C L I N I C A L A N D L A B O R A T O R Y I N V E S TI G A T I O N S
BJD
British Journal of Dermatology
The role of BRAF mutations in primary melanoma growth rate and survival* V.J. Mar,1,2,3 W. Liu,1 B. Devitt,4 S.Q. Wong,3 A. Dobrovic,5 G.A. McArthur,3 R. Wolfe2 and J.W. Kelly1 1
Victorian Melanoma Service, Alfred Hospital, Melbourne, Vic. 3181, Australia Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic. 3181, Australia 3 Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Vic. 3002, Australia 4 Department of Oncology, St Vincent’s Hospital, Fitzroy, Vic. 3065, Australia 5 Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Heidelberg, Vic. 3084, Australia 2
Summary Correspondence Victoria J. Mar. E-mail: [email protected]
Accepted for publication 24 February 2015
Funding sources This project was supported by the Victorian Government through the Victorian Cancer Agency Translational Research Program Grant. V.J.M. was supported by a National Health and Medical Research Council of Australia (NHMRC) PhD Scholarship.
Conflicts of interest None declared. This work has been presented, in part, at the Melanoma World Congress, Hamburg, 2013, and the Australasian College of Dermatology Annual Scientific Meeting, Melbourne, 2014 *Plain language summary available online. DOI 10.1111/bjd.13756
Background The clinical behaviour and prognosis of primary melanomas harbouring BRAF mutations is not fully understood. Objectives To investigate the effect of mutation status on primary melanoma growth rate and melanoma-specific survival (MSS). Methods A prospective cohort of 196 patients with stage I–III primary cutaneous melanoma were followed for a median of 92 months, pre-dating the institution of BRAF inhibitor therapy. Clinicopathological variables were correlated with mutation status and hazard ratios (HRs) estimated for MSS. Results Of 196 tumours, 77 (392%) were BRAF V600E, 10 (51%) BRAF V600K and 33 (168%) were NRAS mutant. BRAF V600E mutant melanomas were associated with favourable clinical characteristics and tended to be slower growing compared with BRAF V600K, NRAS mutant or BRAF/NRAS wild-type tumours (012 mm per month, 061 mm per month, 036 mm per month and 023 mm per month, respectively; P = 005). There were 39 melanoma deaths, and BRAF mutant melanomas were associated with poorer MSS in stage I–III disease [HR 260, 95% confidence interval (CI) 120–563; P = 002] and stage I–II disease (HR 339, 95% CI 112–1022; P = 003) after adjusting for other prognostic variables. Considered separately, BRAF V600E mutant melanomas were strongly associated with MSS independently of thickness and nodal status (HR 389, 95% CI 167–909; P < 001) but BRAF V600K mutant tumours were not (HR 119, 95% CI 036–392; P = 077). Conclusions The presence of a BRAF mutation does not necessarily ‘drive’ more rapid tumour growth but is associated with poorer MSS in patients with early-stage disease.
What’s already known about this topic?
•
There is some evidence that BRAF mutations are associated with poor prognosis in primary melanoma, although most studies focus on patients with metastatic melanoma.
What does this study add?
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76
Evidence for clinical and behavioural differences between BRAFV600E and BRAFV600K molecular subtypes. BRAF mutations are associated with poorer melanoma-specific survival in patients with stage I–III melanoma. Mutation status should be considered as a prognostic factor in this group, particularly with adjuvant therapies on the horizon.
British Journal of Dermatology (2015) 173, pp76–82
© 2015 British Association of Dermatologists
BRAF mutations in primary melanomas, V.J. Mar et al. 77
Activating mutations in the oncogene BRAF are an early event occurring in approximately 40% of cutaneous melanomas. However, the impact of activated BRAF on early disease progression and survival remains unclear. In a cohort of 814 primary melanomas, we have recently shown that, while BRAF mutant tumours are significantly thinner than BRAF wild-type mutant tumours, they are more likely to present with nodal metastases.1 Long-term follow-up is not yet available for this cohort, and will be confounded by the advent of BRAF inhibitor therapies. In a separate cohort, which pre-dates targeted therapies, we have reported an association between NRAS mutation status and poorer survival, with no significant association for BRAF mutant tumours after a median follow-up period of 47 months.2 The current study extends that follow-up period to 92 months and differentiates BRAF V600E and V600K mutant tumours to better understand the clinical behaviour of these molecular subtypes. When evaluating melanoma prognosis at initial presentation, thickness and ulceration of the primary tumour, as well as nodal status, are established independent prognostic variables, with mitotic rate (MR) recognized and added more recently to the American Joint Committee on Cancer (AJCC) staging system.3,4 There is some evidence that the presence of a BRAF mutation in melanoma is associated with a poor prognosis, although these studies are mostly based on patients with metastatic disease.5–8 One study has shown evidence for poorer disease-free survival but not overall survival in patients with BRAF mutant stage I and II disease.9 Univariate analysis of primary and metastatic cases show no association between mutation status and overall survival.10–16 We have previously reported an association between BRAF mutant primary melanomas and low tumour thickness,1 as well as low levels of cell proliferation markers [phosphorylated histone 3 (PH3)] and cell cycle activity (Ki67).17 Detection of a true association between BRAF mutation status and survival would therefore require longer-term follow-up with consideration of other prognostic variables. The extent to which the presence of a strong ‘driver’ mutation might actually drive more rapid primary tumour growth is unclear. Melanoma