Purpose: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS).

Experimental Design: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan–Meier methods.

Results:BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm.

Conclusions: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF. Clin Cancer Res; 20(12); 3328–37. ©2014 AACR.

Translational Relevance

We present the somatic mutation correlative studies for E2603, a randomized Eastern Cooperative Oncology Group (ECOG) clinical phase III trial investigating the benefit of sorafenib added to carboplatin and paclitaxel in patients with advanced-stage melanoma. Although this trial failed to demonstrate a significant contribution of sorafenib, the observed effects of carboplatin/paclitaxel have resulted in this regimen being considered a standard treatment for metastatic melanoma. Our results suggest patients with NRAS-mutant melanoma do worse with chemotherapy alone, possibly explaining the poor prognosis observed in these patients in other studies. However, there was a trend toward improvement in clinical response and progression-free survival in these patients with the addition of sorafenib, equivalent to patients with BRAF-mutant and WT/WT melanomas. This finding suggests that non-BRAF selective MAPK inhibitors may have a role in targeted therapy for this population, an important insight given the lack of molecularly targeted therapeutic options for NRAS-mutant melanoma.

Cutaneous melanoma is the most aggressive form of skin cancer. Its incidence and mortality are increasing, with approximately 76,690 new cases and 9,480 deaths related to melanoma projected for 2013 (1). In the past few years, on the basis of improvement in survival, new therapies were approved for the treatment of advanced-stage melanoma, including the CTLA4 antagonist ipilimumab (Yervoy, Bristol-Myers Squibb), and the targeted BRAF inhibitors vemurafenib (Zelboraf, Genentech) and dabrafenib (Tafinlar, GlaxoSmithKline), and the MEK inhibitor trametinib (Mekinist, GlaxoSmithKline; refs. 2–7). Before the approval of ipilimimab and BRAF inhibitors, no effective, standard treatment existed for advanced melanoma. Patients were treated with dacarbazine or high-dose interleukin-2, which demonstrated limited objective response rates of 10% to 20% (8, 9). Carboplatin and paclitaxel also were used to treat patients with advanced-stage melanoma with similar results (3). In the absence of treatment, overall median survival for patients with advanced melanoma is between 9 and 12 months (3, 10).

Sorafenib (Nexavar, Bayer Pharmaceuticals) is an oral multikinase inhibitor that targets angiogenesis through VEGF receptor (VEGFR) and platelet-derived growth factor receptor β (PDGFR); RAF kinases, including BRAF and CRAF; and other receptor tyrosine kinases (11, 12). Sorafenib has been demonstrated to be effective in the treatment of renal cell, hepatocellular carcinoma, and most recently differentiated thyroid cancer, mainly through its effects on angiogenesis, and is U.S. Food and Drug Administration (FDA)-approved for these indications (13–16). Phase II clinical trials investigated the effect of sorafenib monotherapy in patients with advanced melanoma and demonstrated limited to no response but a tolerable side effect profile (17, 18). A phase I clinical trial investigated the effect of adding sorafenib to carboplatin and paclitaxel in patients with melanoma to increase the effectiveness of chemotherapy, with favorable response rates independent of BRAF mutation status, including 1 complete response (CR) and 9 partial responses (PR) in patients with melanoma (19). Thus, a randomized, double-blinded, placebo-controlled phase III trial was initiated by Eastern Cooperative Oncology Group (ECOG) to investigate the impact of addition of sorafenib to carboplatin and paclitaxel (ECOG 2603). Although the clinical trial did not demonstrate a difference in overall survival (OS) with addition of sorafenib to chemotherapy, it did establish the combination of carboplatin and paclitaxel as a standard of care in treatment of metastatic melanoma, where evidence had been lacking (20).

Somatic mutations identified in melanoma tumor tissues have been shown to play an integral role in melanoma pathogenesis. Mutations in BRAF are found in up to 50% of melanomas, with the most common mutation occurring at codon 600 resulting in the BRAFV600 mutation (21–25). NRAS also is mutated in 15% to 20% of melanomas, with the most common mutation resulting in substitution for the amino acid glutamine (Q) at position 61 (26–29). Additional mutations have been identified within both BRAF and NRAS, which occur at lower frequencies. The association of somatic mutations with clinical response to chemotherapy and targeted therapy is not clearly defined, with the exception of targeted inhibition of mutant BRAF.

The current study presents the somatic mutation correlative studies for the large E2603 clinical trial. We used the pretreatment tumor samples from patients enrolled on the E2603 to assess the association of somatic mutations identified in tumor samples with clinicopathologic features and clinical outcomes. E2603 was a randomized phase III clinical trial of carboplatin/paclitaxel ± sorafenib. Importantly, patients were included without determination of mutation status in E2603, and this unselected population allowed for the evaluation of the natural history of melanoma, before mutant BRAF–specific targeted therapy. We also determined whether somatic mutations in patient tumors were associated with progression-free survival (PFS) and OS, alone or in conjunction with known prognostic markers in melanoma.

Patients

In the double-blind phase III ECOG 2603 clinical trial, patients were randomized to receive carboplatin/paclitaxel [CP (control arm); area under the curve (AUC) of 6 and 225 mg/m2, respectively, every 3 weeks] or carboplatin/paclitaxel plus sorafenib [CPS (experimental arm); 400 mg per os twice daily for days 2–19 of every 21-day cycle; ref. 20]. To be eligible for the study, patients needed to have confirmed melanoma that was unresectable or metastatic; uveal melanomas and patients with brain metastases were excluded. Additional eligibility criteria included measurable disease, age at least 18 years, ECOG performance status (PS) of 0 or 1, and normal baseline laboratory values. Treatment with prior chemotherapy or MAPK pathway targeted therapy were exclusion criteria. All prior therapies, including radiation, needed to be completed at least 4 weeks before trial enrollment.

Patient demographics and tumor characteristics were collected, which included age at diagnosis, gender, American Joint Committee on Cancer (AJCC) stage, ECOG PS, Breslow depth, Clark level, number of sites involved, primary tumor site, ulceration, and lactate dehydrogenase (LDH) level. In addition, the latitudes of individual recruitment sites were identified to determine whether mutation status was associated with sun exposure; degrees of latitude were stratified in groups of 10,000s (e.g., 10,000–20,000).

Melanoma tumor samples and DNA isolation

Available pretreatment tumor samples were obtained from patients enrolled on the E2603 clinical trial who gave consent to use their tumor samples under the approval of the Institutional Review Boards of participating centers. The most recent tissue sample was preferred, either diagnostic biopsy or resection; if metastatic tissue was unavailable, primary melanoma samples were permitted. Patients had provided written consent. Hematoxylin and eosin (H&E) sections of formalin-fixed, paraffin-embedded (FFPE) tumor samples were evaluated for tumor quality and content. A single pathologist (D.L. Rimm) examined each tumor sample to ensure the presence of at least 70% tumor. Tumor samples were macrodissected to more than 70% tumor. DNA was isolated from FFPE tumors, either scraped from slides or extracted from paraffin rolls. After deparafinization, DNA was isolated with sodium thiocyanate, followed by digestion with proteinase K, and precipitated with ammonium acetate. DNA was resuspended in TE and concentrations were determined using picoGreen (Quant-iT PicoGreen dsDNA Reagent, Invitrogen).

