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There is a need for improved prognostic markers in melanoma. This study tested the prognostic significance and clinicopathologic correlations of BRAF and NRAS mutations in patients with metastatic melanoma.
Clinical and pathologic data were collected retrospectively on melanoma patients clinically tested for BRAF (exon 15) and NRAS (exons 1, 2) mutations at the M. D. Anderson Cancer Center. Analyses were performed to identify significant associations of mutations with tumor and patient characteristics, and with survival from the diagnosis of stage IV disease.
The genotypes of the full cohort (n=677) were 47% BRAF mutation, 20% NRAS mutation, and 32% wild-type for BRAF and NRAS (“WT”). Tumor mutation status was associated (p=0.008) with the risk of CNS involvement at the diagnosis of stage IV disease, with higher prevalence observed in BRAF-mutant (24%) and NRAS-mutant (23%) patients than in WT (12%). Among non-uveal melanoma patients with mutation testing within 6 months of stage IV diagnosis (n=313), patients with NRAS mutations had a median survival of 8.2 months from stage IV diagnosis, which was shorter than WT patients (15.1 months, p=0.004). Multivariate analysis of this population incorporating age, gender, M1 category, serum LDH, and mutation status confirmed NRAS mutations are independently associated with decreased OS (vs WT, p=0.005, HR= 2.05).
Patients with BRAF or NRAS mutations are more likely than WT patients to have CNS involvement at the diagnosis of distant metastatic disease. NRAS mutation status is an independent predictor of shorter survival from the diagnosis of stage IV melanoma.
An estimated 70,230 patients will be diagnosed with cutaneous melanoma, the most lethal skin cancer, and approximately 8,790 will die from this disease in 2011.1 Detailed analyses of clinical outcomes have identified a number of validated clinical and pathological features that correlate with stage-specific survival in this disease.2–4 However, currently there are no validated molecular prognostic markers in melanoma. The most common somatic event in melanoma is mutation of the serine-threonine kinase BRAF, which is a component of the RAS-RAF-MEK-MAPK signaling pathway. Overall, point mutations in the BRAF gene occur in 40–50% of melanomas.5 Over 90% of the mutations in BRAF result in substitution of the valine at position 600, resulting in activation of the downstream effectors of the RAS-RAF-MEK-MAPK pathway.6 This pathway may also be activated by point mutations in NRAS, which codes for a small GTP-binding protein. NRAS is mutated in 15–25% of melanomas, most frequently at hotspots in exon 1 (codon 12) and exon 2 (codon 61).5 The overwhelming majority of activating BRAF and NRAS mutations in melanomas have been mutually exclusive.7
Previous studies have examined associations of BRAF and NRAS mutations with primary tumor characteristics and prognosis in earlier stage disease.8–11 However, limited data is available for patients with metastatic disease.12, 13 An improved understanding of the prognostic significance of mutation status in these patients may help in the appropriate design and interpretation of clinical trials, and add to the understanding of the biology of this disease. We have performed a retrospective analysis of a large cohort of advanced melanoma patients who underwent testing for both BRAF (exon 15) and NRAS (exons 1 and 2) mutations. The results of this analysis provide the first evidence that melanoma patients with an activating mutation in the NRAS gene have significantly shorter survival from stage IV diagnosis than patients without a mutation in either the BRAF or NRAS gene, and the prognostic value of this marker is independent of current validated prognostic markers.