rate of growth (ROG) is an approximation of change in primary tumour volume (using thickness) over time, correlating with MR and other histological markers of proliferation.18–20 One study has shown an association between rapidly growing melanomas and NRAS mutations but no association with BRAF mutations.21 Recent studies have shown that patients presenting with rapidly growing melanomas are more likely to have nodal disease and present earlier with metastatic disease.22–24 Tempo of disease progression together with mutation status in patients with advanced melanoma plays an important role in determining treatment options. Currently, mutation status is routinely tested only in patients with metastatic disease. Improved understanding of the role of different molecular subtypes in driving early melanoma growth and disease progression may have important implications for management, particularly with adjuvant therapies on the horizon. © 2015 British Association of Dermatologists
Patients and methods A prospective cohort of patients referred to either the Victorian Melanoma Service or Peter MacCallum Cancer Centre with stage I–III invasive cutaneous melanoma diagnosed between 1 May 2003 and 30 September 2004 included 208 patients with known BRAF and NRAS mutation status, melanoma ROG and other clinicopathological data. This cohort was reported previously, with a median follow-up of 47 months.2 This followup period has now been extended to 92 months (complete to 31 July 2011). Selective BRAF inhibitor phase I trials for patients with BRAF mutant metastatic disease commenced in July 2009. Given that the exposure to BRAF inhibitor treatment was low in this cohort, it was not considered in this analysis. Ethical approval was obtained from the human research ethics committees at both institutions. Details of assays used for BRAF (exon 15) and NRAS (exon 3) testing have been described previously.2 Further assays using high-resolution melting analysis and confirmed by Sanger sequencing were performed to differentiate BRAF V600E and V600K mutations.25 Tissue was exhausted for 12 cases, which were therefore excluded in the final analysis of 196 patients. The method of assessment of ROG has been described in detail elsewhere and used by others.19,20,26,27 It is based on the date the lesion was first noticed (D1), the date a change was noticed within the lesion (D2) and the date of excision (D3), together with tumour thickness at excision.20 Melanomas were considered to arise from a pre-existing lesion if the interval between D1 and D3 was > 5 years. In these cases, D2 was taken as the time of onset of malignancy. Nodal status was reported as positive if confirmed either by sentinel node biopsy or histological confirmation of clinically detected nodes. Melanoma-specific survival was assessed by linkage with the Victorian Cancer Registry (VCR) database. For cases that were unmatched on the VCR database, contact was made with the original referring doctor. At the time of linkage, Victorian mortality data were complete to 31 July 2011. Deaths from melanoma were coded from death certificates held by the VCR. Statistical analysis Variables included in the initial multivariate analyses included all those with significant univariate hazard ratios (HRs). Where a variable’s measurements followed a heavily skewed distribution, log transformation of the measurements was considered. Log base 2 was used to aid interpretation of results. To maximize the number of events per HR (using 10 as a commonly accepted rule of thumb),28 known prognostic variables (thickness, MR and ulceration) were included with variables of interest (mutation status) after a backward elimination procedure. Use of continuous variables also served to maximize the explanatory capacity of the model rather than categorizing thickness and MR, for example according to AJCC cut-offs.3 British Journal of Dermatology (2015) 173, pp76–82
78 BRAF mutations in primary melanomas, V.J. Mar et al. Table 1 Associations between mutation status and tumour characteristics Total BRAF
Variable
Total cases, n (%) 87 (44) Median age (years) 51 ROG (mm per month) 015 (004–064) Thickness (mm) 09 (06–18) Mitoses per mm2 10 (0–40) PH3 per mm2 10 (0–100) Tumour subtype, n (%) SSM 66 (76) NM 17 (20) LMM 3 (3) Other 1 (1) Node positive, n (%) 9 (10)
BRAF V600E
BRAF V600K
NRAS mutant
BRAF/NRAS WT
P-valuea
P-valueb
77 50 012 08 10 10
(39)
10 56 061 29 88 70
33 56 036 19 15 40
(17)
76 58 023 18 10 65
(39)
– < 001 030 < 001 003 001
– < 001 005 < 001 < 001 001
61 13 2 1 6
(79) (17) (3) (1) (8)
20 9 2 2 4
(61) (27) (6) (6) (12)
33 18 15 10 8
(43) (24) (20) (14) (11)
< 001
< 001
100
020
(004–055) (05–16) (0–20) (0–80)
5 4 1 0 3
(5) (006–123) (10–48) (30–100) (0–250) (50) (40) (10) (0) (30)
(01–10) (10–28) (10–50) (20–170)
(005–05) (07–40) (0–55) (0–150)
Values are given as median (interquartile range) unless otherwise stated. WT, wild-type; ROG, rate of growth; PH3, phosphorylated histone 3; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma. aP-value for association between three mutation groups (BRAF, NRAS, WT); bP-value for association between four mutation groups (BRAF V600E, V600K, NRAS, WT).
Kruskal–Wallis and v2 tests were used for associations between mutation status and other variables. Cox proportional hazards regression was used to estimate HRs for melanomaspecific survival (MSS) with censoring of patients who were alive at the last available mortality linkage date, and with mortality from other causes treated as censoring events. Stata 12 software (StataCorp, College Station, TX, U.S.A.) was used for all statistical analyses.
stage III disease. Seventy-seven (392%) were BRAF V600E, 10 (51%) BRAF V600K and 33 (168%) were NRAS mutant. There were 52 deaths over 1276 person-years of follow-up, 39 of which were due to melanoma. Of the 39 deaths due to melanoma, 19 (49%) were due to BRAF mutant tumours and nine (23%) were due to NRAS mutant tumours.
