Abstract Homozygous deletions (HDs) are a common mechanism by which tumor suppressor genes (TSG) are inactivated in cancer. Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A/p16) is the most commonly deleted TSG, with HDs in 40% of pancreatic ductal adenocarcinomas (PDAC) harboring p16 deletions. HDs create novel junctions, which can be exploited for sensitive early detection and minimal residual disease testing, using the PAtient-specific REarrangement (PARE) method. Here we characterize the underlying structural rearrangements that produce p16 HDs in a panel of PDAC cancer cell lines using pair-end whole genome sequencing (WGS) and high density SNP microarray analyses. Rearrangements were defined as simple interstitial deletions when the segment of a chromosome arm was deleted and the ends joined. The complex rearrangements causing the HD were categorized at both breakpoints as inversions and/or translocations. Sanger sequencing confirmed novel junctions, and breakpoints were assessed for non-templated base insertions and microhomology. We report that complex rearrangements underlie half of p16 HDs in PDAC (3 of 5). Most striking, the combination of an inversion and inter-chromosomal translocation (INV+TRANS) was exclusively responsible for these complex rearrangements. Inversions ranged in size from 47 to 3,373bp and translocations were non-recurrent and were often non-reciprocal (2 of 3). All HDs were resolved (either by interstitial deletion or INV+TRANS) without the addition of any non-templated bases inserted at the novel junction, arguing against the involvement of error-prone non-homologous end joining (NHEJ). Microhomology was seen at most rearrangement junctions (8 of 9), but with a median of only 2 bases. Further work is needed to elucidate the mechanism responsible for INV+TRANS rearrangements. Citation Format: Alexis L. Norris, Hirohiko Kamiyama, Ralph H. Hruban, James R. Eshleman. CDKN2A/p16 is inactivated by the combination of inversion and translocation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2015-3935