The results of CLIA-certified testing for BRAF and NRAS mutations performed by the University of Texas MD Anderson Cancer Center Molecular Diagnostics lab for melanoma patients from February 1, 2007, to September, 13, 2010, were reviewed under an Institutional Review Board approved protocol. The specific mutations detected, patient demographics (age, gender, ethnicity), and primary tumor characteristics (date of diagnosis, anatomic location, histology, Breslow thickness, ulceration, mitotic rate) were recorded for all patients. Characteristics at the time of stage IV diagnosis (date, age, involved sites, serum lactate dehydrogenase) were also collected; stage IV was defined by AJCC 7th edition criteria, including M1a category (Any T, Any N, with distant cutaneous and/or subcutaneous and/or lymph node metastasis and non-elevated serum LDH) or M1b category (Any T, Any N with pulmonary metastasis without other visceral metastasis and non-elevated serum LDH) or M1c category (Any T, Any N with visceral metastasis or elevated serum LDH with any sites of distant metastasis).14 Treatments with the selective BRAF inhibitors vemurafenib (Roche) or GSK2118436 (GlaxoSmithKline) or the selective MEK inhibitors selumitinib (AstraZeneca) or GSK1120212 (GlaxoSmithKline) were also recorded. The most recent clinical follow-up date and vital status (through 1/15/2011) were collected.
Mutation testing was performed by CLIA-certified pyrosequencing assays by the MDACC Molecular Diagnostics Lab. Pyrosequencing of BRAF exon 15 (inclusive of codons 595 to 601) and NRAS exon 1 (codons 12 and 13) and exon 2 (codons 60 and 61) was performed.
The association between continuous parameters and mutational status was assessed by analysis of variance (ANOVA). Fisher’s exact test was used to compare the distribution of categorical variables between mutational groups. Distributions of time to event (time to stage IV and survival from stage IV) were estimated using the Kaplan-Meier method, and distributions were compared using the log-rank test. Cox proportional hazards regression analysis was used to assess the association between multiple covariates and the two survival parameters. For each parameter, an overall comparison was first made between the three mutational groups, followed by each of the three pairwise comparisons. To adjust for multiplicity when making pairwise comparisons, Tukey’s HSD method was used within the ANOVA framework for continuous variables, and the method of Holm and Bonferroni was used for categorical variables. Except as noted above, all statistical tests were performed two-sided with a 5% Type I error rate. Software suite R (http://www.r-project.org) version 2.12.0 was used.
Overall, 677 melanoma patients with successful testing for both BRAF and NRAS were identified (Table 1). Mutations in exon 15 of BRAF (only) were found in 320 (47.3%), and NRAS mutations (only) in 136 (20.1%). The majority of the BRAF mutations were represented by the V600E (71.9% of BRAF mutations) and V600K (22.5%) substitutions. Substitutions at position 60–61 accounted for 82.4% of NRAS mutations, most frequently Q61R/K/L. Four (0.6%) patients had activating mutations in both BRAF and NRAS; 217 (32.1%) of the patients did not have a mutation in either BRAF exon 15 or NRAS exons 1–2, and are referred to as wild-type (WT) for subsequent analyses. Due to the rarity and uncertain functional significance of non-V600 BRAF exon 15 mutations (n=6) and dual BRAF/NRAS mutations (n=4), patients with these genotypes were excluded from further analyses.
At the time of initial diagnosis of melanoma, patients with a BRAF mutation (“BRAF”) were significantly younger (median age, 49.8 years) than the NRAS-mutant (“NRAS”) (55.7 years, p=0.0008) or the WT (59.5 years, p<0.0001) patients (Table 2). There was also a significant association for mutation with race (p=0.042), although there were very few non-Caucasian patients in this cohort (4.1%). NRAS mutations were less common in Hispanic patients (8.8%) compared to the Caucasians (21.1 %) while Asian/Black/Unknown patients had an increased frequency of being WT (61.1% versus 31.1% of Caucasians).
Mutation status was strongly associated with the anatomic type of melanoma (p<0.0001) (Table 2). Among patients with a cutaneous primary (n=516), 49% were BRAF, 21% were NRAS, and 30% were WT. Patients with a mucosal melanoma (n=28) had a similar prevalence of NRAS (18%), but fewer BRAF (7%), and thus more WT (75%). None of the uveal melanoma patients (n=11) had a mutation in either BRAF or NRAS. Patients with an unknown primary tumor (n=110) had a prevalence of BRAF (53%), NRAS (19%), and WT (28%) genotypes that was very similar to the patients with a cutaneous melanoma, but differed significantly from those with a mucosal primary (p<0.0001). Among the patients with documented cutaneous primary tumor characteristics, there was no significant association observed between mutation status and Breslow thickness (n=424, p=0.40), mitotic rate (n=311, p=0.16), or ulceration status (n=344, p=0.44) (Table 3). Mutation status was significantly associated with primary tumor site and histology. BRAF mutations were most prevalent in truncal melanomas (63.9%) and with superficial spreading morphology (63.2%); NRAS mutations were most prevalent in melanomas arising from the arm/leg (34.7%) and with nodular histology (28.3%) (Table 3).