Results
The median growth rate for all BRAF mutant melanomas (015 mm per month) was slower than both NRAS mutant (036 mm per month) and wild-type (023 mm per month) tumours, although this did not reach statistical significance (P = 020) (Table 1). BRAF V600E mutant tumours had a slower median growth rate than BRAF V600K mutant
Descriptive statistics At presentation, of the 196 patients, 114 (581%) had stage I disease, 61 (311%) had stage II disease and 21 (107%) had
Rate of growth and mutation status
Table 2 Uni- and multivariate hazard ratios (HRs) for melanoma-specific survival in patients with stage I–III disease Multivariate 1a
Univariate BRAF/NRAS status Thickness Ulceration Mitoses Subtype SSM NM LMM Other Nodes WT BRAF V600E V600K NRAS
Multivariate 2b
Events (n)
HR
95% CI
P-value
HR
95% CI
P-value
HR
95% CI
P-value
178–260 269–701 154–210
< 001 < 001 < 001
185 139 119
122–280 066–291 085–166
< 001 040 030
228
168–310
< 001
16
215 435 180
17 14 2 6 16 11 19 15 4 9
100 307 102 317 777 100 145 127 298 203
181–521 039–264 150–671 464–1303
< 001 100 < 001 < 001
100 092 069 172 500
043–200 009–531 053–561 223–1119
080 070 040 < 001
291–1179
< 001
030 050 006 001
315 434 141 373
128–781 167–1131 037–544 136–1023
001 < 001 060 001
586 100 260 389 119 320
120–563 167–909 036–392 129–793
002 < 001 080 001
069–304 058–277 095–938 085–490
CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; WT, wild-type. One hundred and ninety-three patients including 39 melanoma deaths; adjusted for nodal status, thickness, mitotic rate, ulceration and subtype. bAs for multivariate 1 but adjusted for thickness and nodal disease only, with nonsignificant variables removed. a
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BRAF mutations in primary melanomas, V.J. Mar et al. 79
0·50
Univariate analysis for melanoma-specific survival
0·00
0·25
MSS (%)
0·75
1·00
active, as measured by both MR and PH3 [MR median 9 vs. 1 mitoses per mm2 (P < 001), PH3 median 7 vs. 1 per mm2 (P = 001)]. They were also more commonly of nodular melanoma subtype (40%; P < 001) (Table 1).
0
2
4 Time (years)
8
BRAF V600E/K
0·50 0·00
0·25
MSS (%)
0·75
1·00
BRAF/NRAS WT NRAS
6
Age, sex and tumour site were not significantly associated with MSS in this cohort. As expected, tumour thickness, MR and ulceration were strongly associated with MSS, as was tumour subtype (nodular melanoma and acral lentiginous melanoma). Patients with NRAS mutant melanomas were observed to have shorter survival times than those with wildtype tumours [HR 20, 95% confidence interval (CI) 08– 49], which seemed plausible yet may have been a chance finding (P = 010) (Table 2). BRAF mutant tumours had little evidence of univariate association with MSS (HR 14, 95% CI 07–30; P = 030). If considered separately, BRAF V600K mutant tumours tended towards an association with poorer MSS, although this was still not significant (HR 298, 95% CI 095–938; P = 006). Multivariate analysis for melanoma-specific survival
0
2
4 Time (years) BRAF/NRAS WT BRAF V600K
6
8
BRAF V600E NRAS
Fig 1. Mutation status and multivariate analysis for melanoma-specific survival (MSS). (a) Kaplan–Meier MSS graph among patients with stage I–III disease with BRAF, NRAS and BRAF/NRAS wild-type (WT) mutant tumours (adjusted for thickness and nodal status; plotted at mean log thickness and no nodal involvement). (b) Kaplan–Meier MSS graph among patients with stage I–II disease with BRAF V600E and BRAF V600K, NRAS and BRAF/NRAS WT mutant tumours (adjusted for thickness and nodal status; plotted at mean log thickness and no nodal involvement).
tumours (012 mm per month vs. 060 mm per month) (P = 005). Mutation status and other clinical characteristics Correlations between mutation status and clinical characteristics have been described previously;2 however, they are presented briefly here for this subset of 196 patients with differentiation of BRAF V600E and V600K mutations (Table 1). BRAF mutant tumours were significantly thinner at diagnosis (median thickness 09 mm) compared with NRAS mutant tumours (19 mm) and wild-type tumours (18 mm) (P < 001). BRAF mutant tumours also had a lower median MR (P = 003) and lower levels of PH3 staining (P = 001), a more sensitive marker of mitosis (Table 1). Although the number of BRAF V600K mutant tumours was small, they were thicker than BRAF V600E mutant tumours (median 28 mm vs. 08 mm; P < 001) and more mitotically © 2015 British Association of Dermatologists
The initial multivariate Cox model included mutation status together with significant covariates from the univariate analyses: nodal status, thickness, MR, ulceration and subtype (Table 2, ‘Multivariate 1’). The HRs for nodal status, thickness and mutation status were statistically significant but were not significant for ulceration, MR or subtype. Nonsignificant variables were removed (Table 2, ‘Multivariate 2’) in order to optimize the number of events per HR, and the likelihood ratio test comparing nested models was not significant. The presence of either a BRAF or NRAS mutation was strongly related to outcome for patients with stage I–III disease [BRAF: HR 260, 95% CI 120–563 (P = 002); NRAS: HR 320, 95% CI 129–730 (P = 001)] (Table 2; Fig. 1a). If BRAF V600E and V600K were considered separately, BRAF V600E mutant melanomas remained strongly associated with MSS independently of thickness and nodal status (HR 389, 95% CI 167– 909; P < 001) but BRAF V600K mutant tumours did not (HR 119, 95% CI 036–392; P = 077) (Table 2; Fig. 1b). If only stage I and II patients were considered (175 patients, 23 deaths), survival times were shorter for patients with BRAF mutant tumours than for those with wild-type tumours after adjusting for thickness (HR 294, 95% CI 107– 807; P = 004) (Table 3). There was much weaker evidence of an association between the presence of an NRAS mutation and survival in this group (HR 320, 95% CI 071–732; P = 020). As reflected in the wide CIs, this model lacked sufficient events per HR to draw strong conclusions. However, interestingly, of the four patients who died from melanomas < 1 mm in thickness, all had BRAF mutant tumours (three V600E and one V600K). In contrast to results shown in Table 2, a significant association between BRAF V600K mutant tumours and poorer survival British Journal of Dermatology (2015) 173, pp76–82
