J. A. Reza1, S. Litherland2, X. Zhu5, P. Veldhuis4, A. J. Almodovar6, N. Fanaian3, J. P. Arnoletti1,2,4 1Florida Hospital,Center For Specialized Surgery,Orlando, FLORIDA, USA 2Florida Hospital Cancer Institute,Translational Research,Orlando, FLORIDA, USA 3Florida Hospital,Center For Diagnostic Pathology,Orlando, FLORIDA, USA 4Florida Hospital,Institute For Surgical Advancement,Orlando, FLORIDA, USA 5Florida Hospital,Center For Interventional Endoscopy,Orlando, FLORIDA, USA 6Florida Hospital,Translational Research Core,Orlando, FLORIDA, USA
Introduction:
Mutations in the KRAS oncogene (K-RASmut) have been associated with pancreatic ductal adenocarcinoma (PDAC) progression and aggressive behavior. We have previously reported the presence of K-RASmut transcriptionally active circulating tumor cells (CTC) in the portal circulation of PDAC patients. We hypothesized that active chromatin K-RASmut DNA from tumor cells may be detected in pancreatic juice following pancreatico-duodenectomy and indicative of risk for progression among surgically treated patients with peri-ampullary neoplasms.
Methods:
We collected portal venous blood and pancreatic juice samples from 17 patients who underwent pancreatico-duodenectomy for surgical treatment of various peri-ampullary neoplasms (PDAC=6, ampullary cancer=5, neuroendocrine tumor=3, cholangiocarcinoma=1, IPMN=1), and chronic pancreatitis (n=1). Portal vein circulating tumor cells (CTC) were isolated by high-speed FACS and analyzed by quantitative RT-PCR for K-RAS exon 12 mutant gene expression (K-RASmut). DNA, chromatin, and histone acetylated active chromatin were isolated from pancreatic juice samples (collected post-operatively from temporary external pancreatic duct stents) by chromatin immunoprecipitation (ChIP) and analyzed for the presence K-RASmut and other cancer related gene sequences by quantitative PCR and ChIP SEQ.
Results:
DNA, chromatin, and acetylated chromatin containing mutated K-RAS gene were detectable in pancreatic juice secreted after surgical resection of pancreatic, ampullary and bile duct carcinomas. The detection of pancreatic juice K-RASmut in chromatin and acetylated chromatin directly correlated with the number of CTC found in the portal venous blood (p=0.0108, p=0.0405, respectively). ChIP SEQ analyses indicated the presence of acetylated chromatin in pancreatic juice from PDAC patients, including RET and two other candidate chromatin loci, not found in similar analysis of non-malignant pancreatic juice from pancreatitis. PDAC patients with K-RASmut+ DNA in their portal blood CTC exhibited K-RASmut mRNA expression, indicative of transcriptionally active CTC surviving after primary tumor resection. K-RASmut gene activation in CTC correlated positively with progression free survival (p=0.0267, r2=0.4827, n=13, median follow-up of 4.5 months).
Conclusion:
Detectable K-RASmut chromatin in pancreatic juice suggests that viable cancer cells remain in the pancreatic duct following surgical resection. Analysis of pancreatic juice and portal venous blood CTC may be useful for stratification of tumor recurrence potential and identification of molecular therapeutic targets in peri-ampullary neoplasms.