Development of highly specific and sensitive PNA-clamp PCR assay for the detection of common mutations (BRAF V600E and NRAS) in CRC using FFPE tissue sample

REGER R MIKAEEL; JAMES HOWARD PRINGLE

doi:10.26682/sjuod.2018.21.2.9

REGER R MIKAEEL Dept. of Biology, College of Science, Kurdistan Region-Iraq
JAMES HOWARD PRINGLE Dept. of Cancer Studies and Molecular Medicine, University of Leicester-England

DOI: https://doi.org/10.26682/sjuod.2018.21.2.9

Keywords: Colorectal cancer;, , BRAF;, NRAS;, MAPK pathway;, anti-EGFR therapies;, PNA and PCR;

Abstract

Background: Colorectal cancer (CRC) is a frequent and widespread malignancy. Commonly mutated KRAS, BRAF and NRAS genes lead to constant activation of the MAPK pathway; consequently transforming basal stem cells into adenocarcinoma. Selective inhibitors of the MAPK pathway, such as Cetuximab and Panitumumab, have been implemented in clinical practice since 2004. Sanger sequencing and pyro sequencing have been commonly used for detection of BRAF and NRAS mutation in the UK. Nevertheless, sometimes the analytical sensitivity of these methods is not high enough to detect a low percentage of cells containing the mutation. Methods: a highly sensitive and specific Peptide nucelic acide (PNA)-clamp PCR was developed and validated firstly on cell lines that were known to harbour the mutations in BRAF^V600E or NRAS codons 12 and 13. The wild type and mutant DNA was serially diluted to test the sensitivity of the assay. Total DNA was extracted from 84 formalin fixed paraffin embedded (FFPE) tissues including adenomas, carcinoma and hyperplastic polyps; and screened for BRAF^V600E or NRAS mutations. Results: the highly sensitive PNA-clamp QUASAqPCR assay significantly detected 1:2000 ratio of mutant to wild type background (P < 0.01). The assay on FFPE tissue showed 15.47% (13/84) BRAF^V600E and 8.33% (7/84) NRAS mutants, of which NRAS c.38G>A (G13D) was the most common. BRAF^V600E mutation was significantly associated to right side tumour (P< 0.014), but was not related to any other clinical features. However, there was no significant association between NRAS mutation and any clinicopathological features. Conclusion: this study demonstrates the sensitivity, specificity and utility of PNA-clamp PCR assay in detecting the common point mutations in CRC in a low percentage of cells containing the mutation, providing an attractive tool for future research and therapeutic applications.

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