Variation of mutant allele frequency in NRAS Q61 mutated melanomas

Cutaneous melanoma is a highly aggressive and treatment-resistant human cancer. The most frequent genetic alterations involve genes of the MAP kinase signaling pathway [1‐3]. Activating hot-spot mutations are mainly found in BRAF (codon V600) and in NRAS (codon Q61, and less frequently in the codons G12 and G13) genes, in 35–50% and 15–25% of cutaneous melanoma, respectively [4, 5]. Among BRAF alterations, the BRAF V600E mutation in exon 15 is predominant (85%) and due to a substitution of a valine to a glutamic acid (c.1799 T > A, p.V600E) [6, 7]. BRAF and NRAS mutations are almost always mutually exclusive [8, 9].

Mutant NRAS melanomas have been reported to have more aggressive clinical features than other subtypes, with thicker lesions, elevated mitotic activity, and higher rates of lymph node metastasis [10‐12]. Additionally, NRAS mutation status was reported as a predictor of poorer outcomes with lower median survival compared to non-NRAS mutated melanoma [10, 13].

The discovery of BRAF mutations led to the development of targeted treatments [14, 15]. However despite major clinical benefit in melanomas with BRAF mutation, secondary resistance occurs in most patients during the first year of treatment. Thus combinations of BRAF and MEK inhibitors have been developed, and were shown to induce longer progression free survivals (PFS) of patients with BRAF mutated melanomas [16‐18]. By contrast, targeted treatment of patients with NRAS mutated melanomas is still a challenge, although an international phase 3 prospective study with the MEK inhibitor binimetinib recently provided promising results [19].

We recently studied the frequency of BRAF mutant alleles (M%BRAF) and showed that M%BRAF is highly heterogeneous and frequently increased in BRAF mutated melanomas [20]. Interestingly, a recent clinical study showed that the increased BRAF V600 mutation level was significantly associated with a better response rate to vemurafenib during the first 10 months of treatment [21]. These observations highlighted the importance of quantitative evaluation of BRAF mutation before melanoma treatment.

Although biological and clinical implication of the frequency of mutant alleles of BRAF in melanomas are currently under investigation, no data are available concerning the variation of M%NRAS. Accordingly, we conducted this study to investigate NRAS Q61 mutations and M%NRAS in a series of 199 melanomas wild type for BRAF V600. The mechanisms of the M%NRAS variations were then studied by fluorescence in situ hybridization (FISH) and by amplified fragment length polymorphism (AFLP).

In this study, we reported the prevalence of NRAS Q61 mutation and, for the first time, the variations of NRAS mutant alleles (M%NRAS), in a large series of human melanoma samples. We have demonstrated that M%NRAS was highly heterogeneous; indeed, only 61% of NRAS mutated melanomas were heterozygous, while 30% of cases had a significantly increased M%NRAS (≥60%). Our results were confirmed by analysis of the cases of the TCGA database.

NRAS pyrosequencing assays used in this study were developed to identify all hot-spot mutations in the codon 61 of NRAS gene. The specificity of these assays for different mutations was confirmed by Sanger sequencing. Additionally, the genotyping accuracy of 40 NRAS mutated melanomas, 27 of which were p.Q61R was confirmed by immunohistochemistry with an antibody against Q61R [27]. In a recent study, we have demonstrated that pyrosequencing was a robust molecular technique for oncogenic mutant allele quantification, by comparing it with quantitative real time PCR and picodroplet digital PCR [20]. This previous study was focused on BRAF mutations, and similar M%BRAF heterogeneity was demonstrated in melanomas, with 19% of cases having an increased M%BRAF. Altogether, we estimate from both series that 36.2% of melanomas with BRAF/NRAS mutations have a non-heterozygous oncogenic allele.

Few studies have investigated M%NRAS in melanomas. Recently, we reported two cases with an increase of M%NRAS during metastatic melanoma progression; suggesting that M%NRAS may enhance metastatic capacities of melanomas [28]. Additionally, a large screening study of 833 cells lines from the database of Cancer Genome Project, Sanger Institute, focused on frequently mutated genes (six suppressor gene and five oncogenes), has identified NRAS homozygous mutation in 10% of cell lines [29]. However, the zygosity status was only determinate by manual examination of sequencing electropherograms.