Mutation analysis/genotyping

Genotyping was performed using a custom iPlex Sequenom panel interrogating 74 mutations in 13 genes, described in detail in Supplementary Methods. Primers and multiplex design are available upon request. Genotyping was done in the Perelman School of Medicine Molecular Profiling Facility (Philadelphia, PA). Additional details are provided in Supplementary Methods. Sequencing of BRAF exon 11 mutations was performed on all tumor samples that were wild-type (WT) for BRAF, NRAS, KIT, and GNA11/GNAQ. Primers and PCR conditions are found in Supplementary Methods.

Statistical analysis

The Fisher exact test was used to compare BRAF and NRAS mutation by patients' demographic and disease characteristics, and multivariable logistic regression was used to calculate adjusted ORs for BRAF and NRAS mutations for these variables. Fisher exact test and multivariable logistic regression were also used to compare the clinical response (CR plus PR) between treatment arms by BRAF/NRAS mutation status. The distribution of OS and PFS was estimated using the Kaplan–Meier method and was compared between groups using log-rank test. Data from 4 patients with GNA11/GNAQ mutations were excluded from survival analysis as these samples most likely originated from metastases from uveal melanomas. Cox proportional hazards models were used to assess the prognostic value of BRAF and NRAS mutation on OS after adjustment for other variables. OS was defined as time from randomization to death from any cause, and patients who were still alive were censored at the date of last known alive. PFS was defined as time from randomization to disease progression or death from any cause (whichever occurred first), and patients who were still alive and had no disease progression were censored at the date of last disease assessment. All P values were for 2-sided tests and P < 0.05 was considered statistical significance. Because of the exploratory nature of the study, no adjustment was made for multiple comparisons. All analysis was conducted using STATA 11.2 (30).

Mutation frequency

From the 823 patients enrolled on the E2603 clinical trial, 620 archival pretreatment samples were available for analysis. H&E stains of tumor samples were analyzed to determine tumor presence and adequacy for analysis. Of the available samples, 76 (12%) samples had no demonstrable tumor, 241 (39%) samples had insufficient tumor, 22 (4%) samples with H&E slides did not have tumor sample available for processing, and DNA extraction was not performed on one duplicate sample. A total of 280 samples underwent DNA extraction, 243 (87%) of which had adequate DNA for genotyping. Accounting for multiple tumor samples from 31 patients, 179 unique patient samples were analyzed. Eighty-one tumor samples were from patients on the control arm (CP) and 98 tumor samples were from patients on the experimental arm (CPS). No significant difference in patient demographic and disease characteristics was found between the 179 patients and the remaining 643 patients were not included in the present analysis (data not shown). The OS was similar as well (Supplementary Fig. S1). Of the 179 patients included in the current analysis, there was no significant difference between 2 treatment arms regarding known prognostic factors and clinical outcomes (data not shown).

In the 179 analyzable patients, the overall BRAF mutation rate was 45% and NRAS mutation rate was 23% (Table 1). Nearly 33% tumors lacked BRAF or NRAS mutations and are termed WT/WT. Mutations in BRAF and NRAS were mutually exclusive, with the exception of one tumor sample that harbored BRAFV600K and NRASA146P mutations. The 3 observed BRAF exon 11 mutations were p.G464E, p.G469A, and p.G469V. The AKT1E17K mutation was coincident with a BRAFV600E mutation and the AKT3E17K mutation with NRASQ61K mutation. The 6 CDK4R24 mutations, with different amino acid substitutions, were coincident with BRAFV600E mutations. Four of the 5 CTNNB1 (β-catenin) mutations were concurrent with BRAF mutations. One CTNNB1S37F mutation was observed in BRAFV600E-mutant tumor, whereas the other sample was WT/WT. Two CTNNB1S45Y mutations were seen in tumors with BRAFV600R mutations, and one, CTNNB1S45F, with BRAFV600E mutation.

Table 1.

Frequency of somatic mutations in tumor samples

GeneMutationNo. of mutated% mutateda
AKT1 p.E17K 0.56 
AKT3 p.E17K 0.56 
BRAF Exon 11 1.7 
 p.L597P 0.56 
 p.V600 all 78 43.82 (36.4–51.4) 
 p.V600D 0.56 
 p.V600E 56 31.50 (24.7–38.8) 
 p.V600K 16 8.99 (5.2–14.2) 
 p.V600R 2.25 
 p.K601E 1.15 
 Allb 80 44.70 (37.3–52.3) 
CDK4 p.R24C/G/H 3.41 
CTNNB1 p.S37F 1.16 
 p.S45F/Y 1.78 
GNA11 p.Q209L 0.71 
GNAQ p.Q209L/P 1.83 
KIT p.L576P 0.58 
 p.D816V 0.56 
 D820Y 0.57 
NRAS p.G13C/D/R/V 2.96 
 p.Q61 all 35 19.77 (14.2–26.4) 
 p.Q61H 1.13 
 p.Q61K 10 5.65 (2.7–10.1) 
 p.Q61L 2.26 
 p.Q61P 0.56 
 p.Q61R 18 10.17 (6.1–15.6) 
 p.A146P 0.64 
 Allc 41 22.90 (17.0–29.8) 
WT/WT  59 33 (26.1–40.4) 
GeneMutationNo. of mutated% mutateda
AKT1 p.E17K 0.56 
AKT3 p.E17K 0.56 
BRAF Exon 11 1.7 
 p.L597P 0.56 
 p.V600 all 78 43.82 (36.4–51.4) 
 p.V600D 0.56 
 p.V600E 56 31.50 (24.7–38.8) 
 p.V600K 16 8.99 (5.2–14.2) 
 p.V600R 2.25 
 p.K601E 1.15 
 Allb 80 44.70 (37.3–52.3) 
CDK4 p.R24C/G/H 3.41 
CTNNB1 p.S37F 1.16 
 p.S45F/Y 1.78 
GNA11 p.Q209L 0.71 
GNAQ p.Q209L/P 1.83 
KIT p.L576P 0.58 
 p.D816V 0.56 
 D820Y 0.57 
NRAS p.G13C/D/R/V 2.96 
 p.Q61 all 35 19.77 (14.2–26.4) 
 p.Q61H 1.13 
 p.Q61K 10 5.65 (2.7–10.1) 
 p.Q61L 2.26 
 p.Q61P 0.56 
 p.Q61R 18 10.17 (6.1–15.6) 
 p.A146P 0.64 
 Allc 41 22.90 (17.0–29.8) 
WT/WT  59 33 (26.1–40.4) 

aCalculated as percentage of patients with data available; exact binomial 95% CIs included when numbers sufficient.

bBRAF_all includes all BRAF V600 and BRAF K601 mutations and excludes BRAF exon 11 mutations.

cNRAS_all includes all NRAS mutations, NRAS G13, NRAS A146, and NRAS Q61.

