For the development and implementation of personalized medicine, a sensitive, specific, low-cost and easy-to-implement assay to identify various genetic alterations such as BRAF V600E mutation is needed. By selecting primer pair and optimizing PCR cycling conditions, we achieved this goal by using ARMS based PCR. One of the advantages of ARMS-PCR is that the assay is designed to amplify a relative larger common fragment of DNA that flanks the mutation site in all samples regardless of their mutation status. This common fragment conveniently serves as an internal control for template DNA quality as well as potential PCR inhibition. The mutant or wild-type specific PCR amplifications take place in the same reaction tube, thereby allowing the mutant or wild-type specific PCR primers to compete for binding to very limited templates. To increase the sensitivity for detecting this mutation, we tried to change the ratio of four primers and found that the combination of 400 nM primer Fo, 200 nM primer Ro and Fiwt, 800 nM primer Rimut resulted the strongest mutant band. The primer binding to template varies based on their annealing conditions, but relative high Fo and the highest Rimut primer concentrations might favor the production of mutant product in our design. In addition, relative low concentration for Fiwt and Ro primers might reduce full length and wild type product formation by the competition effect of each primer. By such a design, the three products were produced at reasonably comparable levels. Otherwise, the wild type or whole length bands might overshadow the weak mutant band if the mutant BRAF V600E abundance is low, which is frequently the case for clinical specimens. With this study, we improved assay sensitivity to as low as 0.5% mutant allele level in the background of wild-type DNA, which is quite sensitive among ARMS-PCR methods . This sensitivity is very difficult to reach with direct dideoxy sequencing which has the sensitivity of 15-20%, and with pyrosequencing which has greatly increased sensitivity to about 2% [22, 23]. Considering DNA samples obtained from FFPE tissue is often fragmented due to damage by formalin fixation, we designed our PCR product size to be small (no more than 200 bp) to maximize the chance of successful amplification. This was proven to be helpful since all 72 FFPE samples had successful amplification.
The reported prevalence of BRAF V600E mutation for conventional PTC varied from 36% to 67% in the US and European studies [7, 8, 24–27], and was 83% in a Korean study . The difference in prevalence could be due to different study population or different methodologies used. The prevalence of BRAF V600E mutation in the tall-cell variant PTC is in general higher than that of the conventional type, with a mean prevalence of 77% in several studies . On the contrary, the follicular variant of PTC is rarely involved by BRAF V600E mutation, with a mean prevalence of 12% . Using ARMS-PCR, we tested 72 thyroid tumors and determined that the frequencies of BRAF V600E mutation in the conventional, tall-cell and follicular variants of PTC were 66%, 75% and 0%, respectively. Our frequency of BRAF V600E mutation in conventional PTC was at the high end of the reported results, possibly reflecting our improved assay sensitivity compared with automated DNA sequencing, which was the method used in many of the reported studies. Our study showed that DNA sequencing method missed 3 out of 30 PTC samples with BRAF V600E detected by ARMS-PCR, resulting in a false negative rate of 10%. In our validation study, the assay specificity is 100% (14/14) based on negative mutation in follicular adenoma. This is consistent with previous reports since benign follicular adenoma has not been found to harbor the BRAF mutation .
BRAF V600E mutation testing has demonstrated utility in helping select CRC patients who are considering monoclonal antibody therapy as wild-type BRAF is required for response to anti-EGFR antibodies  and improve diagnostic accuracy in thyroid FNA samples [12, 13]. In addition, BRAF V600E mutation is associated with sporadic microsatellite instable CRC, but not hereditary non-polyposis colorectal cancer (HNPCC) syndrome [29, 30]. Therefore, the presence of BRAF V600E mutation is an exclusion criteria for HNPCC genetic testing [29, 30]. BRAF V600E mutation testing can also help facilitate clinical studies of BRAF-targeted therapies . The improved understanding of the role BRAF mutations in cancer diagnosis, prognosis and treatment has increased the need for BRAF mutation testing [4, 32].
New methods are constantly being developed for BRAF V600E mutation detection. Shifted termination assay (STA) developed by TrimGen Corporation declared the sensitivity of 1-5% . Lang et al. developed an allele-specific, also known as ARMS, real-time PCR using Taqman probe to increase the sensitivity to 1% and using internal control to exclude false negative results . Morandi et al. developed an allele specific locked nucleic acid (LNA) quantitative PCR assay using LNA-modified allele specific primers and LNA-modified beacon probes to achieve sensitivity of 0.1% . Dual-priming oligonucleotide (DPO)-based multiplex PCR was commercially available from Seegene (Seoul, Korea) and has a sensitivity of 2% . Qiagen developed a BRAF mutation detection PCR kit with a sensitivity of 1.27%. Our method using ARMS-PCR therefore appears to have high sensitivity (0.5%) for BRAF V600E mutation detection.