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Decreased 53BP1 expression predicts improved survival in sporadic ovarian carcinoma
Abstracts / Gynecologic Oncology 130 (2013) e1–e169
Methods: Following pathologic review and laser capture microdissection to enrich for tumor cells, whole exomes were prepared from DNA of 2 independent SBTs (SBT-s2 and SBT-s5) and subjected to massively parallel DNA sequencing, using the Illumina Genome Analyzer IIx. Image analysis and base calling were performed using the standard Genome Analyzer pipeline software, Sequencing Control Studio (SCS), and Real-Time Analysis (RTA), respectively. Publicly available software tools, such as BWA aligner, the SamTools package, Picard, and GATK were used for bioinformatical analysis of data. Validation of potential somatic mutations was performed using standard Sanger sequencing techniques. Results: A small number of somatic mutations were identified in these SBTs. Both tumors contained an activating mutation of the BRAF gene. A total of 15 additional somatic mutations were identified, 9 in one tumor and 6 in the other. Eleven were missense mutations and four were nonsense or deletion mutations. Fourteen of the 16 genes found to be mutated in this study have been reported to be mutated in other cancers. Furthermore, 12 of these genes are mutated in invasive ovarian cancers. The FBXW7 and KIAA1462 genes are noteworthy candidates for a genetic role in serous borderline tumorigenesis, insofar as inactivating mutations of these 2 genes have been reported in low-grade serous ovarian carcinomas. Conclusions: A small number of somatic genetic mutations characterize SBTs of the ovary. The mutant genes described herein represent candidates for the pathogenesis of SBT of the ovary; further studies are required to determine their biological relevance. doi:10.1016/j.ygyno.2013.04.363
305 Tumor-specific targeting with novel LXY-30 peptide-linked nanoparticles for paclitaxel delivery in ovarian cancer mouse xenograft model N. Suby1, K. Xiao2, Y. Yi2, W. Xiao2, L. Tiglao2, K. Lam2. 1UC Davis Medical Center, Sacramento, CA, 2University of California - Davis, Sacramento, CA. Objective: Boronate cross-linked micelles (BCM) allow for drug delivery to the tumor and retain the encapsulated drug under physiologic conditions and release due to lower pH values or by a trigger release such as mannitol. In previous work, we demonstrated that nanocarriers linked with the LXY-30 ligand increases in vitro cellular uptake compared to nanocarriers alone in SKOV-2 ovarian cancer cells. In this study, we investigated the therapeutic efficacy and toxicity profiles of the BCMs carrying paclitaxel (PTX) in ovarian cancer xenograft-bearing mice. Methods: The intraperitoneal (IP) xenograft model was established by injecting SKOV-3/LUC cells intra-abdominally into female athymic nude mice at 6-8 weeks of age. SKOV-3/Luciferase is a line that stably expresses the firefly luciferase gene, which was used for intraabdominal imaging of tumor intensity. After 2 months of tumor growth, on day 0, the mice were randomly divided into 5 groups (PBS, PTX, BCM-PTX, BCM-LXY30-PTX, and BCMLXY30- PTX followed by mannitol trigger) and were treated with IP injections of the formulations twice weekly for a total of 3 doses. PTX was given at a dose of 10 mg/kg, which is nearly the maximum tolerated dose (MTD). At 24 hours following the administration of the BCM-PTXLXY-30 nanoparticle, the drug release was triggered by injection of a 20% mannitol solution per mouse. The mice were followed weekly with imaging. Tumor burden was assessed by the relative intensity of the tumor. Toxicities were assessed by analyzing the effects on animal behavior and body weight change. Results: Compared with the PBS and PTX groups, mice in the nanoparticle treatment groups showed significant inhibition of tumor growth (P b 0.05). At the dose of 20 mg PTX/kg, the BCM-PTX and
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BCM-LXY30-PTX exhibited superior tumor growth inhibition and longer survival time compared to PTX. The BCM-LXY30-PTX and BCM-LXY30-PTX with mannitol trigger showed improved tumor growth inhibition and longer survival times than the BCMPTX group. The median survival time was 35 days for PBS, 45.5 days for 10 mg/ kg PTX, 49 days for 20 mg/kg BCM-PTX, 63 days for 20 mg/kg BCMLXY30- PTX, and 66 days for 20 mg/kg BCM-LXY30-PTX with mannitol trigger. Conclusions: We demonstrated that the novel use of the BCM linked with LXY30 was more efficacious than both the free drug at the MTD and BCM without the use of a ligand at equivalent doses of PTX in the ovarian cancer xenograft mouse model. doi:10.1016/j.ygyno.2013.04.364