Association of mutations with patient demographics and disease characteristics

At the time of initial diagnosis, patients with BRAF mutant melanomas trended toward earlier age of diagnosis (median age cutoff = 58), whereas those with WT/WT melanomas trended toward older age of diagnosis (Table 2). Patients with BRAF-mutant melanomas had a worse ECOG PS than those patients with NRAS-mutant or WT/WT melanomas (P = 0.038). Patients with BRAF-mutant melanoma had an increased number of disease sites; 41% of patients with BRAF-mutant melanoma had ≥4 sites involved as compared with patients with NRAS-mutant (15%) or WT/WT melanomas (22%; P = 0.026). To assess the effect of sun exposure on mutation status, we used proximity to the equator as a surrogate and stratified recruitment locations by latitude. No mutation was associated with latitude (Table 2).

Table 2.

Correlation of mutation status with patient demographics and disease characteristics

WT/WTMutant BRAFMutant NRAS
Demographics and disease characteristicsn (%)n (%)n (%)P value
Age at diagnosisa 
 Young 23 (39.66) 42 (57.53) 19 (50) 0.127 
 Old 35 (60.34) 31 (42.47) 19 (50)  
Gender 
 Male 40 (67.80) 51 (64.56) 22 (55) 0.416 
 Female 19 (32.20) 28 (35.44) 18 (45)  
AJCC stage 
 Unresectable stage III 5 (8.48) 11 (13.92) 7 (17.50) 0.747 
 M1a/M1b 21 (35.59) 25 (31.65) 13 (32.50)  
 M1c 33 (55.93) 43 (54.43) 20 (50)  
ECOG PS 
 0 38 (64.41) 38 (48.10) 28 (70) 0.038 
 1 21 (35.59) 41 (51.90) 12 (30)  
Number of involved sites 
 1 13 (22.03) 13 (16.46) 12 (30) 0.026 
 2–3 33 (55.93) 34 (43.04) 22 (55)  
 ≥4 13 (22.03) 32 (40.51) 6 (15)  
Primary site 
 Lower limb 12 (20.34) 9 (11.39) 9 (22.50) 0.107 
 Trunk 11 (18.64) 29 (36.71) 13 (32.50)  
 Other 36 (61.02) 41 (51.90) 18 (45)  
Latitudeb 
 40–50 35 (59.32) 44 (55.70) 26 (65) 0.751 
 30–40 22 (37.29) 29 (36.71) 12 (30)  
 20–30 2 (3.39) 6 (7.60) 2 (5)  
Ulceration 
 No 21 (35.59) 34 (43.04) 18 (45) 0.880 
 Yes 22 (37.29) 25 (31.65) 12 (30)  
 Unknown 16 (27.12) 20 (25.32) 10 (25)  
LDH level 
 Normal 36 (62.07) 41 (52.56) 23 (60.53) 0.493 
 Elevated 22 (37.93) 37 (47.44) 15 (39.47)  
Breslow depth 
 Low 17 (28.81) 27 (34.18) 18 (45) 0.370 
 High 21 (35.59) 21 (26.58) 12 (30)  
 Missing 21 (35.59) 31 (39.24) 10 (25)  
Clark levelc 
 SubQ fat 13 (36.11) 8 (16) 7 (25.93) 0.076 
 Reticular 19 (52.78) 34 (68) 12 (44.44)  
 Other 4 (11.11) 8 (16) 8 (29.63)  
WT/WTMutant BRAFMutant NRAS
Demographics and disease characteristicsn (%)n (%)n (%)P value
Age at diagnosisa 
 Young 23 (39.66) 42 (57.53) 19 (50) 0.127 
 Old 35 (60.34) 31 (42.47) 19 (50)  
Gender 
 Male 40 (67.80) 51 (64.56) 22 (55) 0.416 
 Female 19 (32.20) 28 (35.44) 18 (45)  
AJCC stage 
 Unresectable stage III 5 (8.48) 11 (13.92) 7 (17.50) 0.747 
 M1a/M1b 21 (35.59) 25 (31.65) 13 (32.50)  
 M1c 33 (55.93) 43 (54.43) 20 (50)  
ECOG PS 
 0 38 (64.41) 38 (48.10) 28 (70) 0.038 
 1 21 (35.59) 41 (51.90) 12 (30)  
Number of involved sites 
 1 13 (22.03) 13 (16.46) 12 (30) 0.026 
 2–3 33 (55.93) 34 (43.04) 22 (55)  
 ≥4 13 (22.03) 32 (40.51) 6 (15)  
Primary site 
 Lower limb 12 (20.34) 9 (11.39) 9 (22.50) 0.107 
 Trunk 11 (18.64) 29 (36.71) 13 (32.50)  
 Other 36 (61.02) 41 (51.90) 18 (45)  
Latitudeb 
 40–50 35 (59.32) 44 (55.70) 26 (65) 0.751 
 30–40 22 (37.29) 29 (36.71) 12 (30)  
 20–30 2 (3.39) 6 (7.60) 2 (5)  
Ulceration 
 No 21 (35.59) 34 (43.04) 18 (45) 0.880 
 Yes 22 (37.29) 25 (31.65) 12 (30)  
 Unknown 16 (27.12) 20 (25.32) 10 (25)  
LDH level 
 Normal 36 (62.07) 41 (52.56) 23 (60.53) 0.493 
 Elevated 22 (37.93) 37 (47.44) 15 (39.47)  
Breslow depth 
 Low 17 (28.81) 27 (34.18) 18 (45) 0.370 
 High 21 (35.59) 21 (26.58) 12 (30)  
 Missing 21 (35.59) 31 (39.24) 10 (25)  
Clark levelc 
 SubQ fat 13 (36.11) 8 (16) 7 (25.93) 0.076 
 Reticular 19 (52.78) 34 (68) 12 (44.44)  
 Other 4 (11.11) 8 (16) 8 (29.63)  

NOTE: P values were from Fisher exact test for categorical and binary variables.

Abbreviation: SubQ fat, subcutaneous fat.

aMedian age cutoff = 58 years.

bLatitudes are presented in quintiles, representing degrees of latitude in 10,000s from the equator (e.g., 20–30, 20,000–30,000 degrees of latitude).

cFor Clark level, other includes interface papillary-reticular dermis, extension into papillary dermis, and above basal lamina.

