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Human leukocyte antigen polymorphisms predict NY-ESO-1 tumor antigen serology and overall survival in epithelial ovarian cancer
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Abstracts / Gynecologic Oncology 130 (2013) e1–e169
methylation in ovarian cancer initiation cells and tested its potential as a prognostic biomarker. Methods: We compared the methylomic profiles between an ovarian cancer-initiating cell line (CP70sps) and its parental line (CP70) by bead arrays (Illumina). Quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR), quantitative methylation-specific PCR (QMSP), and pyrosequencing were used for the validation of gene expressions and DNA methylation in cell lines and clinical samples. We assessed the clinicopathologic correlation of potential DNA methylation sequences as markers. Results: We identified gene H as a potential candidate. Methylation status of H gene by QMSP was tested in 113 ovarian cancer tissues, which revealed an independent prognostic significance of H gene methylation for progression-free survival (PFS) and overall survival (OS). Kaplan-Meier analysis and multivariate Cox regression analysis showed that low levels of H gene methylation conferred a poor PFS (HR 5.02, 95% CI 1.19-21.27, P b 0.05) and OS (HR 7.85, 95% CI 1.0757.63, P b 0.05), which suggests the existence of a more stemlike phenotype defined by H gene with reduced therapeutic responses. Conclusions: Our data demonstrate that the methylation of H gene is an independent prognostic biomarker for ovarian cancer patients. H gene may provide a novel therapeutic target for ovarian cancer. doi:10.1016/j.ygyno.2013.04.381
323 Human leukocyte antigen polymorphisms predict NY-ESO-1 tumor antigen serology and overall survival in epithelial ovarian cancer S. Daudi, M. Brady, A. Miliotto, J. Matsuzaki, S. Lele, K. Odunsi. Roswell Park Cancer Institute, Buffalo, NY. Objective: The generation of antitumor immune responses, spontaneously or by vaccination, determines the outcome of patients with epithelial ovarian cancer (EOC). HLA genes are crucial to tumor surveillance, whereby tumor antigens are presented by Human leukocyte antigen (HLA) class I and II molecules to T cells, resulting in an orchestrated cellular and humoral immunity. NY-ESO-1 (ESO), a highly immunogenic member of the “cancer testes” antigen family, elicits spontaneous immune responses in a subset of patients with EOC. We hypothesized that the generation of spontaneous immunity to ESO and the clinical outcome of EOC patients are determined by HLA genetic polymorphisms. Methods: A total of 116 patients with ESO-expressing tumors, as determined by immunohistochemistry, were included in this study. Matched serum samples were assessed by enzyme-linked immunosorbent assay for the ESO antibody. Low-resolution HLA typing was performed using sequence-specific polymerase chain reaction. The relative odds of the ESO serologic response correlating with HLA type was analyzed using a multivariate logistic model. A proportional hazards model was used to assess the association between HLA allele groups and survival. Results: ESO humoral immunity was detected in 47% (54/116) and was absent in 53% (62/116) of EOC patients. The HLA class II alleles, DRB1*13 and DRB4*01, were significantly more frequent in ESO-seropositive patients (70% [16/23] and 63% [19/30], respectively) than in ESOseronegative patients (30% [7/23] and 37% [11/30], respectively) (odds ratio [OR] 0.223, 95% CI 0.08-0.619, P = 0.008 and OR 0.293, 95% CI 0.119-0.722, P = 0.004, respectively). HLA class I allele frequencies were similar in seropositive and seronegative patients. Improved survival was observed in patients with the A*01 subtype as compared to other HLA-A subtypes (HR 0.491, 95% CI 0.226-0.832). Conclusions: HLA polymorphisms are associated with antitumor immunity and may influence clinical outcomes in EOC. The HLA-
DRB1*13 and HLA-DRB4*01 alleles are significantly associated with the development of humoral immunity to ESO but not necessarily with survival in EOC patients, most likely as a result of immunologic tolerance. Although a relationship between the HLA-A*01 subtype and survival was seen, the underlying mechanism(s) will need to be determined. This study supports innovative vaccination strategies targeting HLA class I and II ESO epitopes, portending a broad immunologic response.