306 Decreased 53BP1 expression predicts improved survival in sporadic ovarian carcinoma K. Pennington1, A. Wickramanayake1, B. Norquist1, S. Kaufmann2, C. Pennil1, R. Garcia1, K. Agnew1, P. Welcsh1, E. Swisher1. 1University of Washington Medical Center, Seattle, WA, 2Mayo Clinic, Rochester, MN. Objective: 53BP1 is a critical regulator of the balance between homologous recombination (HR) and the more error-prone nonhomologous endjoining (NHEJ) DNA repair. Deletion of 53BP1 in brca1(but not brca2-) null cells rescues embryonic lethality, partially restores HR, and reverses sensitivity to poly-ADP ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression in a large number of primary and recurrent ovarian carcinomas to determine if 53BP1 expression is associated with clinical outcomes in sporadic and inherited cases. Methods: We evaluated 53BP1 protein expression using immunohistochemistry in 248 ovarian carcinomas comprehensively characterized for germline mutations in BRCA1 and BRCA2, including 54 paired primary and recurrent samples. We evaluated relative 53BP1 and BRCA1 mRNA expression in a subset of 85 cases with quantitative reverse transcriptase-polymerase chain reaction. Results: Both primary and recurrent BRCA1-mutated (but not BRCA2mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wild type carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wild type and in BRCA1-mutated but not in BRCA2-mutated ovarian carcinomas. In wild type carcinomas, lower 53BP1 message predicted improved survival (median survival 74 vs. 41 months, HR 0.49, 95% CI 0.27-0.88, P b 0.02). Survival was not significantly impacted by BRCA1 message level. Neither 53BP1 protein nor mRNA expression was associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. Conclusions: BRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wild type or BRCA2-mutated carcinomas, in direct contrast to previous findings in breast carcinomas. Higher 53BP1, which promotes NHEJ DNA repair, could explain the high aneuploidy that is characteristic of BRCA1- mutated ovarian carcinomas. In wild type ovarian carcinomas, decreased 53BP1 mRNA expression predicts improved overall survival, an opposite effect to our prediction. We speculate that if 53BP1 expression correlates with a predominant NHEJ phenotype, then low 53BP1 could be a good prognostic factor in sporadic cases secondary to overall decreased aneuploidy and more ordered DNA repair. doi:10.1016/j.ygyno.2013.04.365
Methods: Following pathologic review and laser capture microdissection to enrich for tumor cells, whole exomes were prepared from DNA of 2 independent SBTs (SBT-s2 and SBT-s5) and subjected to massively parallel DNA sequencing, using the Illumina Genome Analyzer IIx. Image analysis and base calling were performed using the standard Genome Analyzer pipeline software, Sequencing Control Studio (SCS), and Real-Time Analysis (RTA), respectively. Publicly available software tools, such as BWA aligner, the SamTools package, Picard, and GATK were used for bioinformatical analysis of data. Validation of potential somatic mutations was performed using standard Sanger sequencing techniques. Results: A small number of somatic mutations were identified in these SBTs. Both tumors contained an activating mutation of the BRAF gene. A total of 15 additional somatic mutations were identified, 9 in one tumor and 6 in the other. Eleven were missense mutations and four were nonsense or deletion mutations. Fourteen of the 16 genes found to be mutated in this study have been reported to be mutated in other cancers. Furthermore, 12 of these genes are mutated in invasive ovarian cancers. The FBXW7 and KIAA1462 genes are noteworthy candidates for a genetic role in serous borderline tumorigenesis, insofar as inactivating mutations of these 2 genes have been reported in low-grade serous ovarian carcinomas. Conclusions: A small number of somatic genetic mutations characterize SBTs of the ovary. The mutant genes described herein represent candidates for the pathogenesis of SBT of the ovary; further studies are required to determine their biological relevance. doi:10.1016/j.ygyno.2013.04.363
