Abstract 224: Establishing de novo platform for whole genome sequencing of pancreatic cyst fluid for early detection and diagnosis of pancreatic cancer
Authors: Adekeye Adenike , Haque Farzana , Ettelaie Camille , Nikitenko Leonid , Maraveyas Anthony 
Published: April 2023

Abstract
Background: Pancreatic cancer (PC) is a leading cause of cancer-related deaths globally. Accurate PC detection at early or premalignant stage, when surgery is effective, would increase survival rates and prevent unnecessary surgery or surveillance. About 10% of PC cases arise from pancreatic cystic (PCy) lesions, including intraductal papillary mucinous neoplasms (IPMNs), which constitute up to 30% of resective pancreatic surgery. Current diagnostic methods cannot accurately predict which cysts are malignancy-associated. Molecular diagnostic attempts using PCy fluid (PCyF), as a liquid biopsy, to analyze common mutations associated with PC development are yet to contribute to early diagnosis. Whole genome sequencing (WGS) is utilized in various cancers, PC inclusive, for detecting genetic changes associated with carcinogenesis, but not in PCyF due to technical limitations, including isolated DNA purity.
Aim: To establish a de novo platform for WGS of PCyF.
Methods: Three PC cell lines (PANC1, AsPC1 and MiaPACA2) were cultured in vitro. Five PCyF samples, including one from PC patient (based on histopathology report) were obtained from the University of Hull (UoH)-based ethically approved study (TEM-PAC, NCT03536793, REC 18/LO/0736). Genomic DNA (gDNA) isolation protocol was optimized using magnetic- and column-based kits (MagMAX DNA Multi-sample and PureLink Genomic DNA; ThermoFisher) and varying number of cultured cells (100,000-500,000; to determine detection limits) and used for PCyF samples (35 - 70 μl). gDNA purity (A260/280 ≥ 1.8 and A260/230 ≥ 2) and concentration were determined using NanoDrop2000, and amount per cell was calculated. WGS of PC sample was done using 200ng of gDNA on Illumina NovaSeq6000 platform (Novogene Ltd, Cambridge, UK) at 50x sequencing depth. Sequencing reads were mapped to reference human genome (hg38) with the Burrows-Wheeler Aligner to detect disease-related genomic variants in each chromosome covered - insertion-deletions (InDels), single nucleotide polymorphisms (SNPs), copy number variants (CNVs) and structural variants (SVs) with subsequent analysis using UoH High Performance Computer Viper.
Results: Pure gDNA was isolated from all cell lines (0.03 ± 0.01 ng/cell) and from PCyF samples (217 ng - 13.8 μg; equivalent to 7,000 - 690,000 cells and 8 - 346 ng/µl). WGS of PC patient PCyF revealed several cancer-related changes/variants including 106,776 (12.31%) novel InDels, 181,000 more SNPs, CNVs (337 gains; 149 losses) and 10,024 SVs.
Conclusions: We established a de novo platform for WGS analysis of gDNA isolated from small volumes of PCyF samples, including estimation of previously unknown cellular density in these liquid biopsies. Our proof-of-concept study provides a foundation for a large-scale analysis of PCyF samples from TEM-PAC and other clinical studies using WGS for early detection and diagnosis of PC.
Citation Format: Adenike Adekeye, Farzana Haque, Camille Ettelaie, Leonid Nikitenko, Anthony Maraveyas. Establishing de novo platform for whole genome sequencing of pancreatic cyst fluid for early detection and diagnosis of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 224.