After excluding non-V600 BRAF (n=6) mutants, patients with concurrent BRAF and NRAS mutations (n=4), and one patient with incomplete data, a total of 519 patients in the cohort that developed stage IV melanoma (48.6% BRAF, 20.0% NRAS, 31.4% WT) were further analyzed. The interval from the diagnosis of melanoma to the diagnosis of stage IV showed a trend for shorter duration for the NRAS patients, but this difference was not statistically significant using 3-group or 2-group comparisons (Table 4). BRAF patients were younger than the NRAS (p=0.0047) and WT (p<0.0001) patients at stage IV diagnosis.
Serum LDH showed a non-significant trend for association with mutation status (p=0.09), with NRAS (15.4%) and WT (14.7%) less frequently having elevated LDH then BRAF patients (22.6%). M1 category, as defined by the anatomic sites of involvement only (i.e. excluding serum LDH), at the time of diagnosis of stage IV disease was significantly associated with tumor mutation status (p=0.0018). Two-group comparison identified a significant difference (p=0.0007) between BRAF and WT patients, with a higher rate of M1a and lower rate of M1b patients in the BRAF group. Pair-wise comparison of M1 category found no significant difference for NRAS patients versus either BRAF or WT.
Analysis of anatomic sites that characterize M1c disease (i.e., non-pulmonary visceral) identified a significant association of mutation status with the rate of CNS involvement at the time of the diagnosis of stage IV disease (p=0.0076). There was a higher rate of CNS involvement among the BRAF (24.4%, p=0.01) and NRAS (23.1%, p=0.056) patients as compared to WT (12.4%).
In contrast, there was no association with mutation status and the prevalence of liver (p=0.79) bone (p=0.43), skin (p=0.42) or lymph node (p=0.83) metastases. There was a significant association with lung involvement (p=0.049), with a lower rate of lung metastases among the BRAF (55.2%) and NRAS (56.7%) patients compared to WT (66.9%) at stage IV presentation (Table 4).
Tumor mutation status correlated with overall survival from the diagnosis of stage IV disease in the full cohort (n=519) (Figure 1A). NRAS patients (n=104) had significantly shorter median overall survival (15.5 months) than WT patients (n=163, 23.5 months; p = 0.02). The median overall survival of BRAF patients (n=252, 24.2 months) did not differ from the WT patients.
The analysis of survival by mutation status could be biased by the inclusion of patients who survived with stage IV disease for several years prior to the implementation of molecular testing. The relevance of this potential confounder in this cohort is supported by the atypically long survival from stage IV diagnosis detected in each of the groups.4 Therefore, an additional analysis was performed among patients with mutation testing within 6 months of the diagnosis of stage IV. As recent reports support that targeted therapies against the RAS-RAF-MEK-MAPK may have marked clinical activity in patients with activating BRAF mutations,15–17 the BRAF patients who had enrolled on non-randomized clinical trials of selective BRAF or MEK inhibitors (n=41) were analyzed separately from BRAF patients who had not (n=112). No BRAF patients in this study were enrolled on randomized protocols of selective BRAF or MEK inhibitors. Patients with uveal melanoma (n=11) were also excluded due to the lack of BRAF or NRAS mutations in these patients and the distinctive clinical course of this disease.