80 BRAF mutations in primary melanomas, V.J. Mar et al. Table 3 Uni- and multivariate hazard ratios (HRs) for melanoma-specific survival in patients with stage I–II disease only Multivariate 1a
Univariate BRAF/NRAS status Thickness Ulceration Mitoses Subtype SSM NM LMM Other WT BRAF V600E V600K NRAS
Multivariate 2b
HR
95% CI
P-value
HR
95% CI
P-value
HR
95% CI
P-value
152–289 105–586 134–227
< 001 004 < 001
183 069 143
106–316 025–192 095–215
003 050 009
240
169–341
< 001
8
210 248 174
11 8 1 3 7 11 9 2 5
100 058 059 409 100 128 114 291 171
104–641 008–457 114–1468
001 07 < 001
100 097 064 213
033–283 008–511 040–1138
100 070 040
050–330 042–306 060–1399 054–538
060 080 020 040
339 312 622 235
112–1022 100–973 107–3611 072–773
003 005 004 020
100 294 269 507 320
107–807 094–768 101–2539 071–732
Events
004 006 005 020
CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; WT, BRAF and NRAS wild-type. aOne hundred and seventy-five patients including 23 melanoma deaths; adjusted for thickness, mitotic rate, ulceration and subtype. bAs for multivariate 1 but adjusted for thickness only, with nonsignificant variables removed.
is shown for patients with stage I–II disease (Table 3). Of the two deaths from BRAF V600K mutant tumours in this group, one tumour was thick with many mitoses and the other thin with few mitoses; hence, mutation status was best able to explain the deaths. However, death from BRAF V600K mutant tumours among all cases was explained by virtue of them being thicker and more mitotically active. Date of diagnosis of distant metastases was known for 33 of the 39 patients who died of melanoma. While there was no univariate association between mutation status and distant disease-free survival (DDFS), patients with BRAF mutant tumours had significantly poorer DDFS on multivariate analysis [HR 271, 95% CI 118–617 (P = 002) for patients with stage I–III disease; HR 318, 95% CI 107–947 (P = 004) for patients with stage I–II disease only]. NRAS mutant tumours were associated with poorer DDFS on multivariate analysis for patients with stage I–III disease (HR 280 95% CI 108–727; P = 004) but this association was not significant for patients with stage I–II disease alone (HR 240, 95% CI 066–864; P = 020). There was no significant association between mutation status and the time from diagnosis of distant metastatic disease to time of death on either uni- or multivariate analysis, although numbers were too small to draw strong conclusions from.
Discussion In this cohort, long-term follow-up revealed that patients with stage I–III primary melanomas harbouring BRAF mutations had approximately threefold the risk of death than patients with BRAF/NRAS wild-type tumours. There was a similar trend for patients with early stage I–II disease; however, the number of deaths in this group was too low to draw strong conclusions from. Importantly, this cohort pre-dates the use of BRAF inhibitors, the use of which does not therefore confound outcomes. British Journal of Dermatology (2015) 173, pp76–82
These findings support results from a larger cohort of 814 primary melanomas showing that BRAF mutant tumours were more likely than BRAF wild-type tumours to present with nodal metastases despite being significantly thinner.1 This study also provides an explanation for recent findings that thin melanomas are responsible for a disproportionate number of melanoma deaths,29 whereby deaths from thin tumours occurred after approximately 6 years compared with just 2 years following diagnosis of thick melanoma.29 Interestingly, melanomas harbouring a BRAF V600K mutation were associated with other poor prognosis tumour characteristics, such as increased tumour thickness, proliferative markers (MR and PH3) and nodular subtype. In accordance with this, they also tended to be more rapidly growing than BRAF V600E mutant tumours. Compared with BRAF V600E melanomas, which are associated with younger age and location on the trunk, BRAF V600K mutant melanomas have been shown to be more common in elderly patients, and on the head and neck (associated with chronic sun damage).30 As the number of tumours with this mutation was small (n = 10) in this cohort, no strong conclusions can be drawn; however, the results suggest that BRAF V600E and BRAF V600K mutant tumours differ in their clinical presentation, as well as their biological behaviour. Recent studies in metastatic disease have suggested that BRAF V600K mutant tumours have a more aggressive phenotype.30,31 The presence of a BRAF V600K mutation was not an independent predictor of outcome in this group once thickness was taken into account. Lack of distinction between these two mutations in other studies may lead to underestimation of the strength of association between the presence of BRAF V600E mutation and outcome. It is important to acknowledge that the measurement of melanoma ROG is an imperfect approximation, which relies on patient recall. However, subjective measurements of ROG have been shown to reflect objective measures of primary © 2015 British Association of Dermatologists
BRAF mutations in primary melanomas, V.J. Mar et al. 81
melanoma growth rate closely, particularly for more rapidly growing tumours.32 In the context of this study, ROG is useful for providing insight into the longitudinal clinical behaviour of different molecular subtypes in addition to proliferative markers, which are assessable only at one time point. We have previously reported an association between NRAS mutant melanomas and poorer MSS, although the presence of a BRAF mutation was not a significant prognostic factor in that study.2 The current cohort has the advantage of longer followup time (median 92 vs. 47 months) and more events (39 vs. 28 deaths), thereby extending the horizon over which these associations are assessed, and improving statistical power. In particular, Figure 1 shows separation in the survival curve for patients with BRAF mutant tumours only after the extended follow-up time. The fact that the longer follow-up period in the current study shows BRAF status