Colorectal cancer (CRC) is the third most common cancer in Brazil and globally (1,2). The most prevalent molecular pathways for colorectal cancer development are APC, TP53, and KRAS mutations. The KRAS gene is 47,305 bp long and contains 6 exons. It plays the role of a GTPase in the transduction of signals (3). The activation of KRAS forms a GTP complex, which can then be inactivated by hydrolysis to GDP. The mutated form of KRAS renders the complex less susceptible to hydrolysis, remaining in an activated form which induces the cell to proliferate via several signal pathways, including MAPK (3,4).

The RAS family includes three subunits: Kirsten-RAS (KRAS), Neuroblastome-RAS (NRAS), and Harvey-RAS (HRAS). Mutations of KRAS are found in 34.7%, of NRAS in 7%, and of HRAS in 0.5% of CRC. Mutations in KRAS and NRAS confer a poor prognosis, even at the metastatic stage or early stage colorectal cancers. Additionally, they can lead to the development of resistance against anti-EGFR molecules (5-9). By contrast, EGFR inhibitors confer a positive predictive value response and increased overall survival in KRAS and NRAS wild-type tumors (10). Venook et al suggested that sidedness of the primary tumor greatly affects the clinical outcomes in an advanced or metastatic setting (11). The median survival for primary tumors located on the left side was significantly longer than that for tumors of the right side (overall survival: 33.3 vs. 19.4 months; P<0.0001). Patients treated with cetuximab with wild-type KRAS and left-sided primary tumors had an OS of 37.5 months versus those with right-sided primary tumors who had an OS of 16.4 months (HR = 1.97; 95% CI = 1.56-2.48). This suggests that patients with primary tumors on the right side of the colon should not be treated with anti-EGFRs (11).

DNA sequencing is the gold standard for detecting mutations. Originally, Sanger sequencing constituted standard usage; however, next-generation sequencing (NGS) has allowed for faster and high-throughput screening for mutations in several types of cancer (12). High-resolution melting (HRM) has recently been used as an alternative strategy to DNA sequencing (13). Through differences in DNA melting temperatures and curve profiles, it is possible to distinguish mutant samples from controls. HRM analyzers allow for a more accurate detection of differences in melting temperatures between two samples (13). In the present study, it was hypothesized that HRM could be used as an effective alternative to next-generation sequencing for the detection of KRAS mutations in colorectal cancer.

Personalized oncology claims that biomarkers predict a likely response to chemotherapy. In CRCs, somatic mutations in exon 2 of KRAS are predictive for resistance to anti-EGFR antibodies. Several studies have reported mutations in exon 2, ranging from 36 to 45% (14). Next-generation sequencing, which demonstrates a high concordance with Sanger sequencing methods, has been used for faster detection of mutations in different genes within larger DNA sequences (15).

Ishige et al reported that 25% of the mutations in exon 2 were in codon 12 and 4% in codon 13(6). Neumann et al (16) also reported that 80% of the mutations in exon 2 were in codon 12. In the present study, the prevalence of KRAS mutations was 34%, mainly in exon 2. The mutations on exon 2 were 12% in codon 13, and 54% in codon 12. Although findings of those studies lacked concordance, they did agree that codon 12 at exon 2 is the most common site of mutation in KRAS for colorectal cancer.

High-resolution melting has been used as an alternative strategy for sensitive detection of DNA sequence variations in different genes (17). This method is based on the different DNA melting temperatures for specific targets. This method cannot, however, be used to identify changes in the DNA sequence, although wild-type sequences and mutant sequences correlate with specific, and different, melting curves. In CRC, HRM has been evaluated to validate its routine use compared with both direct sequencing and NGS in various studies (18-23).

The HRM assay with DNA concentrations ranging from 20 to 100 ng, were able to evaluate the exon 2 of KRAS with a sensitivity and specificity of 83.3 and 96.6%, respectively, compared to NGS. Demonstrating a high concordance between the two methods used (ĸ = 0.816). Negru et al reported concordance of approximately 99% between HRM and DNA sequencing for exon 2(20). The large capacity for detecting mutations in exon 2 of KRAS in our study was similar to that in other studies with different populations (17,24-26).

The short number of samples is a limitation of this study. However, this study proved that this technique had a high sensitivity and specificity that may be validated in a high number of tumors. This assay, different from the others that was carried out in formalin-fixed paraffin-embedded (FFPE) tissue, was elaborate in fresh tumor samples that can increase the sensitivity of detection of mutations. A possible explanation is a possible DNA degeneration during the preparation and maintenance of the archived samples in FFPE. Another possible advantage when compared to other published articles is that the sequencing of KRAS mutation was by NGS and not by Sanger or pyrosequencing, which both have a low sensitivity.

Two advantages had to be considered in the diagnosis of KRAS mutations in CRC by HRM. This method is less expensive than NGS and may be an alternative method to diagnose KRAS mutation This is particularly important in view of the growing need to identify mutant profiles for different target genes in many types of cancer. Another advantage of HRM is the time spent for performance of the test, i.e., approximately 3.5 h compared to NGS, which requires some days.

In conclusion, in our cohort, HRM had a high index of comparison with NGS, which was the gold standard for detecting DNA mutations of KRAS in colorectal cancer.