Interestingly, in vitro studies of mutant RAS family members had demonstrated a high oncogenic potential of increased mutant allele frequency. The oncogenic potential of NRAS ^G12D/G12D was highly increased as compared to heterozygous or hemizygous NRAS cells in NRAS-driven hematopoietic transformation [30]. Additionally, progenitors of hematopoietic cells expressing the highest levels of NRAS ^G12D demonstrated cytokine-independent CFU-GM colony growth and exhibited an increased level of pAkt, pErk and pS6 proteins. Endogenous expression of HRAS ^G12V promotes papilloma and angiosarcoma development and these neoplasm initiations have been strongly associated with HRAS ^G12V allelic and gene copy number imbalances [31, 32].

Mutation in one allele of an oncogene is sufficient for activation of its targets and M%NRAS is expected to be around 50% in diploid cells. However, in tumours with high chromosome instability, chromosome number is rarely disomic and M%NRAS could widely exceeded 50%. To better understand the chromosome mechanisms leading to NRAS mutant allele increase in the proportion of NRAS mutated melanoma, we firstly performed FISH analyses with 2 BAC probes covering NRAS region and another region of chromosome 1, telomeric to this gene, in a large series of 104 melanomas. Different types of NRAS/chromosome 1 status were observed. Polysomy was mainly observed in NRAS mutated tumours and disomic and/or disomic but rare polysomic cells were less frequent in High non-HET M%NRAS than in M%NRAS WT tumours. Amplification and deletion of NRAS gene were rarely observed and were seen in both NRAS WT and NRAS mutated melanomas. Genomic analysis of human cutaneous melanoma genomes have been described in several studies. However, in most of them, only melanoma cell lines were studied. The analysis of 60 melanoma cell lines by Gast [33] have revealed targeted focal amplifications of NRAS genes in 11% of them (n = 7/60) and amplification were detected in both NRAS mutated and NRAS WT melanomas. This frequency is higher that the frequency of NRAS amplification detected in the present series and in other reports. In a subset of cutaneous melanocytic lesions, NRAS amplification was found to be restricted to a few cases with NRAS mutations [34]. Additionally, Stark and colleagues reported rare instances of focal amplification including NRAS gene in two cell lines with NRAS mutation; however, a poor correlation between copy number increase and concomitant mutation in this oncogene was described [35]. Polysomy of chromosome 1 and intra-tumour NRAS/chromosome 1 heterozygosity were frequently found in our series and was preferentially observed in NRAS mutated cancers. Correlation between mutant burden and gene copy gains have already been described for KRAS [36] and BRAF gene [20]. To our knowledge, NRAS/chromosome 1 copy number variations have never been described in melanomas with regard to the NRAS mutant allele burden.

Secondly, we analyzed chromosome 1 microsatellite polymorphism in normal and tumor DNA in a group of 29 NRAS mutated cancers by ALFP method. As expected, LOH with WT allele loss was mostly restricted to the High non-HET M%NRAS group. However, unlike in haematological malignancy [37, 38], acquired copy-neutral LOH was not a predominant mechanism of mutant allele imbalance in NRAS Q61 mutated melanomas; indeed this aberration was detected in only 23% of our samples. Other mechanisms of High non-HET M%NRAS were amplification and gain of NRAS gene (14%) and polysomy of chromosome 1 (23.8%). In 9 melanomas (38.1%), an intra-tumor copy number variation of NRAS/chromosome 1 was detected. As most melanomas have copy number variations of whole chromosomes and of chromosome segment, NRAS mutant allele increase could be a consequence of chromosome instability and clonality in these tumors.

Correlation of M%NRAS with clinical data revealed no association with age, sex, histological melanoma subtypes, nor with histoprognostic markers of the patients with NRAS Q61 mutated melanomas. Moreover, no differences in patient survival outcomes were observed between patients with <60% and ≥60% of M%NRAS. However, this cohort is a retrospective monocentric study, and the analyses were limited by small number of patients. Furthermore, the value of M%NRAS has to be investigated for prediction of response to targeted therapy, as done for M%NRAS with promising results. We hypothesize that High non-HET M%NRAS could have an oncogenic addiction effect, which could improve the sensitivity of targeted therapy in this subgroup of NRAS Q61 mutated melanoma.

We report herein for the first time that 30% of cutaneous NRAS mutant melanomas have a high M%NRAS. Chromosome instability, (chromosome 1 polysomy, intra-tumor copy number variation of chromosome1/NRAS) rather than the acquired copy neutral LOH seems to be responsible for most of the cases with high M%NRAS. Histoprognostic markers and survivals were not different when comparing patients with <60% and ≥60% of M%NRAS; however this should be checked in a larger and multicentric series.