to be an independent predictor of survival points towards these tumours growing and progressing more slowly, and is perhaps the reason why previous studies with shorter follow-up periods have not found any association between BRAF status and overall survival in early stage disease.2,9,12,13 Our finding that the presence of a BRAF mutation in earlystage disease is associated with poor outcome is supported by other studies of metastatic disease,5–8 and a recent study in early-stage disease that showed poorer disease-free survival , but not MSS, in 147 patients, with 17 deaths over a median followup of 48 months.9 The relative survival probability between patients with BRAF vs. BRAF/NRAS wild-type melanomas is pronounced after adjustment for thickness and nodal status, illustrating that, in early-stage disease, it is important to take other prognostic variables into account. This might explain why other studies reporting a univariate association only have not found a significant association between BRAF status and outcome.10,11,13 In our study the shorter MSS among patients with BRAF and NRAS mutant tumours was more likely to be attributable to shorter DDFS rather than a more rapid progression from stage IV disease to death. However, larger studies of stage IV cohorts have shown poorer overall survival following diagnosis of metastatic disease.5,33 Poorer survival among patients with BRAF mutant tumours is therefore likely to be attributable to both poorer DDFS and progression from stage IV disease to death. Although BRAF is a key oncogene in the mitogen-activated protein kinase pathway, which ultimately induces cell proliferation, the exact role of BRAF mutations in tumour progression remains complex. We have shown that proliferation markers are more likely to be reduced in BRAF V600E mutant tumours, and, indeed, the prognostic significance of MR is lost once mutation status is considered in this study. A few previous studies have reported a reduction in MR among BRAF mutant tumours,11,17 while others have not.5,13 It would be interesting to assess the prognostic significance of MR in larger cohorts with known mutation status. While BRAF and NRAS mutations are early initiating events in melanoma, it is likely that other molecular events are © 2015 British Association of Dermatologists
required for progression, to overcome such phenomena as melanocyte senescence (growth arrest) and suppression of apoptosis.34 Mutations in BRAF are found in benign melanocytic naevi, which proliferate after oncogene activation and then become senescent.35 Activated BRAF may induce senescence by upregulating p16 and other pathways early in melanoma formation.34,35 However, in late-stage disease, BRAF (unlike NRAS) mutations are often accompanied by loss of phosphatase and tensin homolog,36 resulting in reduced melanocyte apoptosis. This suggests that, acting alone, BRAF mutations are less efficient drivers of tumour proliferation and metastasis than NRAS.34 This may explain why the former often present as thinner lesions and are slower to progress. We have previously shown that BRAF mutant primary melanomas are more likely to show RAC1 immunoreactivity, which may be important in the early migration of these tumours.1 Our results show that the presence of a BRAF mutation increases the risk of melanoma death in patients with stage I–III disease. Evidence is growing that an assessment of mutation status along with other prognostic variables is important in assessing the risk of progression from early-stage disease, particularly as we approach an era of adjuvant therapies.
Acknowledgments We would like to acknowledge the Victorian Cancer Council for their assistance in data linkage for mortality follow-up and the contributions of Dr William Murray, Professor John Dowling, Dr Jill Magee and Dr Graham Mason, dermatopathologists involved in the reporting of cases in this cohort.
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82 BRAF mutations in primary melanomas, V.J. Mar et al. 9 Nagore E, Requena C, Traves V et al. Prognostic value of BRAF mutations in localized cutaneous melanoma. J Am Acad Dermatol 2014; 70:858–62. 10 Edlundh-Rose E, Egyhazi S, Omholt K 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. 11 Akslen LA, Angelini S, Straume O et al. BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival. J Invest Dermatol 2005; 125:312–17. 12 Shinozaki M, Fujimoto A, Morton DL et al. Incidence of BRAF oncogene mutation and clinical relevance for primary cutaneous melanomas. Clin Cancer Res 2004; 10:1753–7. 13 Ellerhorst JA, Greene VR, Ekmekcioglu S et al. Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res 2011; 17:229–35. 14 Houben R, Becker JC, Kappel A et al. Constitutive activation of the Ras-Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 2004; 3:6. 15 Kumar R, Angelini S, Czene K et al. BRAF mutations in metastatic melanoma: a possible association with clinical outcome. Clin Cancer Res 2003; 9:3362–8. 16 Maldonado JL, Fridlyand J, Patel H et al. Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst 2003; 95:1878–90. 17 Liu W, Kelly JW, Trivett M et al. Distinct clinical and pathological features are associated with the BRAF(T1799A(V600E)) mutation in primary melanoma. J Invest Dermatol 2007; 127:900–5. 18 Liu W, McArthur GA, Trivett M et al. Correlation of subjective selfreported melanoma growth rate with objective tumor proliferation markers. Arch Dermatol 2008; 144:555–6. 19 Grob JJ, Richard MA, Gouvernet J et al. The kinetics of the visible growth of a primary melanoma reflects the tumor aggressiveness and is an independent prognostic marker: a prospective study. Int J Cancer 2002; 102:34–8. 20 Liu W, Dowling JP, Murray WK et al. Rate of growth in melanomas: characteristics and associations of rapidly growing melanomas. Arch Dermatol 2006; 142:1551–8. 21 Nagore E, Hacker E, Martorell-Calatayud A et al. Prevalence of BRAF and NRAS mutations in fast-growing melanomas. Pigment Cell Melanoma Res 2013; 26:429–31. 22 Martorell-Calatayud A, Nagore E, Botella-Estrada R et al. Defining fast-growing melanomas: reappraisal of epidemiological, clinical, and histological features. Melanoma Res 2011; 21:131–8.
