title: 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.affiliation: Department of Medical Epidemiology and Biostatistics and.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Huang
authors.affiliation: Center for Digestive Diseases, Division of Surgery, CLINTEC, and.
authors.forename: Johannes-Matthias
authors.initials: JM
authors.lastname: Löhr
authors.affiliation: Center for Digestive Diseases, Division of Surgery, CLINTEC, and.
authors.forename: Magnus
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: Center for Digestive Diseases, Division of Surgery, CLINTEC, and.
authors.forename: Ralf
authors.initials: R
authors.lastname: Segersvärd
authors.affiliation: Department of Biosciences and Nutrition and Center for Innovative Medicine (CIMED), Karolinska Institutet, Huddinge, Sweden;
authors.forename: Hans
authors.initials: H
authors.lastname: Matsson
authors.affiliation: Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Clinical Pathology/Cytology, Karolinska University Hospital, Stockholm, Sweden;
authors.forename: Caroline
authors.initials: C
authors.lastname: Verbeke
authors.affiliation: Center for Digestive Diseases, Division of Surgery, CLINTEC, and.
authors.forename: Rainer
authors.initials: R
authors.lastname: Heuchel
authors.affiliation: Department of Biosciences and Nutrition and Center for Innovative Medicine (CIMED), Karolinska Institutet, Huddinge, Sweden;
authors.forename: Juha
authors.initials: J
authors.lastname: Kere
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, MA;
authors.forename: A John
authors.initials: AJ
authors.lastname: Iafrate
authors.affiliation: Department of Medical Epidemiology and Biostatistics and Department of Pathology, Massachusetts General Hospital, Boston, MA; Current address: Department of Biomedical Sciences, City University of Hong Kong, Hong Kong. zhengzongli@gmail.com weimin.ye@ki.se.
authors.forename: Zongli
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Department of Medical Epidemiology and Biostatistics and zhengzongli@gmail.com weimin.ye@ki.se.
authors.forename: Weimin
authors.initials: W
authors.lastname: Ye
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D021441:Carcinoma, Pancreatic Ductal / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D005787:Gene Frequency; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010183:Pancreatic Ducts / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D051901:Smad4 Protein / Q000235:genetics; D012907:Smoking / Q000453:epidemiology; D016019:Survival Analysis; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D016159:Tumor Suppressor Protein p53; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins