warranted to determine if STMN expression is a marker for progression to endometrial carcinoma. doi:10.1016/j.ygyno.2012.07.013
Inhibition of enhancer of zeste homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines R. Eskander, T. Ji, B. Huynh, R. Wardeh, B. Hoang, L. Randall. UCI Medical Center, Orange, CA, USA. Objectives: To investigate the potential role of EZH2 on endometrial cancer cell line proliferation, migration and invasion and to examine the impact of EZH2 expression on Wnt pathway inhibitors. Methods: EZH2 expression levels were compared between the nonmalignant immortalized human endometrial cell line T-HESC, and 3 endometrial cancer cell lines, ECC-1, RL95-2 and HEC1-A using Western blot and PCR. Following confirmation of differential expression, stable EZH2 knockdown cell lines were created using RNA interference of EZH2. Appropriate clones were selected for all 3 cell lines. Cell proliferation was analyzed using MTT assays. In addition, effects on cell migration and invasion were determined. Fluorescent activated cell sorting was used to examine effects of shEZH2 on cell cycle progression. The impact of EZH2 knockdown on Wnt pathway inhibitor expression was evaluated using Western blot analysis. Lastly, EZH2 expression in endometrial cancer tissue specimens and controls was examined using immunohistochemistry. Comparison of differences between control and shEZH2 cell lines was performed using Student's t test and Fischer's exact test. Results: EZH2 protein expression was increased in all 3 endometrial cancer cell lines, and human endometrial cancer tissue specimens relative to control. RNA interference of EZH2 expression in ECC-1, RL95-2, and HEC1-A significantly decreased cell proliferation, migration and invasion. Furthermore, down regulation of EZH2 expression resulted in a significant increase in the proportion of cells arrested in the G2/M phase of the cell cycle. Furthermore, RNA interference of EZH2 expression was associated with an increase in the expression of Wnt pathway inhibitors sFRP1 and Dkk3, and a concomitant decrease in beta catenin. Western blot analysis also illustrated increased p53 expression following EZH2 interference. Lastly, EZH2 expression in human tissue samples was significantly associated with increased stage, grade, depth of invasion and nodal metastasis. Conclusions: Our results suggest that high EZH2 expression is associated with tumor cell proliferation, migration and invasion in 3 endometrial cancer cell lines. In addition, RNA interference of EZH2 was shown to increase the expression of Wnt pathway inhibitors SFRP1 and DKK3, likely impacting cell proliferation. Further investigation into this potential therapeutic target is warranted. doi:10.1016/j.ygyno.2012.07.014
S5
Expression patterns of the Wnt pathway inhibitor dickKOPF-3 (Dkk3) and secreted frizzled-related proteins (sFRP) 1 and 4 in endometrial endometrioid adenocarcinoma: A gynecologic oncology group study R. Eskandera, S. Alib, H. Lankesb, T. Dellingera, B. Hoanga, N. Ramirezc, B. Monkd, J. Walkere, E. Eisenhauerf, L. Randalla. aUCI Medical Center, Orange, CA, USA, bGOG Statistical and Data Center, Roswell Park, NY, USA, cThe Research Institute at Nationwide Children's Hospital, Columbus, OH, USA, dCreighton University School of Medicine, Phoenix, AZ, USA, eUniversity of Oklahoma, Oklahoma City, OK, USA, fOSU, Columbus, OH, USA. Objectives: This study aimed to determine the differential expression patterns of the Wnt pathway inhibitors Dkk3, sFRP1 and sFRP4 between normal Mullerian tissue and endometrial endometrioid adenocarcinoma specimens collected on GOG protocol 210. Methods: A total of 87 specimens were obtained from 60 women (60 cancer specimens with 27 of these women providing control Mullerian tissues). Given the exploratory nature of this study, Pvalues b 0.1 were considered statistically significant. Messenger RNA (mRNA) and protein levels of the Wnt pathway inhibitors Dkk3, sFRP1 and sFRP4 were evaluated by real time reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. The level of expression was dichotomized by the median expression level for each marker (less than = low; greater than or equal to = high). Associations between marker expression, clinico-pathologic features, and patient outcome were evaluated. Results: The median age for this cohort was 60 years, with a median BMI of 32 kg/m2. Eighty-one percent of patients had grade 1 or 2 tumors; 19% had grade 3 disease. Low Dkk3 expression was associated with higher tumor grade (p = 0.07), deeper myometrial or serosal invasion (p = 0.1), and obesity (p = 0.04). In addition, there was a significant down regulation of sFRP1 and sFRP4 expressions in patients with higher tumor grade (p = 0.07 and 0.03, respectively). Patients with disease recurrence had significantly lower sFRP1 and sFRP4 expression levels (p = 0.03 and 0.1, respectively). Lastly, although the study was not powered to compare patient outcome by expression level, ad hoc analysis, including Kaplan–Meier estimates, indicated a notable progression free survival advantage in patients with increased Dkk3, sFRP1 and sFRP4 expressions. Conclusions: Wnt pathway inhibitor (Dkk3, sFRP1 and sFRP4) expression was significantly down regulated in patients with high grade disease, deep myometrial invasion and disease recurrence. Furthermore, a progression free survival advantage was seen in patients with increased marker expression levels. These results are consistent with pre-clinical studies and support a potential role for Wnt targeting in endometrial cancer. Larger trials are warranted to validate these findings.
doi:10.1016/j.ygyno.2012.07.015
Utility of routine surveillance methods in detecting recurrence in high grade endometrial cancer patients J. Hunna, M. Tenneya, E. Bishopb, K. Mooreb, A. Tergasc, E. Lengyela, N. Leea, S. Yamadaa. aUniversity of Chicago Medical Center, Chicago, IL, USA, bUniversity of Oklahoma, Oklahoma City, OK, USA, cJohn Hopkins Medical Center, Baltimore, MD, USA. Objectives: To evaluate surveillance patterns and follow-up practices for women with high grade endometrial cancer and analyze the utility of pap tests, imaging studies, and tumor markers in detecting recurrence of disease and evaluating site of recurrence. Methods: This is a multi-institution retrospective chart review of women diagnosed with grade 3 endometrioid (G3), clear cell (CC),
complex hyperplasia without atypia, 3 were simple hyperplasia with atypia, 5 were complex hyperplasia with atypia, and 11 were endometrial adenocarcinoma. STMN and pSTMN expression levels had a statistically significant difference between groups (p = 0.0156 and 0.0456 respectively). When evaluating for PTEN expression, there was no difference in PTEN copy number between groups when analyzing in comparison to STMN and pSTMN expressions (p N 0.05). However, when excluding complex hyperplasia without atypia and simple hyperplasia with atypia due to low sample size in these groups, there was a statistical difference between the remaining three groups (p = 0.046). Conclusion: Stathmin (STMN) expression could identify aggressive endometrial hyperplasia and be used as a marker for physicians to identify those patients needing increased surveillance. Further studies are warranted to determine if STMN expression is a marker for progression to endometrial carcinoma. doi:10.1016/j.ygyno.2012.07.013
Inhibition of enhancer of zeste homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines R. Eskander, T. Ji, B. Huynh, R. Wardeh, B. Hoang, L. Randall. UCI Medical Center, Orange, CA, USA. Objectives: To investigate the potential role of EZH2 on endometrial cancer cell line proliferation, migration and invasion and to examine the impact of EZH2 expression on Wnt pathway inhibitors. Methods: EZH2 expression levels were compared between the nonmalignant immortalized human endometrial cell line T-HESC, and 3 endometrial cancer cell lines, ECC-1, RL95-2 and HEC1-A using Western blot and PCR. Following confirmation of differential expression, stable EZH2 knockdown cell lines were created using RNA interference of EZH2. Appropriate clones were selected for all 3 cell lines. Cell proliferation was analyzed using MTT assays. In addition, effects on cell migration and invasion were determined. Fluorescent activated cell sorting was used to examine effects of shEZH2 on cell cycle progression. The impact of EZH2 knockdown on Wnt pathway inhibitor expression was evaluated using Western blot analysis. Lastly, EZH2 expression in endometrial cancer tissue specimens and controls was examined using immunohistochemistry. Comparison of differences between control and shEZH2 cell lines was performed using Student's t test and Fischer's exact test. Results: EZH2 protein expression was increased in all 3 endometrial cancer cell lines, and human endometrial cancer tissue specimens relative to control. RNA interference of EZH2 expression in ECC-1, RL95-2, and HEC1-A significantly decreased cell proliferation, migration and invasion. Furthermore, down regulation of EZH2 expression resulted in a significant increase in the proportion of cells arrested in the G2/M phase of the cell cycle. Furthermore, RNA interference of EZH2 expression was associated with an increase in the expression of Wnt pathway inhibitors sFRP1 and Dkk3, and a concomitant decrease in beta catenin. Western blot analysis also illustrated increased p53 expression following EZH2 interference. Lastly, EZH2 expression in human tissue samples was significantly associated with increased stage, grade, depth of invasion and nodal metastasis. Conclusions: Our results suggest that high EZH2 expression is associated with tumor cell proliferation, migration and invasion in 3 endometrial cancer cell lines. In addition, RNA interference of EZH2 was shown to increase the expression of Wnt pathway inhibitors SFRP1 and DKK3, likely impacting cell proliferation. Further investigation into this potential therapeutic target is warranted. doi:10.1016/j.ygyno.2012.07.014
