Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: Heteroduplex analysis of limiting dilution PCRs

Abstract

We have developed a simple, robust, highly-sensitive assay for identifying gene mutations in clinical samples. We applied this assay to detect p53 and p16 mutations in pancreatic juice obtained from patients undergoing evaluation and treatment of pancreatic disease. The assay strategy involves PCR amplifying DNA at limiting dilution (LD-PCR) followed by screening PCR products for mutations using temperature gradient capillary electrophoresis (TGCE). Compared to conventional TGCE, TGCE after LD-PCR significantly increased the number of detectable mutations in pancreatic duct juice. Using LD-PCR, mutations in p53 and/or p16 were found in the pancreatic juice of 12 of 20 individuals with pancreatic cancer compared to only 1 of 8 patients with chronic pancreatitis, 0 of 8 individuals without evidence of pancreatic disease (p < 0.02).We conclude that limiting dilution PCR is an effective strategy for improving the detection of mutations in clinical samples and when applied to pancreatic juice to detect mutations of p53 and/or p16, it can help distinguish patients with pancreatic cancer from those without evidence of pancreatic neoplasia.

Genetic and Epigenetic Alterations of Familial Pancreatic Cancers

“…Because KRAS2 gene mutation detection has some predictive value for distinguishing pancreatic cancer from other non-cancerous conditions of the pancreas and because it is frequently mutated in both familial and sporadic pancreatic cancers, the quantification of KRAS2 gene mutations could potentially serve as one of a panel of molecular markers that could be used to help predict the presence of an underlying pancreatic neoplasm. Similarly, since the TP53 gene is mutated in ~75% of sporadic pancreatic adenocarcinomas and in a significant fraction of familial pancreatic cancers, it too is a potential molecular marker of pancreatic neoplasia (57–59). …”

DNA Methylation Alterations In Endoscopic Retrograde Cholangiopancreatography Brush Samples of Patients With Suspected Pancreaticobiliary Disease

“…Ultimately, a combination of highly specific markers may provide the best diagnostic utility. Newer assays that can detect low concentrations of these mutations in pancreatic juice 41, and novel assays and technologies are likely to improve the detection of low concentrations of mutant DNA for cancer diagnosis in the future.…”

Precursors to Invasive Pancreatic Cancer

“…65,66 Other common somatic mutational events, such as TP53 and CDKN2A/p16 mutations are also amenable to detection in biospecimens like pancreatic juice, as are epigenetic alterations such as aberrant promoter methylation. 68,69 For pancreatic cystic lesions, EUS and aspiration of cyst fluid contents provides an opportunity for molecular analysis, such as quantitative assessment of mutant DNA using next generation sequencing, 37 or multiplex microRNA profiling for aberrant biomarker expression. 70 Ultimately, there is unlikely to be one biomarker that proves to be the panacea for early detection, and most likely, we will utilize a multianalyte panel (encompassing DNA, RNA, proteins, or any combinations thereof) for the early diagnosis and therapeutic stratification of pancreatic neoplasia.…”