Original Article
KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance

https://doi.org/10.1016/j.prp.2014.01.011Get rights and content

Abstract

Mutations of KRAS are detectable in 70–90% of pancreatic duct adenocarcinomas (PDAC), using direct sequencing. We used a highly sensitive molecular method in order to investigate: (a) the frequency and prognostic significance of different KRAS mutations and, (b) whether the presence of KRAS mutations in histologically-negative resection margins of PDAC could explain local tumor recurrence after surgery.

Twenty-seven patients with histologic diagnosis of PDAC, radical pancreaticoduodenectomy and histologically-negative margins were evaluated. KRAS mutations were searched for mutant-enriched PCR in tumor and negative resection margins.

KRAS mutations were detected in 85.2% of the cases; the most frequent mutation was G12D (48.1%). Shorter OS was found in patients with G12D (25 months; 95% CI, 20.5–29.5), vs patients with other mutations (31.5 months; 95% CI, 25.6–37.1) (N.S.). KRAS mutation in histologically-negative margins was detected in one patient who died of locoregional recurrence; six patients had tumor recurrence but no mutations in surgical margins.

The high frequency of KRAS mutations suggests a search for KRAS status to improve the diagnosis in suspected cases; the G12D mutation could be related to poor prognosis, but without statistical significance. No correlation was found between the frequency of cancer recurrence and KRAS mutations in surgical margins.

Introduction

Pancreatic duct adenocarcinoma (PDAC) is one of the most aggressive epithelial tumors in both males and females worldwide [1]. The short overall five-year survival rate (less than 5%) is mainly due to the late tumor stage at the time of diagnosis [6], but, even for the small group of patients with resectable tumors, the prognosis remains poor [7]. This could reflect the inadequate removal of the tumor or the presence of precancerous cells in surgical margins that, despite normal morphology, may harbor molecular abnormalities responsible for local cancer recurrence. Molecular genetic analyses indicated that activating mutations of KRAS oncogene are early events in pancreatic carcinogenesis, and occur in 70–90% of PDAC [3], [10]. Point mutations involve most of all the codon 12 [5], in contrast to colorectal cancer (CRC) in which mutations in codon 13 are not rare, occurring in about 20% of the cases [23]. The role of KRAS mutation in predicting response to target therapies in CRC is widely accepted [2], whereas its prognostic significance is doubtful. A recent methanalysis conducted in 1261 CRCs seems to indicate a poor prognosis for patients harboring G12V mutation vs patients with KRAS wild-type or harboring other KRAS mutations, suggesting the importance of an accurate molecular characterization of KRAS gene in order to establish its real prognostic significance [11].

Frequency and type of KRAS mutations in PDAC have been analyzed in few studies, all using low-sensitive direct sequencing, obtaining conflicting results concerning statistical correlation with mutation and survival or local recurrence [12], [13], [16], [18], [20].

In this study, the prognostic significance of KRAS mutation status was analyzed in 27 patients with PDAC by using a highly sensitive mutant-enriched PCR that is able to recognize one copy of mutated DNA among 1000 copies of wild-type DNA. KRAS status was also searched for in histologically-negative surgical margins in order to evaluate whether KRAS mutations may correlate with local tumor recurrence independently to the histologic evidence of cancer or dysplasia.

Section snippets

Patients and tissue specimens

Twenty-seven patients who underwent radical pancreaticoduodenectomy for pancreatic adenocarcinoma from 1996 to 2010 were investigated.

The patients were selected on the basis of the following criteria: radical surgical procedure with complete tumor ablation, clinical follow-up of at least 12 months and resection margins histologically negative. All operations were performed by the same surgeon (M.G.), and all patients underwent radical pancreaticoduodenectomy following the Whipple – Child

Clinical material

Fourteen patients were males (52%) and 13 were females (48%), with a mean age of 67 years at surgery (range 51–80; median 68). No perioperative mortality occurred. The postoperative morbidity rate was 11.11% (one case of peritoneal bleeding needing re-operation, one case of postoperative gastrointestinal bleeding and one case of pancreatic fistula, treated conservatively).

In Table 2 the clinico-pathological findings of the series are summarized. No statistical correlation was found, comparing

Discussion

In this study, the frequency and the type of KRAS mutations were searched for in tumor samples of pancreatic adenocarcinoma in order to investigate whether they could have prognostic significance; in addition, KRAS status was also investigated in histologically-negative surgical margins in order to evaluate whether KRAS mutations may correlate with local tumor recurrence. The study was performed by using, for the first time in routine samples, a highly sensitive molecular method (ME-PCR) that

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