Abstract 4876: Proteomic analysis of pelvic tissues at the time of risk-reducing surgery in BRCA mutation carriers

Abstract

Abstract Objective: To develop a workflow for collecting specimens for proteomic analysis from the peritoneum, fallopian tubes (FT), and ovarian surface epithelium (OSE) at the time of risk-reducing surgery in women at increased risk for developing malignancies due to a germline BRCA mutation. Methods: A pilot set of 6 subjects with BRCA2 mutations were consented for an IRB-approved protocol for surgical specimen collection. During laparoscopic removal of the FT and ovaries, peritoneal washings, OSE brushings, and FT lavage specimens were collected without interfering with standard pathological analysis of the tissues. After immunodepletion, samples were loaded in a stacking gel and digested in-gel with trypsin. Digests were analyzed in duplicate by nanoflow reversed-phase liquid chromatography coupled online to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database from the European Bioinformatics Institute using SEQUEST. Differences in protein abundance between samples were derived by spectral counting. Immunohistochemical (IHC) staining for p53 and loss of heterozygosity (LOH) testing for p53 at the D17S-1844, D17S-516, and D17S-786 loci were performed on all paraffin-embedded FT specimens. Results: A total of 265, 328, and 241 different proteins were identified by 2 or more unique peptides each from the any of the OSE, FT and peritoneal samples, respectively. The number of proteins identified on both the right and left side in individual subjects ranged from 16-33% for OSE and 31-36% for FT. 37 common proteins involved in cell signaling, cell growth, cellular assembly, acute phase response, and DNA replication and repair were found in at least 1 specimen from all 6 subjects. 23 of the common proteins are involved in cancer pathways. All surgical pathology was negative both for malignancy and possible precursor lesions such as tubal intraepithelial carcinoma. MUC16 (CA-125) was elevated by proteomic analysis in the right FT of 1 subject; the left FT lavage in this subject could not be collected due to tubal scarring. Although IHC for p53 was normal in both of these FT, LOH for p53 was present in both. IHC was also negative for all remaining FT specimens. LOH was absent in both FT in 3 subjects, present in 1 FT but not the other in 2 subjects, and present bilaterally in 1 subject mentioned above. Conclusions: This workflow allows analysis of the proteomes of peritoneal, FT, and OSE specimens collected at the time of laparoscopic risk-reducing surgery, a technique which has not been described previously. It presents a unique opportunity for closer examination of the tissues at risk for malignant transformation in women with BRCA mutations. Harvesting proteins from the environment where ovarian and related cancers arise will inform current attempts to use the “p53 signature” to evaluate the FT as the potential source of many pelvic serous tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2011-4876