A total of 313 non-uveal metastatic melanoma patients with molecular testing within 6 months of stage IV diagnosis were identified (median time to testing 43 days). The median follow up duration from stage IV diagnosis was 12 months. The NRAS patients (n=66) had the worst outcomes, with a median survival of only 8.2 months from the diagnosis of stage IV, which was significantly shorter than the WT patients (15.1 months, p=0.004) (Figure 1B). The BRAF patients treated with a BRAF or MEK inhibitor had significantly longer survival (n=41, median OS not reached, median follow up 15.6 months) compared to the WT (p=0.0145) patients. BRAF patients who did not receive a BRAF inhibitor (n=112, median OS 10.3 months) had a non-significant trend for shorter survival compared to the WT patients (p=0.10). BRAF patients treated with BRAF inhibitors survived longer than those who were not (p=0.0002).
Multivariate Cox regression modeling of these data was performed for the cohort of 313 patients (Table 5). Consistent with their status as validated prognostic markers in stage IV melanoma, elevated serum LDH (HR 2.75 versus not elevated, p<0.0001), M1b category (HR 3.29 versus M1a, p=0.03) and M1c category (HR 4.02 versus M1a, p=0.007) were independent predictors of survival in this group. In contrast, age (p=0.37) and gender (p=0.91) did not predict survival. The observed differences in survival for NRAS patients (HR 2.05, p=0.005) and for BRAF patients treated with BRAF or MEK inhibitors (HR 0.45, p=0.01) compared to WT patients remained significant on multivariate analysis.
This study represents one of the largest single-institution cohorts of melanoma patients characterized for activating BRAF and NRAS mutations to date, and to our knowledge the largest cohort of stage IV patients. The study is strengthened by the use of a clinically certified mutation detection method for all patients, and by the inclusion of testing for mutations in exon 1 (as well as exon 2) of NRAS, which was not performed in some previous studies in melanoma.12, 18 We report here the novel findings that the presence of an NRAS mutation correlates with shorter survival from the diagnosis of stage IV melanoma, and that the presence of either a BRAF or NRAS mutation is associated with an increased risk of CNS involvement at initial stage IV diagnosis. Our study has also confirmed several previously reported associations with BRAF mutations, including age19, primary tumor site20, and improved survival with targeted therapies against the RAS-RAF-MEK-MAPK pathway.15
Studies of primary tumor characteristics in which both BRAF and NRAS testing were performed have been discordant. Two recent consecutive series of >200 primary melanomas both reported that primary tumors with NRAS mutations were associated with increased Breslow thickness.8, 12 These studies, however, had conflicting findings regarding the relationship for NRAS mutation status with mitotic rate and ulceration. In our study we did not find significant associations between mutation status and primary tumor Breslow thickness, mitotic rate, or ulceration (Table 3). However, as our patient population was selected by retrospectively identifying patients who had undergone clinically indicated mutation testing and thus overwhelmingly consisted of patients who developed distant metastases, it is possible that these results are not representative of all patients with primary melanoma. Of note, the striking similarity of the mutation profile between patients with unknown primary melanomas with those who have cutaneous primary tumors supports the hypothesis that the majority of unknown primary patients had a regressed or occult cutaneous primary rather than an occult mucosal melanoma.
Previous analyses comparing NRAS mutations to patient survival have also reported discordant results. A recent prospective study identified no significant difference in overall survival from initial melanoma diagnosis among NRAS-mutant melanoma patients.8 Another recent prospective study of 249 Australian melanoma patients reported shorter melanoma specific survival from initial melanoma diagnosis for NRAS patients compared to WT.12 This difference was attributable to shorter relapse-free survival (RFS) after initial treatment of local/regional disease among the NRAS patients, a trend also observed in our study (Table 4). The study that identified shorter RFS did not detect a significant difference in survival by NRAS mutation status after the diagnosis of metastatic disease, but its power was limited by a low prevalence of patients with distant metastases and few relevant events for analysis.12 Our subset of 313 non-uveal metastatic melanoma patients with molecular testing within 6 months of stage IV had a 45.4% mortality rate during the examined follow-up period, allowing for improved sensitivity in survival analyses. An additional retrospective study found that the NRAS-mutated tumor genotype was associated with increased overall survival (compared to the BRAF-mutated and WT tumor genotypes).13 However, this study included only 82 patients with stage IV disease. In our study, compared to WT patients, NRAS mutation was associated with shorter survival from stage IV diagnosis, in both the full cohort of all metastatic melanoma patients (n=519), as well as in the cohort who underwent molecular testing within 6 months of the diagnosis of stage IV (n=313). This difference remained significant on multivariate analysis using validated prognostic markers for this disease.
