BRAF and NRAS Mutations are Heterogeneous and Not Mutually Exclusive in Nodular Melanoma

Abstract

Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there’s a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas

“…This evidence has already been reported [16]. As previously observed [15,[17][18][19], in some samples we have detected co-occurrence of BRAF and NRAS mutations. At the single cell level, mutations in BRAF or NRAS are mutually exclusive [17].…”

NRASQ61R immunohistochemistry detects both NRASQ61R and KRASQ61R mutations in colorectal cancer

“…Furthermore, intratumoural heterogeneous staining of NRASQ61R IHC is common in melanomas. 5,7,8,19,20 Intratumoural heterogeneity resulted from subclonal evolution within the tumour 8,[19][20][21] and existed between primary and metastatic melanomas. 5,7,22 Heterogeneous expression for NRASQ61R IHC was not observed in this study or in the study of follicular carcinomas conducted by Oishi et al 16 Diffuse and homogeneous expression for NRASQ61R IHC supports the concept that the RAS mutation is generally an early molecular aberration in the adenomacarcinoma pathway of CRCs.…”

Co-targeting BRAF and cyclin dependent kinases 4/6 for BRAF mutant cancers

“…Yancovitz et al reported the presence of distinct clonal populations with BRAF wild type or NRAS mutant cells together with cells containing BRAF V600E mutation within the same primary melanoma lesions (Yancovitz et al, 2012). Chiappetta et al described that NRAS and BRAF mutations in melanoma are not mutually exclusive as previously understood but instead co-occur at varying detectible frequencies within the same lesion (Chiappetta et al, 2014) (Sensi et al, 2006;Wilmott et al, 2012;Chiappetta et al, 2014). As highly selective therapies such as vemurafenib deplete the bulk of the tumors, the subpopulations of resistant cells survive, get enriched and promote tumor regrowth.…”