Frequency of RAS variants in Bulgarian patients with metastatic colorectal cancer

One common genetic factor affecting the colorectal cancer (CRC) development and the outcome is the mutational status of the RAS gene family. Somatic variants in HRAS, KRAS, and NRAS genes are influential in reference to colorectal cancerogenesis. Numerous studies show that RAS variants are predictors of resistance to anti-EGFR (epidermal growth factor receptor) therapy for CRC. The aim of the present study was to evaluate the frequency of RAS variants and their association with tumor localization and clinicopathological characteristics of Bulgarian patients with CRC. 228 Bulgarian patients with advanced and metastatic CRC were investigated prospectively. The patients were selected from August 2017 to August 2020. Genomic DNA was extracted from FFPE tumor tissue samples. The commercially available “Easy® KRAS” and “Easy® NRAS” kits were used for detection of somatic mutations of the KRAS and NRAS genes by Real-Time PCR. In our cohort, right-sided tumors were a more common event for women than for men (OR=2.08, 95% CI:1.09-3.99, p=0.027). Right-sided tumors were also more regularly mucinous adenocarcinoma (OR=3.43, 95%CI:1.24-9.52, p=0.018) and well-differentiated tumors (OR=4.32, 95 % CI:1.18-15.86, p=0.027), while it was not the case for the left-sided tumors. 123/228 (53.95%) of the patients were positive for RAS variants and 105/228 (46.05%) had wild-type RAS mutational status. 103 of CRC patients were positive for KRAS variants. 84 (77.78%) KRAS variants in exon 2, 5 (4.63%) in exon 3 and 19 (17.59%) in exon 4 were detected in our study. Within KRAS exon 2 variants in codon 12 were more prevalent than in codon 13 (87.10% vs. 11.10%). The most commonly registered mutation in exon 2 for our cohort was c.35G>T p.Gly12Val (n=28, 33.33%). Whereas the variant c.35G>T p.Gly12Cys (n=4, 4.76%) was the least represented and it was not established in patients with right-sided cancer. KRAS variants in exon 2 were predominant in all three different tumor localizations – right-sided, left-sided, and in transverse colon – were prevailed. In patients with right-sided tumors, we did not establish variants in KRAS exon 3. Tumor localization did not relate to patients` RAS mutational status (p=0.458) at all or to the presence of a variant in KRAS exon 2 or outside the exon 2 (p=0.393). Patients with variants in KRAS had 3.18 times more often simultaneously distant and lymph node metastasis in comparison to patients with wild-type KRAS (OR=3.18, 95% CI:1.15-8.81, p=0.026). Three of 20 positive for NRAS variants patients had simultaneously two variants, therefore 11 (47.83%) samples had NRAS variants in exon 2 (codons 12 or 13), 12 (52.17%) samples - in exon 3 (codons 59 or 61), and none in exon 4 (codons 146 or 117). Our study confirmed that the most commonly mutated site is G12 in KRAS and Q61 in NRAS amongst Bulgarian patients too. As well as that tumor localization does not associate with harboring of RAS variants at all. However, in patients with right-sided tumors, there were no KRAS variants in exon 3, which have to be confirmed in a larger cohort.