In the present study, we carried out a molecular profile of 50 cancer-related genes in precursor lesions of colorectal cancer (CRC). Adenomas exhibited mutations in genes already known to be involved in colorectal carcinogenesis, such as APC, KRAS, TP53, and FBXW7. On the other hand, serrated polyps showed low frequency in APC and TP53 genes and a high frequency of BRAF gene mutations.
Our findings corroborate the molecular differences previously reported in these two major distinct pathways of carcinogenesis7. According to the classic adenoma-carcinoma progression model, the progressive accumulation of genetic alterations leads to carcinoma development from the normal mucosa21. Recent studies have added complexity to this model, demonstrating the presence of molecular heterogeneity in the early stages of the development of colorectal lesions and mutations in several genes considered drivers for CRC22–25. As expected, we found a higher average of driver mutations in advanced adenomas than in early adenomas. The rate of acquisition of mutations is increased in adenomas than normal tissue, and the mutational burden in advanced adenomas has been reported to be similar to tumors, even when only driver mutations are analyzed22,23,26.
We also reported a lower frequency of mutations among serrated polyps when compared to adenomas. Few studies addressed this issue25,27. When comparing only SSL and adenomas, these authors show no difference between the frequency of mutations among these groups, likewise our study. Further, we observed a higher frequency of mutation in SSLs than hyperplastic polyps, which are lesions with lower malignancy potential. Recently, hyperplastic polyps and serrated sessile lesions were associated with the molecular subtype CMS1, which often has MSI and hypermutation28,29.
Additionally, we found a significant difference in the WNT, MAPK, PI3K, and p53 signaling pathways between adenomas and serrated lesions. Alterations in the WNT pathway are an initial event in the adenoma-carcinoma progression, predominantly due to mutations in the APC (40.3–80.0%) followed by the CTNNB1 gene (11.9–20.0%)30–32. In our study, we found a lower frequency of APC (41.8%) and CTNNB1 (3.3%) mutations, which can be because we did not analyze the whole coding sequence, but the major hotspot regions of both genes. In the serrated polyps pathway, the WNT signaling is reported to be less targeted27,33, following our findings.
Activation of the MAPK pathway is also observed in CRC, with mutations mainly found in KRAS and BRAF oncogenes34,35. We found 33.3% of our samples harboring mutations in this pathway, with mutations in the KRAS gene slightly more frequent in the adenoma group (22.4%) and BRAF predominantly present in the SSL group. In the adenoma group, the KRAS mutation frequency is within the variation observed in other studies (10.7–60.0%)25,30,36,37. For the Brazilian population, previous reports on the frequency of KRAS mutation in adenomas have reported lower frequency than we found (13.6%) 19. This difference could be explained by the higher sensitivity of NGS used in this study compared to Sanger sequencing to detect low-frequency variants38,39. We also observed a higher frequency of mutations in the KRAS gene in advanced adenomas than early adenomas, similar to previous studies25,40, including reports on the Brazilian population19.
In serrated polyps, a high frequency of mutations in the MAPK pathway genes was observed, mainly due to the activating BRAF gene mutations in SSLs. This is consistent with previously reported frequency of BRAF mutations in our population19. The presence of mutations in the BRAF gene has been consistently related to SSL with a high frequency of samples (8.7–88%) harboring mutations19,27,32,37,41. Interestingly, the main activating mutation BRAF V600E was found only in serrated polyps, as previously reported19,36.
Mutations in TP53 are generally observed during the transition from adenoma to carcinoma22,42,43. Recent studies reported a lower frequency of TP53 mutations in early or low growth rate adenomas and higher mutation frequency during the progression of early to advanced adenomas25,36. In our data, no significant difference was observed between early and advanced adenomas. Nevertheless, our results agree with the Vogelstein model, where TP53 is associated with the adenoma-carcinoma transition. The frequency of mutations in adenomas was lower when compared to the frequency of mutations in CRC cases previously reported in our population (25.4% in adenomas vs. 56.0% in cancer)16. Besides, mutations in TP53 in the serrated polyps were found only in SSLs, which was already described27,44.
Genes of the PI3K-AKT pathway were also mutated in our samples according to previous reports34,45. This pathway may present mutations in tumors with a focus of advanced adenomas or traditional sessile adenomas46, suggesting a role in late steps of both adenoma-carcinoma and serrated pathways progression. In agreement with these data, we observed a slightly higher frequency of mutations in genes of this pathway in advanced adenomas than in early adenomas. In the advanced adenomas, we found 3.6% of samples harboring mutations in PIK3CA. Mutations in this gene are found in regions of in situ tumors23. However, it is not an initial event during the process of clonal diversification in tumorigenesis, as observed in studies of clonal evolution in CRC22,43,47. Also, PIK3CA mutations are found in tumor-associated adenomas (20.0–30.0%)23,30, or in lower frequency in advanced adenomas (3.2%)48, similar to the frequency observed in our study.
The presence of mutations in the GNAS gene is frequent in CRC7 and has been reported in advanced adenomas22,41,49. Although mutations in GNAS in serrated polyps have already been reported, its frequency is not high and is related to more advanced lesions50. Corroborating these data, we identified mutations in this gene only in advanced adenomas samples and absent in serrated polyps.
Previous studies have shown that polyps and colorectal cancer are more frequent among African Americans than non-Hispanic Whites51–53. In the present study, as expected54–56, we observed a high heterogeneity of the ancestry proportions in our study population, yet, we did not found any difference between genetic ancestry and the groups of precursor lesions evaluated. This result could be due to the small number of cases within each group analyzed.
Despite major findings, our study’s limitations lie in the absence of paired normal tissue samples compared to the profile mutation of lesion samples. To overcome this issue, the variants identified were filtered in data bases, such as ABraOM (Brazilian population) and ExAC (international population). Also, the nature of the target sequencing, which does not cover the whole coding sequencing of the tumor suppressor genes, could underestimate the mutation frequencies. Finally, the absence of critical CRC-related genes, such as TCF7L2 and FAM123B45, could limit our results’ interpretation.
In summary, our study reports for the first time the mutation profile of colorectal precursor lesions in Brazilian patients. We observed mutations in known CRC drivers, such as APC, TP53, and KRAS, with differences according to the type of lesion analyzed, with a higher rate of mutations in adenomas. Moreover, a higher number of mutations were found in advanced adenomas compared to early adenomas and in SSL compared to hyperplastic polyps. These results will add to the already existing knowledge of the different molecular alterations present in the adenoma and serrated pathways and may contribute to the molecular CRC screening in the Brazilian population.