e16265 Background: Adenosquamous carcinoma of the pancreas (ASCP) is a rare histological subtype of pancreatic cancer with high metastasis rate and poor prognosis, which is characterized by a variable proportion of adenocarcinoma and squamous carcinoma components with at least 30% of the tumor showing squamous differentiation. To get further insight into the tumorigenesis and progression, we profiled the genetic characterization and explored the evolutionary model of this subtype. Methods: A total of 10 patients with ASCP were enrolled. For each sample, glandular and squamous carcinoma tissues were separated by microdissection. Whole exome sequencing for 20 samples with matched peri-carcinomatous tissues were performed by next-generation sequencing. Results: Genetic profiling revealed that top 5 mutated genes of adenocarcinoma and squamous components were highly consistent including TP53 (100% vs 100%), KRAS (100% vs 90%), CDKN2A (40% vs 40%), KDM6A (20% vs 20%) and LRP1B (20% vs 20%). KEGG pathway analysis showed that PI3K-Akt, MAPK and FoxO signaling pathways were the most frequently mutated pathways in adenocarcinoma. In contrast, Apelin and Rap1 signaling pathways were highly mutated in squamous components, advocating that these pathways may play important roles in the transformation of squamous carcinomas. A higher proportion of indels were identified in adenocarcinoma than squamous components (12.1% vs 3%). The chromosome regions of 3p21.3, 9p21.3, 11p15.5, 16p13.11 and 21q22.3 were the most commonly deleted regions in the adenocarcinoma components, while losses of 1p36.21, 3p25.3, 3q13.13, 6p22.3 and 11p15.5 had been observed in the squamous components. Some of these regions had important effects on development and progression of cancer. For example, 9p21.3 had well-established impact on cell proliferation and apoptosis containing two crucial tumor suppressor genes, CDKN2A and CDKN2B. Additionally, the C > T mutation was predominant among the 6 types of single nucleotide variations. Most common signatures were related to aging (Single base substitutions, SBS1), BRCA1/2 mutations and responders to platinum therapy (SBS3), and defective DNA mismatch repair (SBS6). Tumor evolution analysis showed that the glandular and squamous components shared a common origin. There were two evolutionary scenarios in the early stages of tumor evolution, with a third of nine patients ware parallel evolution and the remaining patients were linear evolution. KRAS (100%), TP53 (66.7%) and TTN (33.3%) were frequently identified as clonal mutations. Conclusions: This is the first study to elucidate the genomic alteration as well as the clonal evolution using whole-exome sequencing in ASCP patients. It provides evidence for more comprehensive understanding of pathophysiology of ASCP at genomic level, which could contribute to the amelioration for ASCP patients’ management.