Multivariable logistic regression demonstrated that the BRAF mutation is associated with younger age of diagnosis [OR, 2.29; 95% confidence interval (CI), 1.10–4.77], ≥4 sites of disease (OR, 4.1; 95% CI, 1.36–12.38), and disease involvement in the reticular dermis for primary tumors (OR, 3.49; 95% CI, 1.01–12.10; Supplementary Table S1). Truncal primary melanomas tended to have a BRAF mutation (OR, 3.10; 95% CI, 0.95–10.08). Patients were more likely to have an NRAS mutation if they were female (OR, 2.79; 95% CI, 1.10–7.07) and less likely to have an NRAS mutation with ≥4 sites of disease (OR, 0.31; 95% CI, 0.08–1.13; Supplementary Table S1).

We further evaluated disease characteristics of patients whose melanoma had specific BRAFV600 mutations (Table 3) as prior studies have demonstrated different clinicopathologic characteristics (31). Patients with melanomas carrying V600E or V600K mutations demonstrated differences in ages at diagnosis and PS. Patients with BRAFV600E mutants tended to be a younger at diagnosis (P = 0.051) and had a significantly worse PS than patients whose melanomas carried BRAFV600K (P < 0.001).

Table 3.

Correlation of specific BRAFV600 mutations with select patient demographics and disease characteristics

WT BRAFMutant BRAFV600EV600K
Demographics and disease characteristicsn (%)n (%)P valuen (%)n (%)P value
Age at diagnosisa 
 Young 42 (43.8) 43 (58.1) 0.063 34 (68) 6 (40) 0.051 
 Old 54 (56.3) 31 (41.9)  16 (32) 9 (60)  
ECOG PS 
 0 66 (66.7) 39 (48.8) 0.016 19 (34.6) 13 (81.3) <0.001 
 1 33 (33.3) 41 (51.3)  36 (65.5) 3 (18.8)  
Ulceration 
 No 39 (39.4) 35 (43.8) 0.836 24 (43.6) 6 (37.5) 0.906 
 Yes 34 (34.3) 25 (31.3)  19 (34.6) 6 (37.5)  
 Unknown 26 (26.3) 20 (25)  12 (21.8) 4 (25)  
LDH level 
 Normal 59 (61.5) 42 (53.2) 0.269 29 (52.7) 7 (46.7) 0.677 
 Elevated 37 (38.5) 37 (46.8)  26 (47.3) 8 (53.3)  
Breslow depth 
 Low 35 (35.4) 28 (35) 0.486 21 (38.2) 3 (18.8) 0.196 
 High 33 (33.3) 21 (26.3)  16 (29.1) 4 (25)  
 Missing 31 (31.3) 31 (38.8)  18 (32.7) 9 (56.3)  
Clark levelb 
 SubQ fat 20 (31.8) 8 (15.7) 0.083 4 (10.5) 3 (37.5) 0.095 
 Reticular 31 (49.2) 35 (68.6)  27 (71.1) 5 (62.5)  
 Other 12 (19.1) 8 (15.7)  7 (18.4) 0 (0)  
WT BRAFMutant BRAFV600EV600K
Demographics and disease characteristicsn (%)n (%)P valuen (%)n (%)P value
Age at diagnosisa 
 Young 42 (43.8) 43 (58.1) 0.063 34 (68) 6 (40) 0.051 
 Old 54 (56.3) 31 (41.9)  16 (32) 9 (60)  
ECOG PS 
 0 66 (66.7) 39 (48.8) 0.016 19 (34.6) 13 (81.3) <0.001 
 1 33 (33.3) 41 (51.3)  36 (65.5) 3 (18.8)  
Ulceration 
 No 39 (39.4) 35 (43.8) 0.836 24 (43.6) 6 (37.5) 0.906 
 Yes 34 (34.3) 25 (31.3)  19 (34.6) 6 (37.5)  
 Unknown 26 (26.3) 20 (25)  12 (21.8) 4 (25)  
LDH level 
 Normal 59 (61.5) 42 (53.2) 0.269 29 (52.7) 7 (46.7) 0.677 
 Elevated 37 (38.5) 37 (46.8)  26 (47.3) 8 (53.3)  
Breslow depth 
 Low 35 (35.4) 28 (35) 0.486 21 (38.2) 3 (18.8) 0.196 
 High 33 (33.3) 21 (26.3)  16 (29.1) 4 (25)  
 Missing 31 (31.3) 31 (38.8)  18 (32.7) 9 (56.3)  
Clark levelb 
 SubQ fat 20 (31.8) 8 (15.7) 0.083 4 (10.5) 3 (37.5) 0.095 
 Reticular 31 (49.2) 35 (68.6)  27 (71.1) 5 (62.5)  
 Other 12 (19.1) 8 (15.7)  7 (18.4) 0 (0)  

NOTE: P values were from Fisher exact test for categorical and binary variables.

Abbreviation: SubQ fat, subcutaneous fat.

aMedian age cutoff = 58 years.

bFor Clark level, other includes interface papillary-reticular dermis, extension into papillary dermis, and above basal lamina.

Association of mutations with clinical outcomes

In patients with BRAF-mutant melanoma, the clinical response rate (CR plus PR) was 15.6% (5 of 32) and 19.2% (9 of 47) for control and experimental arms, respectively (P = 0.771; Table 4). In patients with NRAS-mutant melanoma, the clinical response rates were 5.6% (1 of 18) and 22.7% (5 of 22) for the control and experimental arms, respectively (P = 0.197). In WT/WT melanoma, the clinical response rate was similar in the control and experimental arms, 16.7% (5 of 30) and 20.0% (5 of 25), respectively (P > 0.05). The treatment-by-mutation interaction test was not statistically significant in logistic model for clinical response (Table 4).

Table 4.

Correlation of response rates with mutation status and study arm

Control armaExperimental arma
Mutated geneNon-responseResponseNon-responseResponseORb (95% CI)
BRAF 84.4 15.6 80.9 19.2 1.99 (0.48–8.30) 
 V600E 81.0 19.0 85.3 14.7 0.73 (0.17–3.11) 
 V600K 83.3 16.7 70.0 30.0 2.14 (0.17–27.10) 
NRAS 94.4 5.6 77.3 22.7 4.26 (0.36–49.74) 
WT/WT 83.3 16.7 80.0 20.0 1.28 (0.28–5.91) 
Control armaExperimental arma
Mutated geneNon-responseResponseNon-responseResponseORb (95% CI)
BRAF 84.4 15.6 80.9 19.2 1.99 (0.48–8.30) 
 V600E 81.0 19.0 85.3 14.7 0.73 (0.17–3.11) 
 V600K 83.3 16.7 70.0 30.0 2.14 (0.17–27.10) 
NRAS 94.4 5.6 77.3 22.7 4.26 (0.36–49.74) 
WT/WT 83.3 16.7 80.0 20.0 1.28 (0.28–5.91) 

NOTE: Response = CR + PR; Non-response = stable disease (SD) + progression (PD) + unevaluable.

aP > 0.05 from Fisher exact test for all comparisons of response rate between 2 treatment arms.

bORs of experimental arm/control arm for tumor response, calculated using multivariable logistic model with treatment-by-mutation interaction term, adjusting for other demographic and disease characteristics. P > 0.05 for the treatment-by-mutation interaction tests.

