Abstract Benign Brenner tumors (BTs) are composed of nests of epithelial cells resembling urothelium, which are surrounded by a dense fibromatous stroma and are considered the benign counterpart of ovarian transitional cell tumors. Their origin remains enigmatic. We have previously described the junction between the fallopian tubal epithelium and the mesothelium of the tubal serosa, and reported that transitional cell metaplasia (TM) is frequently found in this location. We hypothesize that BTs arise from TM epithelium that, when embedded in the ovary as Walthard cell nests (WCs), may develop into BTs, some of which may progress to so-called “atypical proliferative BTs”. The aim of the present study was to evaluate the immunohistochemical (IHC) and molecular genetic features of BTs in an attempt to confirm this hypothesis. A total of 17 ovaries, 25 peritoneal mesothelia, 38 fallopian tubes, 10 TMs, 56 WCs, 48 BTs and 7 atypical proliferative BTs were included in this study. IHC for AKR1C3, alpha-inhibin (INH), AR, β-catenin, calretinin (CRT), CEA, CYP19, ER, p16, PAX2, PAX8, PR, SF1, and WT1, and fluorescence in situ hybridization (FISH) for CDKN2A (p16 encoding gene) were performed. Genomic DNA from 20 BTs was extracted for sequence analysis of BRAF, CTNNB1, ERBB2, FOXL2, KRAS, PIK3CA, and PPP2R1A mutations. A somatic mutation of PIK3CA was detected in the stromal component in one BT. No other mutations were detected in either the epithelial or the stromal component in the remaining BTs. IHC analysis revealed that fallopian tubal secretory cells, TM, WC and epithelial component of BTs and atypical proliferative BTs shared the same IHC profile, consistently expressing AKR1C3, AR, but not CRT, as opposed to mesothelium and ovarian surface epithelium. The stromal component of the tumors showed weak expression of AKR1C3 in 23/26 (88%), AR in 26/29 (90%), ER in 32/42 (74%), PR in 38/43 (88%), and WT1 in 13/41 (32%), and strong expression of CRT in 34/41 (83%), INH in 33/41 (80%), and SF1 in 38/40 (95%) BTs. Notably, CRT, INH, and SF1 showed particularly strong staining in the stromal cells that immediately surrounded the epithelial nests. In contrast, expression of these biomarkers was absent in the BT epithelium. p16 was intensely and diffusely positive in the epithelial component of 9/13 (69%), patchy in 3/13 (23%), and negative in 1/13 (8%) benign BTs. In contrast all 7 atypical proliferative BTs were completely negative for p16 in the epithelial component. FISH identified homozygous deletion of CDNK2A in all atypical proliferative BTs, but CDKN2A was retained in benign BTs (Fisher exact test two sided p-value<0.0001). In summary, our findings suggest: 1) tubal origin of BTs through TM and WC, based on similar IHC profile, 2) an active role of the ovarian stroma in the pathogenesis of BT, based on the periepithelial IHC pattern of CRT, INH, and SF1, markers of steroidogenic cells, along with the epithelial expression of AR, 3) the role of CDKN2A loss in the progression of BT to atypical proliferative BT. Citation Format: Elisabetta Kuhn, Ayse Ayhan Ayhan, Jeffrey D. Seidman, Robert J. Kurman. The pathogenesis of ovarian Brenner tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2013-4764 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.