305 Tumor-specific targeting with novel LXY-30 peptide-linked nanoparticles for paclitaxel delivery in ovarian cancer mouse xenograft model N. Suby1, K. Xiao2, Y. Yi2, W. Xiao2, L. Tiglao2, K. Lam2. 1UC Davis Medical Center, Sacramento, CA, 2University of California - Davis, Sacramento, CA. Objective: Boronate cross-linked micelles (BCM) allow for drug delivery to the tumor and retain the encapsulated drug under physiologic conditions and release due to lower pH values or by a trigger release such as mannitol. In previous work, we demonstrated that nanocarriers linked with the LXY-30 ligand increases in vitro cellular uptake compared to nanocarriers alone in SKOV-2 ovarian cancer cells. In this study, we investigated the therapeutic efficacy and toxicity profiles of the BCMs carrying paclitaxel (PTX) in ovarian cancer xenograft-bearing mice. Methods: The intraperitoneal (IP) xenograft model was established by injecting SKOV-3/LUC cells intra-abdominally into female athymic nude mice at 6-8 weeks of age. SKOV-3/Luciferase is a line that stably expresses the firefly luciferase gene, which was used for intraabdominal imaging of tumor intensity. After 2 months of tumor growth, on day 0, the mice were randomly divided into 5 groups (PBS, PTX, BCM-PTX, BCM-LXY30-PTX, and BCMLXY30- PTX followed by mannitol trigger) and were treated with IP injections of the formulations twice weekly for a total of 3 doses. PTX was given at a dose of 10 mg/kg, which is nearly the maximum tolerated dose (MTD). At 24 hours following the administration of the BCM-PTXLXY-30 nanoparticle, the drug release was triggered by injection of a 20% mannitol solution per mouse. The mice were followed weekly with imaging. Tumor burden was assessed by the relative intensity of the tumor. Toxicities were assessed by analyzing the effects on animal behavior and body weight change. Results: Compared with the PBS and PTX groups, mice in the nanoparticle treatment groups showed significant inhibition of tumor growth (P b 0.05). At the dose of 20 mg PTX/kg, the BCM-PTX and
e127
BCM-LXY30-PTX exhibited superior tumor growth inhibition and longer survival time compared to PTX. The BCM-LXY30-PTX and BCM-LXY30-PTX with mannitol trigger showed improved tumor growth inhibition and longer survival times than the BCMPTX group. The median survival time was 35 days for PBS, 45.5 days for 10 mg/ kg PTX, 49 days for 20 mg/kg BCM-PTX, 63 days for 20 mg/kg BCMLXY30- PTX, and 66 days for 20 mg/kg BCM-LXY30-PTX with mannitol trigger. Conclusions: We demonstrated that the novel use of the BCM linked with LXY30 was more efficacious than both the free drug at the MTD and BCM without the use of a ligand at equivalent doses of PTX in the ovarian cancer xenograft mouse model. doi:10.1016/j.ygyno.2013.04.364
306 Decreased 53BP1 expression predicts improved survival in sporadic ovarian carcinoma K. Pennington1, A. Wickramanayake1, B. Norquist1, S. Kaufmann2, C. Pennil1, R. Garcia1, K. Agnew1, P. Welcsh1, E. Swisher1. 1University of Washington Medical Center, Seattle, WA, 2Mayo Clinic, Rochester, MN. Objective: 53BP1 is a critical regulator of the balance between homologous recombination (HR) and the more error-prone nonhomologous endjoining (NHEJ) DNA repair. Deletion of 53BP1 in brca1(but not brca2-) null cells rescues embryonic lethality, partially restores HR, and reverses sensitivity to poly-ADP ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression in a large number of primary and recurrent ovarian carcinomas to determine if 53BP1 expression is associated with clinical outcomes in sporadic and inherited cases. Methods: We evaluated 53BP1 protein expression using immunohistochemistry in 248 ovarian carcinomas comprehensively characterized for germline mutations in BRCA1 and BRCA2, including 54 paired primary and recurrent samples. We evaluated relative 53BP1 and BRCA1 mRNA expression in a subset of 85 cases with quantitative reverse transcriptase-polymerase chain reaction. Results: Both primary and recurrent BRCA1-mutated (but not BRCA2mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wild type carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wild type and in BRCA1-mutated but not in BRCA2-mutated ovarian carcinomas. In wild type carcinomas, lower 53BP1 message predicted improved survival (median survival 74 vs. 41 months, HR 0.49, 95% CI 0.27-0.88, P b 0.02). Survival was not significantly impacted by BRCA1 message level. Neither 53BP1 protein nor mRNA expression was associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. Conclusions: BRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wild type or BRCA2-mutated carcinomas, in direct contrast to previous findings in breast carcinomas. Higher 53BP1, which promotes NHEJ DNA repair, could explain the high aneuploidy that is characteristic of BRCA1- mutated ovarian carcinomas. In wild type ovarian carcinomas, decreased 53BP1 mRNA expression predicts improved overall survival, an opposite effect to our prediction. We speculate that if 53BP1 expression correlates with a predominant NHEJ phenotype, then low 53BP1 could be a good prognostic factor in sporadic cases secondary to overall decreased aneuploidy and more ordered DNA repair. doi:10.1016/j.ygyno.2013.04.365