In patients with BRAF-mutant melanoma, the median PFS were 2.2 and 5.0 months for the control and experimental arms, respectively, and median OS were 8.8 and 8.9 months for control and experimental arm, respectively (Fig. 1A and D). In patients with NRAS-mutant melanoma, the median PFS were 3.0 and 5.1 months for control and experimental arms, respectively, and the median OS were 9.8 and 10.3 months for control and experimental arms, respectively (Fig. 1B and E). In patients with WT/WT melanoma, the median PFS were 4.5 and 5.8 months for control and experimental arms, respectively, and median OS were 10.0 and 12.1 months for the control and experimental arms, respectively (Fig. 1C and F).

Figure 1.

Kaplan–Meier estimates of PFS and OS based on mutation status. A–C, Kaplan–Meier estimates for PFS for patients with BRAF mutations, NRAS mutations, and WT/WT, respectively, for control and experimental arms. (D–F) Kaplan–Meier estimates for OS for patients with BRAF mutations, NRAS mutations, and WT/WT, respectively, for control and experimental arms.

Figure 1.

Kaplan–Meier estimates of PFS and OS based on mutation status. A–C, Kaplan–Meier estimates for PFS for patients with BRAF mutations, NRAS mutations, and WT/WT, respectively, for control and experimental arms. (D–F) Kaplan–Meier estimates for OS for patients with BRAF mutations, NRAS mutations, and WT/WT, respectively, for control and experimental arms.

Close modal

The results of this study demonstrated no difference between the 2 treatment arms in OS and PFS and no association with treatment outcome and BRAF and NRAS mutations. Consequently, the treatment arms were collapsed as called for in our prospectively defined analysis plan, and we examined the relationship of BRAF and NRAS mutations with OS. Multivariable Cox regression was used to assess the prognostic value of BRAF and NRAS mutations on OS after adjustment for additional prognostic variables known to influence melanoma prognosis (Table 5). Our results demonstrate that BRAF and NRAS mutations are not prognostic factors for OS in E2603 patients. Worse PS (HR, 2.22; 95% CI, 1.46–3.37; P < 0.001) and elevated LDH (HR, 2.16; 95% CI, 1.42–3.28; P < 0.001) predicted for worse survival in patients. The primary site of melanoma also was a significant prognostic marker in this study. Patients with truncal primary melanomas, when compared with lower limb melanomas, had worse survival (HR, 2.41; 95% CI, 1.29–4.49; P = 0.006) as did patients with melanomas at sites other than lower limb and trunk (HR, 1.99; 95% CI, 1.13–3.52; P = 0.017). When years since diagnosis was analyzed as a continuous variable, increased time was associated with an increased survival (HR, 0.94; 95% CI, 0.89–1; P = 0.039).

Table 5.

Cox regression for OS

Demographic and disease characteristicsLevelsHR (95% CI)P
BRAF/NRAS Mutant BRAF vs. WT/WT 1.24 (0.78–1.96) 0.361 
 Mutant NRAS vs. WT/WT 0.91 (0.55–1.51) 0.716 
Treatment CPS vs. CP 0.74 (0.50–1.08) 0.119 
Age at diagnosis,a≥58 vs. <58 1.11 (0.74–1.65) 0.611 
Years since diagnosis Continuous 0.94 (0.89–1.00) 0.039 
Gender Female vs. male 1.14 (0.73–1.76) 0.565 
AJCC stage M1a/M1b vs. unresectable stage III 0.64 (0.34–1.20) 0.163 
 M1c vs. unresectable stage III 0.76 (0.41–1.39) 0.373 
ECOG PS 1 vs. 0 2.22 (1.46–3.37) ≤0.001 
Prior therapy IFN/IL2/GM-CSF vs. none 0.94 (0.65–1.37) 0.754 
 One investigational therapy vs. none 3.14 (0.95–10.43) 0.062 
Number of involved sites 2–3 vs. 1 1.31 (0.80–2.14) 0.283 
 ≥4 vs. 1 1.02 (0.55–1.88) 0.957 
Ulceration Yes vs. no 1.14 (0.72–1.80) 0.571 
 Unknown vs. no 0.98 (0.53–1.82) 0.960 
Clark levelb Reticular vs. subQ fat 0.42 (0.22–0.77) 0.006 
 Other vs. subQ fat 0.65 (0.29–1.49) 0.311 
 Unknown vs. subQ fat 0.82 (0.39–1.73) 0.600 
LDH Elevated vs. normal 2.16 (1.42–3.28) ≤0.001 
Breslow depth High vs. low 0.95 (0.55–1.63) 0.852 
 Missing vs. low 0.85 (0.43–1.70) 0.648 
Primary site Trunk vs. lower limb 2.41 (1.29–4.49) 0.006 
 Other vs. lower limb 1.99 (1.13–3.52) 0.017 
Demographic and disease characteristicsLevelsHR (95% CI)P
BRAF/NRAS Mutant BRAF vs. WT/WT 1.24 (0.78–1.96) 0.361 
 Mutant NRAS vs. WT/WT 0.91 (0.55–1.51) 0.716 
Treatment CPS vs. CP 0.74 (0.50–1.08) 0.119 
Age at diagnosis,a≥58 vs. <58 1.11 (0.74–1.65) 0.611 
Years since diagnosis Continuous 0.94 (0.89–1.00) 0.039 
Gender Female vs. male 1.14 (0.73–1.76) 0.565 
AJCC stage M1a/M1b vs. unresectable stage III 0.64 (0.34–1.20) 0.163 
 M1c vs. unresectable stage III 0.76 (0.41–1.39) 0.373 
ECOG PS 1 vs. 0 2.22 (1.46–3.37) ≤0.001 
Prior therapy IFN/IL2/GM-CSF vs. none 0.94 (0.65–1.37) 0.754 
 One investigational therapy vs. none 3.14 (0.95–10.43) 0.062 
Number of involved sites 2–3 vs. 1 1.31 (0.80–2.14) 0.283 
 ≥4 vs. 1 1.02 (0.55–1.88) 0.957 
Ulceration Yes vs. no 1.14 (0.72–1.80) 0.571 
 Unknown vs. no 0.98 (0.53–1.82) 0.960 
Clark levelb Reticular vs. subQ fat 0.42 (0.22–0.77) 0.006 
 Other vs. subQ fat 0.65 (0.29–1.49) 0.311 
 Unknown vs. subQ fat 0.82 (0.39–1.73) 0.600 
LDH Elevated vs. normal 2.16 (1.42–3.28) ≤0.001 
Breslow depth High vs. low 0.95 (0.55–1.63) 0.852 
 Missing vs. low 0.85 (0.43–1.70) 0.648 
Primary site Trunk vs. lower limb 2.41 (1.29–4.49) 0.006 
 Other vs. lower limb 1.99 (1.13–3.52) 0.017 

Abbreviation: subQ fat, subcutaneous fat.

aMedian age cutoff = 58 years.

bFor Clark level, other includes interface papillary-reticular dermis, extension into papillary dermis, and above basal lamina.