There is no clear etiology for the shorter survival from stage IV for the NRAS-mutant patients. The presence of an NRAS mutation did not correlate with established stage IV prognostic factors such as elevated LDH or advanced M1 category. We did observe an increased rate of CNS involvement at stage IV diagnosis among both BRAF and NRAS patients compared to WT patients. However, survival from stage IV diagnosis for patients without initial CNS involvement was significantly shorter for NRAS (8.3 months) than WT patients (14.3 months, p=0.01) (Figure 2). If an increased prevalence of CNS involvement in BRAF and NRAS patients at stage IV diagnosis is confirmed elsewhere, it could suggest a role for increased CNS surveillance among these patients.
The trend for shorter survival from stage IV diagnosis for BRAF mutant patients who did not receive BRAF or MEK inhibitors compared to BRAF patients treated with these agents is similar to the findings recently reported by Long et al of 197 consecutive advanced melanoma patients.21 The results are also consistent with the results of the recently reported BRIM3 randomized phase III clinical trial of the BRAF inhibitor vemurafenib.15
Our study does have limitations. We included only patients seen at a single tertiary cancer center, and in whom mutation testing was considered clinically indicated. However, our population appears very typical genetically, as the prevalence of BRAF and NRAS mutations in this cohort is nearly identical that that reported in two different meta-analyses of >2,000 patients each.5, 6 In addition, we note the frequencies of V600E and V600K mutations in our cohort are similar to that of the Australian study of 197 advanced melanoma patients21.
The interpretation of the shorter survival among BRAF patients who were not treated with BRAF or MEK inhibitors as compared to WT patients must be viewed with caution, as it is not possible to discern in this study why these patients were not enrolled on clinical trials with BRAF or MEK inhibitors. Finally, additional genetic aberrations may characterize each of the mutation-defined cohorts here, such as PTEN loss22 or activating c-KIT mutations.23
The emergence of BRAF targeted therapies that benefit only patients with activating BRAF mutations will almost certainly lead to clinical trials designed specifically for BRAF wild-type patients in the near future. Identifying effective therapeutic approaches for, and improving outcomes in, melanoma patients with a wild-type BRAF gene is now one of the highest priorities in this disease. Our data support that melanoma patients with an NRAS mutation represent a distinct cohort, with a highly aggressive disease and shorter survival with stage IV disease. Notably, the difference in OS from stage IV between NRAS and WT patients is substantial (~7 months), and is comparable to the OS benefit seen in the recently reported phase III trial of ipilimumab.24 Our results support the need for stratification with respect to NRAS mutation status for the design and interpretation of future melanoma clinical trials for BRAF wild-type patients. In addition, these results highlight the critical need for more effective therapies for melanoma patients with activating NRAS mutations.
Supported by The M. D. Anderson Melanoma Specialized Program in Research Excellence (SPORE) program (P50 CA93459). M.A.D. is supported by an American Society of Clinical Oncology (ASCO) Career Development Award (CDA). Both M.A.D. and A.J.L. are supported by M. D. Anderson Physician-Scientist Program.
Jeffrey Gershenwald, Advisory board(s): Glaxo-Smith-Kline, compensated.
Kevin Kim, Advisory board(s): Genetech and Roche, compensated. Honoraria: Genetech. Research Funding: Astra-Zeneca, Roche, Genetech, and Glaxo-Smith-Kline.
Michael Davies, Consultant: Glaxo-Smith-Kline, compensated. Research Support: AstraZeneca, Glaxo-Smith-Kline, Merck, and Roche.
Alexander Lazar and spouse, Stock Ownership: Roche and Glaxo-Smith-Kline.