Analysis of genotyping methodologies to detect RAS mutations

It is currently known that those patients with metastatic colorectal cancer (CCRm) whose tumors have mutations at the level of certain genes involved in the EGFR receptor pathway (HER-1), such as the RAS gene (KRAS or NRAS exons 2,3 and 4 ), do not benefit from therapy with monoclonal antibodies with antiEGFR activity (Cetuximab or Panitumumab). It is less clear today, however, what may be the role or the real impact of the existence of other low frequency mutations in RAS or at the level of genes equally involved in that same pathway or cell route (BRAF or PIK3CA).

This work published in the Annals of Oncology tries to explore the potential utility of sophisticated or “high sensitivity” genotyping methodologies, comparing them with the standard methodologies commonly used to detect mutations at the RAS level in these tumors, to select patients with CCRm especially sensitive to combination treatment with a standard QT scheme (FOLFIRI) associated with an anti-FRFR monoclonal antibody (Panitumumab). This is one of the first published protocols that investigates this issue in the context of a prospective clinical trial.

To do this, several researchers belonging to the Spanish group for the treatment of digestive tumors (TTD) analyzed in a centralized way with several of these methodologies the tumor samples of 72 patients who received said therapy within a phase II protocol in 12 Spanish hospitals. All cases had been considered “a priori” as carriers of “non-mutated” (wild-type) RAS tumors using standard techniques carried out in each of these centers before beginning treatment.

According to these results, the analysis in search of alterations at the level of RAS, BRAF or PIK3CA genes with quantitative PCR (qPCR) and digital PCR (dPCR) is able to detect a greater number of alterations / mutations than conventional techniques. This is more evident the lower the cut-off point considered, the higher level of sensitivity, to label the tumors as “mutated” (5 to 0.1%). However, in general, the presence of mutations detected by this methodology does not seem to be associated with a worse tumor response (response rate) or lower clinical benefit (progression-free or global survival) associated with the administration of antiEGFR therapy in this population. compared to those cases that do not present them (“ultraselected” population).

There is an inverse correlation between the mutated allelic fraction (qPCR) and the tumor response achieved, with no statistically significant differences being reached between the different levels or cut-off points investigated in this work. More specifically when this value is below 5%, higher level of sensitivity, there are no differences in the response rates achieved between the subgroups, thus applying this percentage as the optimal cut-off point to try to classify patients in this clinical context This is contrary to what was previously suggested by other groups of researchers who propose lower cut-off points (1%), yes, always based on observations derived from retrospective series of patients.

Therefore, according to this work, despite demonstrating in principle these methodologies as more sensitive techniques to detect mutations at the level of the EGFR receptor pathway and its intracellular mediators, however, these tools do not seem to be more useful tools to make a better selection of Candidates to receive these treatments compared to other less sensitive methodologies, usually used so far in this context, with a minimum level of detection of around 5%.

On the other hand, the existence of BRAF level mutations has been confirmed, as in previous studies, as a factor of poor prognosis in these patients. However, since in all cases with this alteration the mutated allelic fraction has been greater than 10%, the prognostic impact of minor subclonal mutations in this gene could not be assessed. Unfortunately, due to the low frequency of detection of mutations at the level of BRAF and PIK3CA in this type of patients even with the use of the most sensitive techniques, it has not been possible to definitively determine the impact of these alterations in predictive terms in relationship with antiEGFR therapy. It would therefore be desirable to try to expand the series with a view to trying to clarify these and other issues of relevance to this population.

Ref. Phase II study of high-sensitivity genotyping of KRAS, NRAS, BRAF and PIK3CA to ultra-select metastatic colorectal cancer patients for panitumumab plus FOLFIRI: the ULTRA trial. Santos C, Azuara D, Viéitez JM, Páez D, Falcó E, Élez E, López-López C, Valladares M, Robles-Díaz L, García-Alfonso P, Bugés C, Durán G, Salud A, Navarro V, Capellá G, Aranda E, Salazar R. Ann Oncol. 2019 May;30(5):796-803. doi: 10.1093/annonc/mdz082. Epub 2019 Dec 4. PMID: 31987347