We investigated the correlation between somatic mutations and clinical outcome in patients with melanoma treated on the E2603 randomized phase III clinical trial. Notably, patients were randomized to treatment arms without prior knowledge of mutation status. Tumor samples from patients on the E2603 clinical trial also provided a large sample population in which to study the natural history of melanoma, before the era of BRAF-targeted therapy. We identified mutations in BRAF, NRAS, and WT/WT melanomas at frequencies consistent with published data (23, 31, 32). We also identified less frequent mutations in BRAF (i.e., BRAFK601E and exon 11 mutations BRAFG464E, BRAFG469A, and BRAFG469V) and NRAS (i.e., G13R, G13V, G13C, and G13D), as well as low-frequency mutations in other genes integral to melanoma pathogenesis. We identified mutations in GNA11/GNAQ in 4 tumor samples. These tumors most likely represent metastatic uveal melanomas as patients with melanoma of unknown primary were permitted on the study, which were excluded in data analysis.

We evaluated the association of mutations with patient demographics and disease characteristics combining data from control and experimental arms, as no differences were identified between the 2 arms. BRAF mutations were associated with worse PS and increased number of sites of disease as compared with NRAS mutations and WT/WT melanomas. Younger age at diagnosis emerged as significant in logistic regression analysis. Truncal primary site of melanoma was marginally associated with higher proportion of BRAF mutation in bivariate analysis and significantly so in multivariable analysis. These results are consistent with observations that have been previously reported (23, 31, 33–35).

Patients with NRAS-mutant melanoma had better PS (PS = 0) and fewer sites of disease involvement (<4) than patients with BRAF-mutant melanoma. The increased number of sites of disease involvement associated with BRAF mutation has not been previously reported in the literature and is a new observation in this set of tumor samples. Also, the presence of an NRAS mutation was associated with female gender in multivariable analysis. Previous studies have not observed an association with female gender but have observed associations of NRAS mutations with increased Breslow depth and older age at diagnosis (25, 26, 32, 33, 36).

Interestingly, patients with BRAFV600K-mutant melanoma had significantly better PS than those with BRAFV600E-mutant melanoma. Moreover, patients with BRAFV600K-mutant melanoma tended to have an older age at the time of diagnosis than those with BRAFV600E-mutant melanoma, which, although not statistically significant, is consistent with prior observations (31). This finding may be due to the double nucleotide change that is observed with BRAFV600K mutation, which could be associated with sun exposure and increased at older ages.

Overall, BRAF and NRAS mutations were not predictive of tumor response to sorafenib in the study. The number of samples with sufficient DNA significantly limited our power to detect potentially meaningful differences in outcome. However, it is important to note that it appears that there was a trend toward a difference in clinical response in patients with NRAS mutations. Patients with NRAS-mutant melanoma demonstrated a response rate similar to BRAF-mutant and WT/WT melanomas when sorafenib was added to chemotherapy (5.6% vs. 22.7% for NRAS; 15.6% vs. 19.2% for BRAF; and 16.7% vs. 20% for WT/WT), a result that remains potentially important in light of the current lack of targeted therapy options for these patients. In addition, patients with NRAS-mutant melanoma experienced increased PFS with the addition of sorafenib to chemotherapy, with PFS similar to those observed with BRAF-mutant or WT/WT melanomas. Similar results were observed in NRAS-mutant melanomas in the phase I trial of carboplatin, paclitaxel, and sorafenib (19); an increased response rate was observed in patients with NRAS-mutant melanoma. Our study was underpowered to draw definitive conclusions regarding outcomes in the NRAS-mutant subset. Sorafenib has activity against multiple kinases, including CRAF (12, 37, 38), and we hypothesize that improved responses observed in patients with NRAS mutations are due to CRAF inhibition (39–41), although effects on angiogenesis cannot be ruled out. However, in light of the lack of targeted therapies for NRAS-mutant melanoma, sorafenib, or other MAPK pathway inhibitors, might warrant investigation in this subset. Of note, there are ongoing clinical trials investigating the effect of MEK inhibitors, either alone or in combination with CDK4/6 or PI3K/mTOR inhibitors, in patients with NRAS mutations (www.clinicaltrials.gov), for which results are eagerly anticipated.

Cox regression for OS demonstrated that neither BRAF nor NRAS mutation is a prognostic biomarker for OS in patients with melanoma on ECOG 2603. Previous findings have demonstrated conflicting reports regarding the prognostic value of NRAS and BRAF mutations (23, 32, 33, 36, 42). No difference in OS was detected in 223 primary melanomas when stratified on the basis of mutational status, specifically BRAF-mutant, NRAS-mutant, and WT melanomas (36). Another study demonstrated no difference in time to development of metastatic disease in patients with melanoma with BRAF mutations versus BRAF wild-type (23); NRAS mutations were not evaluated in this study. In a panel of 249 primary invasive melanomas, the presence of an NRAS mutation was found to be an independent predictor of worse melanoma-specific survival (33). However, in this study, only 14% (36 of 249) of the tumor samples had an NRAS mutation, compared with 45% (112 of 249) BRAFV600E and 40% (101 of 249) WT. Mutation status was not associated with a survival difference once patients had developed metastatic disease (33). In a larger study analyzing 677 melanoma tumor samples, patients with NRAS mutations (20.1%, 136 of 677) had a shorter median OS from time of metastatic diagnosis than patients with WT melanomas (32). Moreover, NRAS mutations were independently associated with decreased survival in multivariate analysis (32). It has not been clear whether the differential survival in patients with NRAS-mutant melanoma is due to intrinsic differences in the biology of NRAS-, BRAF-mutant, and WT/WT melanomas or because NRAS-mutant melanomas respond more poorly to the therapies used. We did not observe a difference in survival based on melanoma mutation status, but the limited size of our sample set may have limited our power to detect a difference. However, it is important to note that we observed worse response rates with chemotherapy alone in patients with NRAS-mutant melanoma than in those with BRAF-mutant and WT/WT melanomas. These data suggest that the reason for the decreased survival in patients with NRAS-mutant melanoma observed in prior studies may be a decreased response to chemotherapy, rather than other differences in the biology of NRAS-mutant melanomas, particularly notable in this sample set, in which patients with NRAS-mutant melanoma had a better PS and fewer sites of disease.

Our study was limited in the number of samples available for analysis for each treatment subgroup. Although 823 patients were enrolled on this clinical trial, biopsy samples were not a requirement for trial entry and were obtained from patients who consented for use of their available tumor samples. Thus, only 179 samples were successfully genotyped and included in the current analysis. However, the individuals from whom samples were genotyped were not significantly different compared with all of the patients on E2603. The issue of insufficient tissue highlights the importance of prospective tissue collection in future clinical trials to empower predictive biomarker investigations.