doi:10.1016/j.ygyno.2013.04.382
324 Dual mTOR inhibition demonstrates antiproliferative and chemosensitizing effects in chemoresistant high-grade papillary serous ovarian cancer F. Musa, I. Giuroiu, S. Blank, S. Korets, J. Curtin, R. Schneider. New York University Medical Center, New York, NY. Objective: To establish the effect of small-molecule mTOR inhibitors on the tumorigenesis of high-grade papillary serous ovarian cancer (OVCA) and to compare the effect of mTOR complex 1 inhibition (mTORiC1) vs. dual mTOR complex 1 and 2 inhibition (mTORCi1 + 2) with and without carboplatin (CPP) in an in vitro model of OVCA. Methods: Clonogenic assays of NIH:OVCAR3 were performed. Treatment conditions included increasing doses of RAD001 (mTORiC1), PP242 (mTORiC1 + 2), carboplatin (CPP; DNA damaging agent), and the combination of PP242 + CPP. Colonies were stained with crystal violet and analyzed using ImageJ64. Cell lysate immunoblots using commercial antibodies probed for key proteins in the mTOR (S6 and pS6, BP1 and p-BP1, mTOR and p-mTOR, Akt and p-Akt) and DNA repair pathways (CHK1 and p-CHK1, CHK2 and p-CHK2, ATM and p-ATM, ATR and p-ATR, p-BRCA1 and others). Colony counts (counts) and average colony diameter (ACD) were compared by ANOVA, with results expressed as mean ± systematic error. Post hoc analyses allowed pair-wise comparisons. Results: One-way ANOVA was significant across treatment arms (F = 17.718, P = .002). PP242-treated cells had a significantly smaller ACD compared to CTL cells (30.5 ± 3.5 vs. 131.5 ± 23.5) and CPPtreated cells (115.5 ± 1.5) (Tukey standard deviation [SD] P = 0.004 and 0.009). Addition of CPP to PP242-treated cells further decreased the ACD (27 ± 4) compared to CTL or compared to CPP alone (Tukey SD P = 0.003 and 0.008). Counts in CPP + PP242-treated cells were decreased vs. CTL (56 ± 9 vs. 129 ± 27) (Tukey SD P = .02). RAD001 (20 nM-5 uM) did not alter counts or ACD when compared to CPP or CTL. Immunoblots of cells treated with PP242 and high-dose RAD001 revealed characteristic inhibition of the mTOR pathway (decreased pmTOR, p-S6, and p-BP1), which was not observed with CPP. Furthermore, PP242 effectively reduced p-Akt (due to mTOR complex 2 inhibition) compared to RAD001 and CPP. Total CHK1 and p-CHK1 were reduced in cells treated with PP242 but not in cells treated with RAD001 or CPP alone. Conclusions: mTOR regulates cap-dependent mRNA translation (CDT), which is upregulated in OVCA. mRNAs with long 5’ untranslated regions, such as CHK1 and other survival mRNAs, have a greater requirement for CDT and are highly sensitive to mTORiC1 + 2. We showed that PP242 has compelling antiproliferative and chemosensitizing effects in OVCA and is superior to RAD001. Inhibition of CHK1 by dual mTOR inhibitors has not yet been investigated in the clinical setting but is likely to be an effective target in control of advanced disease.
doi:10.1016/j.ygyno.2013.04.383
Abstracts / Gynecologic Oncology 130 (2013) e1–e169
methylation in ovarian cancer initiation cells and tested its potential as a prognostic biomarker. Methods: We compared the methylomic profiles between an ovarian cancer-initiating cell line (CP70sps) and its parental line (CP70) by bead arrays (Illumina). Quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR), quantitative methylation-specific PCR (QMSP), and pyrosequencing were used for the validation of gene expressions and DNA methylation in cell lines and clinical samples. We assessed the clinicopathologic correlation of potential DNA methylation sequences as markers. Results: We identified gene H as a potential candidate. Methylation status of H gene by QMSP was tested in 113 ovarian cancer tissues, which revealed an independent prognostic significance of H gene methylation for progression-free survival (PFS) and overall survival (OS). Kaplan-Meier analysis and multivariate Cox regression analysis showed that low levels of H gene methylation conferred a poor PFS (HR 5.02, 95% CI 1.19-21.27, P b 0.05) and OS (HR 7.85, 95% CI 1.0757.63, P b 0.05), which suggests the existence of a more stemlike phenotype defined by H gene with reduced therapeutic responses. Conclusions: Our data demonstrate that the methylation of H gene is an independent prognostic biomarker for ovarian cancer patients. H gene may provide a novel therapeutic target for ovarian cancer. doi:10.1016/j.ygyno.2013.04.381
