Research Article: Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

Date Published: June 7, 2018

Publisher: Public Library of Science

Author(s): Nunthawut Chat-Uthai, Pichpisith Vejvisithsakul, Sutthirat Udommethaporn, Puttarakun Meesiri, Chetiya Danthanawanit, Yannawan Wongchai, Chinachote Teerapakpinyo, Shanop Shuangshoti, Naravat Poungvarin, John Souglakos.


The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

Partial Text

Human v-raf murine sarcoma viral oncogene homologue B1 gene (BRAF), is located on chromosome 7q34. The cytoplasmic serine-threonine kinase encoded by this gene mediates the activation of the mitogen-activated protein kinase (MAPK) signaling pathway involved in cell growth, survival and differentiation. About half of nonacral cutaneous melanomas harbor gain-of-function BRAF mutations, rendering the MAPK pathway constitutively active [1]. The most common mutations in BRAF occur in codon 600. The majority of the BRAF V600 mutations are V600E [2], results in an amino acid substitution at position 600 in BRAF, from a valine (V) to a glutamic acid (E). The next most commonly observed BRAF mutations are V600K, which arises from a double nucleotide change and results in an amino acid substitution of the valine (V) at position 600 by a lysine (K). These mutations account for approximately 95% of BRAF mutations found in melanoma [3]. Other mutations, including V600M, V600R, V600D and V600G, are less common. Mutations of BRAF gene can also be found in colorectal cancer (CRC), papillary thyroid cancer, lung cancer and hairy cell leukemia [4]. Patients with BRAF-mutated CRC have a poor prognosis [5]. Frequency of BRAF mutations in CRC varies widely among different populations around the world [6–11]. The overall frequency of the CRC with BRAF V600 mutations in Asian populations is relatively low in comparison to other ethnic populations [12].

Identification of the mutation status in cancers becomes mandatory on the best way to guide treatment decision in the era of precision medicine. Although Sanger sequencing can be used to identify somatic mutations in DNA from clinical samples, but this well-established techniques can give false detection as a result of relatively poor analytical sensitivity. It reliably detects mutations at a relative concentration of 20–30% [37]. Another problem with this technique is that the test turnaround time is relatively slow. Pyrosequencing and HRM offer better detection limits with shorter assay times but they still suffer from a limited sensitivity for mutant alleles lower than 5 to 10% [23] which are occasionally found in cancer specimens. At this level of LOD, these techniques are not good choices for mutation detection in plasma and body fluids [38]. Lower limit of detection of at least 1% mutant alleles has been generally recommended for liquid biopsy [39]. Allele-specific PCR (AS-PCR) is another commonly used method for the analysis of clinical specimens harboring somatic mutations of oncogenes. This method relies on mutation-specific primers to discriminate wild-type and mutant alleles. Well-designed and optimized AS-PCR assays generally provide ultrasensitive detection of somatic mutations [40].




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