In conclusion, we evaluated tumor samples from patients with advanced melanoma (unresectable stage III or IV) prospectively recruited from a wide range of institutions, representing a varied collection of melanoma tumor samples. We have demonstrated associations between mutation status and clinicopathologic features and patient and disease characteristics. Moreover, while our data are underpowered for the analysis of genetically defined subsets, the observed associations suggest that targeting the MAPK pathway, with sorafenib in this study, may have an effect on clinical outcome in patients with certain mutations, particularly NRAS-mutant melanoma. Ongoing clinical trials also seek to target the MAPK pathway to more effectively treat NRAS-mutant melanoma.

D.L. Rimm is a consultant/advisory board member for Genoptix. J.M. Kirkwood is a consultant/advisory board member for Bristol-Myers Squibb, Celgene, GlaxoSmithKline, Merck, Vical, and Ziopharm and reports receiving a commercial research grant from Prometheus. K.L. Nathanson reports receiving a commercial research grant from GlaxoSmithKline. No potential conflicts of interest were disclosed by the other authors.

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute.

Conception and design: J.M. Kirkwood, S.J. Lee, L.M. Schuchter, K.T. Flaherty, K.L. Nathanson

Development of methodology: K. D'Andrea, J.M. Kirkwood, K.L. Nathanson

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.A. Wilson, R. Letrero, K. D'Andrea, H.M. Kluger, K.T. Flaherty, K.L. Nathanson

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.A. Wilson, F. Zhao, D.L. Rimm, S.J. Lee, K.T. Flaherty, K.L. Nathanson

Writing, review, and/or revision of the manuscript: M.A. Wilson, F. Zhao, J.M. Kirkwood, H.M. Kluger, L.M. Schuchter, K.T. Flaherty, K.L. Nathanson

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.A. Wilson, K. D'Andrea, K.L. Nathanson

