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Clin Chem. 2015 Nov;61(11):1408-16. doi: 10.1373/clinchem.2015.238543. Epub 2015 Sep 16.

Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma.

Author information

1
Department of Medical Epidemiology and Biostatistics and.
2
Center for Digestive Diseases, Division of Surgery, CLINTEC, and.
3
Department of Biosciences and Nutrition and Center for Innovative Medicine (CIMED), Karolinska Institutet, Huddinge, Sweden;
4
Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Clinical Pathology/Cytology, Karolinska University Hospital, Stockholm, Sweden;
5
Department of Pathology, Massachusetts General Hospital, Boston, MA;
6
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.
7
Department of Medical Epidemiology and Biostatistics and zhengzongli@gmail.com weimin.ye@ki.se.

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.

PMID:
26378065
DOI:
10.1373/clinchem.2015.238543
[Indexed for MEDLINE]
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