S5
Expression patterns of the Wnt pathway inhibitor dickKOPF-3 (Dkk3) and secreted frizzled-related proteins (sFRP) 1 and 4 in endometrial endometrioid adenocarcinoma: A gynecologic oncology group study R. Eskandera, S. Alib, H. Lankesb, T. Dellingera, B. Hoanga, N. Ramirezc, B. Monkd, J. Walkere, E. Eisenhauerf, L. Randalla. aUCI Medical Center, Orange, CA, USA, bGOG Statistical and Data Center, Roswell Park, NY, USA, cThe Research Institute at Nationwide Children's Hospital, Columbus, OH, USA, dCreighton University School of Medicine, Phoenix, AZ, USA, eUniversity of Oklahoma, Oklahoma City, OK, USA, fOSU, Columbus, OH, USA. Objectives: This study aimed to determine the differential expression patterns of the Wnt pathway inhibitors Dkk3, sFRP1 and sFRP4 between normal Mullerian tissue and endometrial endometrioid adenocarcinoma specimens collected on GOG protocol 210. Methods: A total of 87 specimens were obtained from 60 women (60 cancer specimens with 27 of these women providing control Mullerian tissues). Given the exploratory nature of this study, Pvalues b 0.1 were considered statistically significant. Messenger RNA (mRNA) and protein levels of the Wnt pathway inhibitors Dkk3, sFRP1 and sFRP4 were evaluated by real time reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. The level of expression was dichotomized by the median expression level for each marker (less than = low; greater than or equal to = high). Associations between marker expression, clinico-pathologic features, and patient outcome were evaluated. Results: The median age for this cohort was 60 years, with a median BMI of 32 kg/m2. Eighty-one percent of patients had grade 1 or 2 tumors; 19% had grade 3 disease. Low Dkk3 expression was associated with higher tumor grade (p = 0.07), deeper myometrial or serosal invasion (p = 0.1), and obesity (p = 0.04). In addition, there was a significant down regulation of sFRP1 and sFRP4 expressions in patients with higher tumor grade (p = 0.07 and 0.03, respectively). Patients with disease recurrence had significantly lower sFRP1 and sFRP4 expression levels (p = 0.03 and 0.1, respectively). Lastly, although the study was not powered to compare patient outcome by expression level, ad hoc analysis, including Kaplan–Meier estimates, indicated a notable progression free survival advantage in patients with increased Dkk3, sFRP1 and sFRP4 expressions. Conclusions: Wnt pathway inhibitor (Dkk3, sFRP1 and sFRP4) expression was significantly down regulated in patients with high grade disease, deep myometrial invasion and disease recurrence. Furthermore, a progression free survival advantage was seen in patients with increased marker expression levels. These results are consistent with pre-clinical studies and support a potential role for Wnt targeting in endometrial cancer. Larger trials are warranted to validate these findings.
doi:10.1016/j.ygyno.2012.07.015
Utility of routine surveillance methods in detecting recurrence in high grade endometrial cancer patients J. Hunna, M. Tenneya, E. Bishopb, K. Mooreb, A. Tergasc, E. Lengyela, N. Leea, S. Yamadaa. aUniversity of Chicago Medical Center, Chicago, IL, USA, bUniversity of Oklahoma, Oklahoma City, OK, USA, cJohn Hopkins Medical Center, Baltimore, MD, USA. Objectives: To evaluate surveillance patterns and follow-up practices for women with high grade endometrial cancer and analyze the utility of pap tests, imaging studies, and tumor markers in detecting recurrence of disease and evaluating site of recurrence. Methods: This is a multi-institution retrospective chart review of women diagnosed with grade 3 endometrioid (G3), clear cell (CC),