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23 Tejera-Vaquerizo A, Nagore E, Herrera-Acosta E et al. Prediction of sentinel lymph node positivity by growth rate of cutaneous melanoma. Arch Dermatol 2012; 148:577–84. 24 Tejera-Vaquerizo A, Lopez-Navarro N, Alcaide-Martin A et al. Correlation of the growth rate of melanoma with the temporal appearance of metastasis. J Eur Acad Dermatol Venereol 2011; 25:366– 7. 25 Richter A, Grieu F, Carrello A et al. A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffinembedded malignant melanoma. Sci Rep 2013; 3:1659. 26 Nagore E, Martorell-Calatayud A, Botella-Estrada R et al. Growth rate as an independent prognostic factor in localized invasive cutaneous melanoma. J Eur Acad Dermatol Venereol 2011; 25:618– 20. 27 Tejera-Vaquerizo A, Barrera-Vigo MV, L opez-Navarro N, HerreraCeballos E. Growth rate as a prognostic factor in localized invasive cutaneous melanoma. J Eur Acad Dermatol Venereol 2010; 24:147–54. 28 Concato J, Peduzzi P, Holford TR et al. Importance of events per independent variable in proportional hazards analysis. I. Background, goals, and general strategy. J Clin Epidemiol 1995; 48:1495– 501. 29 Whiteman DC, Baade PD, Olsen CM. More people die from thin melanomas (≤1 mm) than from thick melanomas (>4 mm) in Queensland, Australia. J Invest Dermatol 2015; 135:1190–3. 30 Menzies AM, Haydu LE, Visintin L et al. Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma. Clin Cancer Res 2012; 18:3242–9. 31 Bucheit AD, Syklawer E, Jakob JA et al. Clinical characteristics and outcomes with specific BRAF and NRAS mutations in patients with metastatic melanoma. Cancer 2013; 119:3821–9. 32 Lin MJ, Mar V, McLean C et al. An objective measure of growth rate using partial biopsy specimens of melanomas that were initially misdiagnosed. J Am Acad Dermatol 2014; 71:691–7. 33 Ekedahl H, Cirenajwis H, Harbst K et al. The clinical significance of BRAF and NRAS mutations in a clinic-based metastatic melanoma cohort. Br J Dermatol 2013; 169:1049–55. 34 Bennett DC. How to make a melanoma: what do we know of the primary clonal events? Pigment Cell Melanoma Res 2008; 21:27– 38. 35 Gray-Schopfer VC, Cheong SC, Chong H et al. Cellular senescence in naevi and immortalisation in melanoma: a role for p16? Br J Cancer 2006; 95:496–505. 36 Curtin JA, Fridlyand J, Kageshita T et al. Distinct sets of genetic alterations in melanoma. N Engl J Med 2005; 353:2135–47.
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BJD
British Journal of Dermatology
The role of BRAF mutations in primary melanoma growth rate and survival* V.J. Mar,1,2,3 W. Liu,1 B. Devitt,4 S.Q. Wong,3 A. Dobrovic,5 G.A. McArthur,3 R. Wolfe2 and J.W. Kelly1 1
Victorian Melanoma Service, Alfred Hospital, Melbourne, Vic. 3181, Australia Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic. 3181, Australia 3 Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Vic. 3002, Australia 4 Department of Oncology, St Vincent’s Hospital, Fitzroy, Vic. 3065, Australia 5 Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Heidelberg, Vic. 3084, Australia 2
Summary Correspondence Victoria J. Mar. E-mail: [email protected]
Accepted for publication 24 February 2015
Funding sources This project was supported by the Victorian Government through the Victorian Cancer Agency Translational Research Program Grant. V.J.M. was supported by a National Health and Medical Research Council of Australia (NHMRC) PhD Scholarship.
Conflicts of interest None declared. This work has been presented, in part, at the Melanoma World Congress, Hamburg, 2013, and the Australasian College of Dermatology Annual Scientific Meeting, Melbourne, 2014 *Plain language summary available online. DOI 10.1111/bjd.13756
Background The clinical behaviour and prognosis of primary melanomas harbouring BRAF mutations is not fully understood. Objectives To investigate the effect of mutation status on primary melanoma growth rate and melanoma-specific survival (MSS). Methods A prospective cohort of 196 patients with stage I–III primary cutaneous melanoma were followed for a median of 92 months, pre-dating the institution of BRAF inhibitor therapy. Clinicopathological variables were correlated with mutation status and hazard ratios (HRs) estimated for MSS. Results Of 196 tumours, 77 (392%) were BRAF V600E, 10 (51%) BRAF V600K and 33 (168%) were NRAS mutant. BRAF V600E mutant melanomas were associated with favourable clinical characteristics and tended to be slower growing compared with BRAF V600K, NRAS mutant or BRAF/NRAS wild-type tumours (012 mm per month, 061 mm per month, 036 mm per month and 023 mm per month, respectively; P = 005). There were 39 melanoma deaths, and BRAF mutant melanomas were associated with poorer MSS in stage I–III disease [HR 260, 95% confidence interval (CI) 120–563; P = 002] and stage I–II disease (HR 339, 95% CI 112–1022; P = 003) after adjusting for other prognostic variables. Considered separately, BRAF V600E mutant melanomas were strongly associated with MSS independently of thickness and nodal status (HR 389, 95% CI 167–909; P < 001) but BRAF V600K mutant tumours were not (HR 119, 95% CI 036–392; P = 077). Conclusions The presence of a BRAF mutation does not necessarily ‘drive’ more rapid tumour growth but is associated with poorer MSS in patients with early-stage disease.