Study supervision: L.M. Schuchter, K.L. Nathanson

This study was coordinated by the Eastern Cooperative Oncology Group (Robert L. Comis, MD, Chair) and supported in part by Public Health Service Grants CA23318, CA66636, CA21115, CA15488, CA14958, CA39229 and from the National Cancer Institute, NIH and the Department of Health and Human Services. This research was funded in part by a NCI Cancer Center Research Training Program Grant T32 CA009615 (PI: Dr. John Maris; to M.A. Wilson), NIH Grant R01 CA115756 (to H.M. Kluger), and NIH GRANT RO1 CA118871 (to K.T. Flaherty and K.L. Nathanson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Howlader
N
,
Noone
AM
,
Krapcho
M
,
Garshell
J
,
Neyman
N
,
Altekruse
SF
, et al
(
eds
) 
SEER Cancer Statistics Review, 1975–2010, National Cancer Institute
.
Bethesda, MD
, http://seer.cancer.gov/csr/1975_2010/,
based on November 2012 SEER data submission, posted to the SEER web site, April 2013
.
2.
Chapman
PB
,
Hauschild
A
,
Robert
C
,
Haanen
JB
,
Ascierto
P
,
Larkin
J
, et al
Improved survival with vemurafenib in melanoma with BRAF V600E mutation
.
N Engl J Med
2011
;
364
:
2507
16
.
3.
Hodi
FS
,
O'Day
SJ
,
McDermott
DF
,
Weber
RW
,
Sosman
JA
,
Haanen
JB
, et al
Improved survival with ipilimumab in patients with metastatic melanoma
.
N Engl J Med
2010
;
363
:
711
23
.
4.
Falchook
GS
,
Lewis
KD
,
Infante
JR
,
Gordon
MS
,
Vogelzang
NJ
,
DeMarini
DJ
, et al
Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial
.
Lancet Oncol
2012
;
13
:
782
9
.
5.
Flaherty
KT
,
Robert
C
,
Hersey
P
,
Nathan
P
,
Garbe
C
,
Milhem
M
, et al
Improved survival with MEK inhibition in BRAF-mutated melanoma
.
N Engl J Med
2012
;
367
:
107
14
.
6.
Hauschild
A
,
Grob
JJ
,
Demidov
LV
,
Jouary
T
,
Gutzmer
R
,
Milward
M
, et al
Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial
.
Lancet
2012
;
380
:
358
65
.
7.
Kim
KB
,
Kefford
R
,
Pavlick
AC
,
Infante
JR
,
Ribas
A
,
Sosman
JA
, et al
Phase II study of the MEK1/MEK2 inhibitor Trametinib in patients with metastatic BRAF-mutant cutaneous melanoma previously treated with or without a BRAF inhibitor
.
J Clin Oncol
2013
;
31
:
482
9
.
8.
Atkins
MB
,
Kunkel
L
,
Sznol
M
,
Rosenberg
SA
. 
High-dose recombinant interleukin-2 therapy in patients with metastatic melanoma: long-term survival update
.
Cancer J Sci Am
2000
;
6
Suppl 1
:
S11
4
.
9.
Comis
RL
: 
DTIC (NSC-45388) in malignant melanoma: a perspective
.
Cancer Treat Rep
1976
;
60
:
165
76
.
10.
Sosman
JA
,
Kim
KB
,
Schuchter
L
,
Gonzalez
R
,
Pavlick
AC
,
Weber
JS
, et al
Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib
.
N Engl J Med
2012
;
366
:
707
14
.
11.
Qi
RQ
,
He
L
,
Zheng
S
,
Hong
Y
,
Ma
L
,
Zhang
S
, et al
BRAF exon 15 T1799A mutation is common in melanocytic nevi, but less prevalent in cutaneous malignant melanoma, in Chinese Han
.
J Invest Dermatol
2011
;
131
:
1129
38
.
12.
Wilhelm
SM
,
Adnane
L
,
Newell
P
,
Villanueva
A
,
Llovet
JM
,
Lynch
M
. 
Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling
.
Mol Cancer Ther
2008
;
7
:
3129
40
.
13.
Escudier
B
,
Eisen
T
,
Stadler
WM
,
Szczylik
C
,
Oudard
C
,
Siebels
M
, et al
Sorafenib in advanced clear-cell renal-cell carcinoma
.
N Engl J Med
2007
;
356
:
125
34
.
14.
Strumberg
D
,
Clark
JW
,
Awada
A
,
Moore
MJ
,
Richly
H
,
Hendlisz
A
, et al
Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: a review of four phase I trials in patients with advanced refractory solid tumors
.
Oncologist
2007
;
12
:
426
37
.
15.
Strumberg
D
,
Richly
H
,
Hilger
RA
,
Schleucher
N
,
Korfee
S
,
Tewew
M
, et al
Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors
.
J Clin Oncol
2005
;
23
:
965
72
.
16.
Brose
MS
,
Nutting
C
,
Jarzab
B
,
Elisei
R
,
Siena
S
,
Bastholt
L
, et al
Sorafenib in locally advanced or metastatic patients with radioactive iodine-refractory differentiated thyroid cancer: the phase III DECISION trial
.
J Clin Oncol
2013
;
31
(suppl; abstr 4).
17.
Eisen
T
,
Ahmad
T
,
Flaherty
KT
,
Gore
M
,
Kaye
S
,
Marais
R
, et al
Sorafenib in dvanced melanoma: a Phase II randomised discontinuation trial analysis
.
Br J Cancer
2006
;
95
:
581
6
.
18.
Ott
PA
,
Hamilton
A
,
Min
C
,
Safarzadeh-Amiri
S
,
Goldberg
L
,
Yoon
J
, et al
A phase II trial of sorafenib in metastatic melanoma with tissue correlates
.
PLoS One
2010
;
5
:
e15588.
19.
Flaherty
KT
,
Schiller
J
,
Schuchter
LM
,
Liu
G
,
Tuveson
DA
,
Redlinger
M
, et al
A phase I trial of the oral, multikinase inhibitor sorafenib in combination with carboplatin and paclitaxel
.
Clin Cancer Res
2008
;
14
:
4836
42
.
20.
Flaherty
KT
,
Lee
SJ
,
Zhao
F
,
Schuchter
LM
,
Flaherty
L
,
Kefford
R
, et al
Phase III trial of carboplatin and paclitaxel with or without sorafenib in metastatic melanoma
.
J Clin Oncol
2013
;
31
:
373
9
.
21.
Chin
L
,
Garraway
LA
,
Fisher
DE
. 
Malignant melanoma: genetics and therapeutics in the genomic era
.
Genes Dev
2006
;
20
:
2149
82
.
22.
Davies
H
,
Bignell
GR
,
Cox
C
,
Stephans
P
,
Edkins
S
,
Clegg
S
, et al
Mutations of the BRAF gene in human cancer
.
Nature
2002
;
417
:
949
54
.
23.
Long
GV
,
Menzies
AM
,
Nagrial
AM
,
Haydu
LE
,
Hamilton
AL
,
Mann
GJ
, et al
Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma
.
J Clin Oncol
2011
;
29
:
1239
46
.
24.
Miller
AJ
,
Mihm
MC
 Jr
. 
Melanoma
.
N Engl J Med
2006
;
355
:
51
65
.
25.
Omholt
K
,
Platz
A
,
Kanter
L
,
Ringborg
U
,
Hansson
J
. 
NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression
.
Clin Cancer Res
2003
;
9
:
6483
8
.
26.
Edlundh-Rose
E
,
Egyhazi
S
,
Omholt
K
,
Mansson-Brahme
E
,
Platz
A
,
Hansson
J
, et al
NRAS and BRAF mutations in melanoma tumours in relation to clinical characteristics: a study based on mutation screening by pyrosequencing
.
Melanoma Res
2006
;
16
:
471
8
.
27.
Goel
VK
,
Lazar
AJ
,
Warneke
CL
,
Reston
MS
,
Haluska
FG
. 
Examination of mutations in BRAF, NRAS, and PTEN in primary cutaneous melanoma
.
J Invest Dermatol
2006
;
126
:
154
60
.
28.
van 't Veer
LJ
,
Burgering
BM
,
Versteeg
R
,
Boot
AJ
,
Ruiter
DJ
,
Osanto
S
, et al
N-ras mutations in human cutaneous melanoma from sun-exposed body sites
.
Mol Cell Biol
1989
;
9
:
3114
6
.
29.
Russo
AE
,
Torrisi
E
,
Bevelacqua
Y
,
Perrotta
R
,
Libra
M
,
McCubrey
JA
, et al
Melanoma: molecular pathogenesis and emerging target therapies (Review)
.
Int J Oncol
2009
;
34
:
1481
9
.
30.
StataCorp
. 
Stata statistical software: Release 11 CSTSL
College Station, TX
:
StataCorp LLC
; 
2009
.
31.
Menzies
AM
,
Haydu
LE
,
Visintin
L
,
Carlino
MS
,
Howe
JR
,
Thompson
JF
, et al
Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma
.
Clin Cancer Res
2012
;
18
:
3242
9
.
32.
Jakob
JA
,
Bassett
RL
 Jr
,
Ng
CS
,
Curry
JL
,
Joseph
RW
,
Alvarado
GC
, et al
NRAS mutation status is an independent prognostic factor in metastatic melanoma
.
Cancer
2012
;
118
:
4014
23
.
33.
Devitt
B
,
Liu
W
,
Salemi
R
,
Wolfe
R
,
Kelly
J
,
Tzen
CY
, et al
Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma
.
Pigment Cell Melanoma Res
2011
;
24
:
666
72
.
34.
Viros
A
,
Fridlyand
J
,
Bauer
J
,
Lasithiotakis
K
,
Garbe
C
,
Pinkel
D
, et al
Improving melanoma classification by integrating genetic and morphologic features
.
PLoS Med
2008
;
5
:
e120.
35.
Curtin
JA
,
Fridlyand
J
,
Kageshita
T
,
Patel
HN
,
Busam
KJ
,
Kutzner
H
, et al
Distinct sets of genetic alterations in melanoma
.
N Engl J Med
2005
;
353
:
2135
47
.
36.
Ellerhorst
JA
,
Greene
VR
,
Ekmekcioglu
S
,
Warneke
CL
,
Johnson
MM
,
Cooke
CP
, et al
Clinical correlates of NRAS and BRAF mutations in primary human melanoma
.
Clin Cancer Res
2011
;
17
:
229
35
.
37.
Adnane
L
,
Trail
PA
,
Taylor
I
,
Wilhelm
SM
. 
Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature
.
Methods Enzymol
2006
;
407
:
597
612
.
38.
Chang
YS
,
Adnane
J
,
Trail
PA
,
Levy
J
,
Henderson
A
,
Xue
D
, et al
Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models
.
Cancer Chemother Pharmacol
2007
;
59
:
561
74
.
39.
Dumaz
N
: 
Mechanism of RAF isoform switching induced by oncogenic RAS in melanoma
.
Small GTPases
2011
;
2
:
289
292
.
40.
Jaiswal
BS
,
Janakiraman
V
,
Kljavin
NM
,
Eastham-Anderson
J
,
Cupp
JE
,
Liang
Y
, et al
Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors
.
PLoS One
2009
;
4
:
e5717.
41.
Sullivan
RJ
,
Flaherty
K
. 
MAP kinase signaling and inhibition in melanoma
.
Oncogene
2013
;
32
:
2373
9
.
42.
Ekedahl
H
,
Cirenajwis
H
,
Harbst
K
,
Carneiro
A
,
Nielson
K
,
Olsson
H
, et al
The clinical significance of BRAF and NRAS mutations in a clinic-based metastatic melanoma cohort
.
Br J Dermatol
2013
;
169
:
1049
55
.