Sensitive And Robust Targeted Sequencing Of Pancreatic Precancer And Tumors Using Microfluidic Single-Molecule Enrichment

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAPancreatic ductal adenocarcinoma (PDAC) arises from two precursor lesions, pancreatic intraepithelial neoplasias and pancreatic cysts, such as intraductal papillary mucinous neoplasias (IPMNs). Since cyst lesions are readily detected on cross sectional imaging, early diagnosis of IPMN-associated PDAC and intervention may be feasible. However, clinical guidelines dictating which IPMNs are high-risk and require surgical resection are suboptimal. Recently, the genomic signature of IPMN-associated carcinomas has been described opening the possibility for targeted genomic analysis. Previous groups have sought to achieve this using conventional methods. However, since IPMN tissue and cyst fluid are limited in quantity and contain inhibitors of PCR, we developed a novel microfluidics-based approach to achieve sensitive and specific targeted amplification and Illumina library construction.We have optimized a novel method to enrich targeted genomic regions for next generation sequencing (NGS) platforms featuring microfluidic partitioning of the sample into uniform picoliter volume droplets containing single molecules of target DNA. All droplets contain all of the primers and PCR reagents, ensuring that every target molecule from the sample is amplified, and after endpoint PCR results in a highly uniform yield that facilitates efficient use of the NGS platform. In addition, the primers contain ‘Illumina tails’ that enable easy sample indexing and loading directly onto a MiSeq without additional library preparation.Here we detail the successful customization and use of the ThunderBolts Cancer Panel, which targets 230 commonly mutated regions in 50 cancer associated genes, with additional primers for commonly mutated genes in PDAC. Addition of the 37 PDAC-relevant primer pairs to the commercially available core panel was very straightforward and resulted in sequencing metrics similar to those of the core panel alone (100% coverage at 100x depth, mean read depth of 2500). Cyst fluid from 30 patients with IPMN resected under Sendai criteria were analyzed. We describe the mutational signature of cyst fluid and relate the presence and quantity of mutations in KRAS, GNAS and PIK3CA to the presence of invasive carcinoma and high grade dysplasia on pathology. Finally, unbiased and high resolution sequencing was obtained using this protocol with as little as 8ng of input DNA.Using picodroplet PCR technology we were able to achieve unprecedented sequencing performance on ultra-low sample inputs. Given the ease of use and customization of the panel, the same platform may be readily adapted to other applications in which samples are limited or are difficult to amplify. Future studies will utilize this platform for an expanded analysis of pancreatic cyst fluid with additional primers.Citation Format: Angelina I. Londono-Joshi, Erica Pratt, Robert W. Cowan, Michael L. Samuels, Steve Kotsopoulos, Jeff Olson, Francis Long, Michelle A. Anderson, Diane Simeone, Andrew D. Rhim. Sensitive and robust targeted sequencing of pancreatic precancer and tumors using microfluidic single-molecule enrichment. [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 1572. doi:10.1158/1538-7445.AM2015-1572