What’s already known about this topic?
•
There is some evidence that BRAF mutations are associated with poor prognosis in primary melanoma, although most studies focus on patients with metastatic melanoma.
What does this study add?
• • •
76
Evidence for clinical and behavioural differences between BRAFV600E and BRAFV600K molecular subtypes. BRAF mutations are associated with poorer melanoma-specific survival in patients with stage I–III melanoma. Mutation status should be considered as a prognostic factor in this group, particularly with adjuvant therapies on the horizon.
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BRAF mutations in primary melanomas, V.J. Mar et al. 77
Activating mutations in the oncogene BRAF are an early event occurring in approximately 40% of cutaneous melanomas. However, the impact of activated BRAF on early disease progression and survival remains unclear. In a cohort of 814 primary melanomas, we have recently shown that, while BRAF mutant tumours are significantly thinner than BRAF wild-type mutant tumours, they are more likely to present with nodal metastases.1 Long-term follow-up is not yet available for this cohort, and will be confounded by the advent of BRAF inhibitor therapies. In a separate cohort, which pre-dates targeted therapies, we have reported an association between NRAS mutation status and poorer survival, with no significant association for BRAF mutant tumours after a median follow-up period of 47 months.2 The current study extends that follow-up period to 92 months and differentiates BRAF V600E and V600K mutant tumours to better understand the clinical behaviour of these molecular subtypes. When evaluating melanoma prognosis at initial presentation, thickness and ulceration of the primary tumour, as well as nodal status, are established independent prognostic variables, with mitotic rate (MR) recognized and added more recently to the American Joint Committee on Cancer (AJCC) staging system.3,4 There is some evidence that the presence of a BRAF mutation in melanoma is associated with a poor prognosis, although these studies are mostly based on patients with metastatic disease.5–8 One study has shown evidence for poorer disease-free survival but not overall survival in patients with BRAF mutant stage I and II disease.9 Univariate analysis of primary and metastatic cases show no association between mutation status and overall survival.10–16 We have previously reported an association between BRAF mutant primary melanomas and low tumour thickness,1 as well as low levels of cell proliferation markers [phosphorylated histone 3 (PH3)] and cell cycle activity (Ki67).17 Detection of a true association between BRAF mutation status and survival would therefore require longer-term follow-up with consideration of other prognostic variables. The extent to which the presence of a strong ‘driver’ mutation might actually drive more rapid primary tumour growth is unclear. Melanoma rate of growth (ROG) is an approximation of change in primary tumour volume (using thickness) over time, correlating with MR and other histological markers of proliferation.18–20 One study has shown an association between rapidly growing melanomas and NRAS mutations but no association with BRAF mutations.21 Recent studies have shown that patients presenting with rapidly growing melanomas are more likely to have nodal disease and present earlier with metastatic disease.22–24 Tempo of disease progression together with mutation status in patients with advanced melanoma plays an important role in determining treatment options. Currently, mutation status is routinely tested only in patients with metastatic disease. Improved understanding of the role of different molecular subtypes in driving early melanoma growth and disease progression may have important implications for management, particularly with adjuvant therapies on the horizon. © 2015 British Association of Dermatologists
Patients and methods A prospective cohort of patients referred to either the Victorian Melanoma Service or Peter MacCallum Cancer Centre with stage I–III invasive cutaneous melanoma diagnosed between 1 May 2003 and 30 September 2004 included 208 patients with known BRAF and NRAS mutation status, melanoma ROG and other clinicopathological data. This cohort was reported previously, with a median follow-up of 47 months.2 This followup period has now been extended to 92 months (complete to 31 July 2011). Selective BRAF inhibitor phase I trials for patients with BRAF mutant metastatic disease commenced in July 2009. Given that the exposure to BRAF inhibitor treatment was low in this cohort, it was not considered in this analysis. Ethical approval was obtained from the human research ethics committees at both institutions. Details of assays used for BRAF (exon 15) and NRAS (exon 3) testing have been described previously.2 Further assays using high-resolution melting analysis and confirmed by Sanger sequencing were performed to differentiate BRAF V600E and V600K mutations.25 Tissue was exhausted for 12 cases, which were therefore excluded in the final analysis of 196 patients. The method of assessment of ROG has been described in detail elsewhere and used by others.19,20,26,27 It is based on the date the lesion was first noticed (D1), the date a change was noticed within the lesion (D2) and the date of excision (D3), together with tumour thickness at excision.20 Melanomas were considered to arise from a pre-existing lesion if the interval between D1 and D3 was > 5 years. In these cases, D2 was taken as the time of onset of malignancy. Nodal status was reported as positive if confirmed either by sentinel node biopsy or histological confirmation of clinically detected nodes. Melanoma-specific survival was assessed