323 Human leukocyte antigen polymorphisms predict NY-ESO-1 tumor antigen serology and overall survival in epithelial ovarian cancer S. Daudi, M. Brady, A. Miliotto, J. Matsuzaki, S. Lele, K. Odunsi. Roswell Park Cancer Institute, Buffalo, NY. Objective: The generation of antitumor immune responses, spontaneously or by vaccination, determines the outcome of patients with epithelial ovarian cancer (EOC). HLA genes are crucial to tumor surveillance, whereby tumor antigens are presented by Human leukocyte antigen (HLA) class I and II molecules to T cells, resulting in an orchestrated cellular and humoral immunity. NY-ESO-1 (ESO), a highly immunogenic member of the “cancer testes” antigen family, elicits spontaneous immune responses in a subset of patients with EOC. We hypothesized that the generation of spontaneous immunity to ESO and the clinical outcome of EOC patients are determined by HLA genetic polymorphisms. Methods: A total of 116 patients with ESO-expressing tumors, as determined by immunohistochemistry, were included in this study. Matched serum samples were assessed by enzyme-linked immunosorbent assay for the ESO antibody. Low-resolution HLA typing was performed using sequence-specific polymerase chain reaction. The relative odds of the ESO serologic response correlating with HLA type was analyzed using a multivariate logistic model. A proportional hazards model was used to assess the association between HLA allele groups and survival. Results: ESO humoral immunity was detected in 47% (54/116) and was absent in 53% (62/116) of EOC patients. The HLA class II alleles, DRB1*13 and DRB4*01, were significantly more frequent in ESO-seropositive patients (70% [16/23] and 63% [19/30], respectively) than in ESOseronegative patients (30% [7/23] and 37% [11/30], respectively) (odds ratio [OR] 0.223, 95% CI 0.08-0.619, P = 0.008 and OR 0.293, 95% CI 0.119-0.722, P = 0.004, respectively). HLA class I allele frequencies were similar in seropositive and seronegative patients. Improved survival was observed in patients with the A*01 subtype as compared to other HLA-A subtypes (HR 0.491, 95% CI 0.226-0.832). Conclusions: HLA polymorphisms are associated with antitumor immunity and may influence clinical outcomes in EOC. The HLA-
DRB1*13 and HLA-DRB4*01 alleles are significantly associated with the development of humoral immunity to ESO but not necessarily with survival in EOC patients, most likely as a result of immunologic tolerance. Although a relationship between the HLA-A*01 subtype and survival was seen, the underlying mechanism(s) will need to be determined. This study supports innovative vaccination strategies targeting HLA class I and II ESO epitopes, portending a broad immunologic response.
doi:10.1016/j.ygyno.2013.04.382
324 Dual mTOR inhibition demonstrates antiproliferative and chemosensitizing effects in chemoresistant high-grade papillary serous ovarian cancer F. Musa, I. Giuroiu, S. Blank, S. Korets, J. Curtin, R. Schneider. New York University Medical Center, New York, NY. Objective: To establish the effect of small-molecule mTOR inhibitors on the tumorigenesis of high-grade papillary serous ovarian cancer (OVCA) and to compare the effect of mTOR complex 1 inhibition (mTORiC1) vs. dual mTOR complex 1 and 2 inhibition (mTORCi1 + 2) with and without carboplatin (CPP) in an in vitro model of OVCA. Methods: Clonogenic assays of NIH:OVCAR3 were performed. Treatment conditions included increasing doses of RAD001 (mTORiC1), PP242 (mTORiC1 + 2), carboplatin (CPP; DNA damaging agent), and the combination of PP242 + CPP. Colonies were stained with crystal violet and analyzed using ImageJ64. Cell lysate immunoblots using commercial antibodies probed for key proteins in the mTOR (S6 and pS6, BP1 and p-BP1, mTOR and p-mTOR, Akt and p-Akt) and DNA repair pathways (CHK1 and p-CHK1, CHK2 and p-CHK2, ATM and p-ATM, ATR and p-ATR, p-BRCA1 and others). Colony counts (counts) and average colony diameter (ACD) were compared by ANOVA, with results expressed as mean ± systematic error. Post hoc analyses allowed pair-wise comparisons. Results: One-way ANOVA was significant across treatment arms (F = 17.718, P = .002). PP242-treated cells had a significantly smaller ACD compared to CTL cells (30.5 ± 3.5 vs. 131.5 ± 23.5) and CPPtreated cells (115.5 ± 1.5) (Tukey standard deviation [SD] P = 0.004 and 0.009). Addition of CPP to PP242-treated cells further decreased the ACD (27 ± 4) compared to CTL or compared to CPP alone (Tukey SD P = 0.003 and 0.008). Counts in CPP + PP242-treated cells were decreased vs. CTL (56 ± 9 vs. 129 ± 27) (Tukey SD P = .02). RAD001 (20 nM-5 uM) did not alter counts or ACD when compared to CPP or CTL. Immunoblots of cells treated with PP242 and high-dose RAD001 revealed characteristic inhibition of the mTOR pathway (decreased pmTOR, p-S6, and p-BP1), which was not observed with CPP. Furthermore, PP242 effectively reduced p-Akt (due to mTOR complex 2 inhibition) compared to RAD001 and CPP. Total CHK1 and p-CHK1 were reduced in cells treated with PP242 but not in cells treated with RAD001 or CPP alone. Conclusions: mTOR regulates cap-dependent mRNA translation (CDT), which is upregulated in OVCA. mRNAs with long 5’ untranslated regions, such as CHK1 and other survival mRNAs, have a greater requirement for CDT and are highly sensitive to mTORiC1 + 2. We showed that PP242 has compelling antiproliferative and chemosensitizing effects in OVCA and is superior to RAD001. Inhibition of CHK1 by dual mTOR inhibitors has not yet been investigated in the clinical setting but is likely to be an effective target in control of advanced disease.
doi:10.1016/j.ygyno.2013.04.383