Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.

Abstract

BACKGROUND:

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Variant profiling is crucial for developing personalized treatment and elucidating the etiology of this disease.

METHODS:

Patients with PDAC undergoing surgery from 2007 to 2012 (n = 73) were followed from diagnosis until death or the end of the study. We applied an anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) method to a panel of 65 selected genes and assessed analytical performance by sequencing a quantitative multiplex DNA reference standard. In clinical PDAC samples, detection of low-level KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations was validated by allele-specific PCR and digital PCR. We compared overall survival of patients according to KRAS mutation status by log-rank test and applied logistic regression to evaluate the association between smoking and tumor variant types.

RESULTS:

The AMP-based NGS method could detect variants with allele frequencies as low as 1% given sufficient sequencing depth (>1500×). Low-frequency KRAS G12 mutations (allele frequency 1%-5%) were all confirmed by allele-specific PCR and digital PCR. The most prevalent genetic alterations were in KRAS (78% of patients), TP53 (tumor protein p53) (25%), and SMAD4 (SMAD family member 4) (8%). Overall survival in T3-stage PDAC patients differed among KRAS mutation subtypes (P = 0.019). Transversion variants were more common in ever-smokers than in never-smokers (odds ratio 5.7; 95% CI 1.2-27.8).

CONCLUSIONS:

The AMP-based NGS method is applicable for profiling tumor variants. Using this approach, we demonstrated that in PDAC patients, KRAS mutant subtype G12V is associated with poorer survival, and that transversion variants are more common among smokers.

• Authors: Jiaqi Huang, Johannes-Matthias Löhr, Magnus Nilsson, Ralf Segersvärd, Hans Matsson, Caroline Verbeke, Rainer Heuchel, Juha Kere, A John Iafrate, Zongli Zheng, Weimin Ye
• Journal: Clinical chemistry (Clin Chem) Vol. 61 Issue 11 Pg. 1408-16 (Nov 2015) ISSN: 1530-8561 [Electronic] England
• PMID: 26378065 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 American Association for Clinical Chemistry.

Chemical References

• KRAS protein, human
• Proto-Oncogene Proteins
• SMAD4 protein, human
• Smad4 Protein
• Tumor Suppressor Protein p53
• Proto-Oncogene Proteins p21(ras)
• ras Proteins

Topics

• Aged
• Aged, 80 and over
• Carcinoma, Pancreatic Ductal (diagnosis, epidemiology, genetics)
• DNA Mutational Analysis
• Female
• Gene Frequency
• Humans
• Male
• Middle Aged
• Mutation
• Pancreatic Ducts (metabolism, pathology)
• Pancreatic Neoplasms (diagnosis, epidemiology, genetics)
• Proto-Oncogene Proteins (genetics)
• Proto-Oncogene Proteins p21(ras)
• Smad4 Protein (genetics)
• Smoking (epidemiology)
• Survival Analysis
• Tumor Suppressor Protein p53 (genetics)
• ras Proteins (genetics)