by linkage with the Victorian Cancer Registry (VCR) database. For cases that were unmatched on the VCR database, contact was made with the original referring doctor. At the time of linkage, Victorian mortality data were complete to 31 July 2011. Deaths from melanoma were coded from death certificates held by the VCR. Statistical analysis Variables included in the initial multivariate analyses included all those with significant univariate hazard ratios (HRs). Where a variable’s measurements followed a heavily skewed distribution, log transformation of the measurements was considered. Log base 2 was used to aid interpretation of results. To maximize the number of events per HR (using 10 as a commonly accepted rule of thumb),28 known prognostic variables (thickness, MR and ulceration) were included with variables of interest (mutation status) after a backward elimination procedure. Use of continuous variables also served to maximize the explanatory capacity of the model rather than categorizing thickness and MR, for example according to AJCC cut-offs.3 British Journal of Dermatology (2015) 173, pp76–82
78 BRAF mutations in primary melanomas, V.J. Mar et al. Table 1 Associations between mutation status and tumour characteristics Total BRAF
Variable
Total cases, n (%) 87 (44) Median age (years) 51 ROG (mm per month) 015 (004–064) Thickness (mm) 09 (06–18) Mitoses per mm2 10 (0–40) PH3 per mm2 10 (0–100) Tumour subtype, n (%) SSM 66 (76) NM 17 (20) LMM 3 (3) Other 1 (1) Node positive, n (%) 9 (10)
BRAF V600E
BRAF V600K
NRAS mutant
BRAF/NRAS WT
P-valuea
P-valueb
77 50 012 08 10 10
(39)
10 56 061 29 88 70
33 56 036 19 15 40
(17)
76 58 023 18 10 65
(39)
– < 001 030 < 001 003 001
– < 001 005 < 001 < 001 001
61 13 2 1 6
(79) (17) (3) (1) (8)
20 9 2 2 4
(61) (27) (6) (6) (12)
33 18 15 10 8
(43) (24) (20) (14) (11)
< 001
< 001
100
020
(004–055) (05–16) (0–20) (0–80)
5 4 1 0 3
(5) (006–123) (10–48) (30–100) (0–250) (50) (40) (10) (0) (30)
(01–10) (10–28) (10–50) (20–170)
(005–05) (07–40) (0–55) (0–150)
Values are given as median (interquartile range) unless otherwise stated. WT, wild-type; ROG, rate of growth; PH3, phosphorylated histone 3; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma. aP-value for association between three mutation groups (BRAF, NRAS, WT); bP-value for association between four mutation groups (BRAF V600E, V600K, NRAS, WT).
Kruskal–Wallis and v2 tests were used for associations between mutation status and other variables. Cox proportional hazards regression was used to estimate HRs for melanomaspecific survival (MSS) with censoring of patients who were alive at the last available mortality linkage date, and with mortality from other causes treated as censoring events. Stata 12 software (StataCorp, College Station, TX, U.S.A.) was used for all statistical analyses.
stage III disease. Seventy-seven (392%) were BRAF V600E, 10 (51%) BRAF V600K and 33 (168%) were NRAS mutant. There were 52 deaths over 1276 person-years of follow-up, 39 of which were due to melanoma. Of the 39 deaths due to melanoma, 19 (49%) were due to BRAF mutant tumours and nine (23%) were due to NRAS mutant tumours.
Results
The median growth rate for all BRAF mutant melanomas (015 mm per month) was slower than both NRAS mutant (036 mm per month) and wild-type (023 mm per month) tumours, although this did not reach statistical significance (P = 020) (Table 1). BRAF V600E mutant tumours had a slower median growth rate than BRAF V600K mutant
Descriptive statistics At presentation, of the 196 patients, 114 (581%) had stage I disease, 61 (311%) had stage II disease and 21 (107%) had
Rate of growth and mutation status
Table 2 Uni- and multivariate hazard ratios (HRs) for melanoma-specific survival in patients with stage I–III disease Multivariate 1a
Univariate BRAF/NRAS status Thickness Ulceration Mitoses Subtype SSM NM LMM Other Nodes WT BRAF V600E V600K NRAS
Multivariate 2b
Events (n)
HR
95% CI
P-value
HR
95% CI
P-value
HR
95% CI
P-value
178–260 269–701 154–210
< 001 < 001 < 001
185 139 119
122–280 066–291 085–166
< 001 040 030
228
168–310
< 001
16
215 435 180
17 14 2 6 16 11 19 15 4 9
100 307 102 317 777 100 145 127 298 203
181–521 039–264 150–671 464–1303
< 001 100 < 001 < 001
100 092 069 172 500
043–200 009–531 053–561 223–1119
080 070 040 < 001
291–1179
< 001
030 050 006 001
315 434 141 373
128–781 167–1131 037–544 136–1023
001 < 001 060 001
586 100 260 389 119 320
120–563 167–909 036–392 129–793
002 < 001 080 001
069–304 058–277 095–938 085–490
CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; WT, wild-type. One hundred and ninety-three patients including 39 melanoma deaths; adjusted for nodal status, thickness, mitotic rate, ulceration and subtype. bAs for multivariate 1 but adjusted for thickness and nodal disease only, with nonsignificant variables removed. a
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BRAF mutations in primary melanomas, V.J. Mar et al. 79
0·50
Univariate analysis for melanoma-specific survival
0·00
0·25
MSS (%)
0·75
1·00
active, as measured by both MR and PH3 [MR median 9 vs. 1 mitoses per mm2 (P < 001), PH3 median 7 vs. 1 per mm2 (P = 001)]. They were also more commonly of nodular melanoma subtype (40%; P < 001) (Table 1).
0
2
4 Time (years)
8
BRAF V600E/K
0·50 0·00
0·25
MSS (%)
0·75
1·00
BRAF/NRAS WT NRAS
6
Age, sex and tumour site were not significantly associated with MSS in this cohort. As expected, tumour thickness, MR and ulceration were strongly associated with MSS, as was tumour subtype (nodular melanoma and acral lentiginous melanoma). Patients with NRAS mutant melanomas were observed to have shorter survival times than those with wildtype tumours [HR 20, 95% confidence interval (CI) 08– 49], which seemed plausible yet may have been a chance finding (P = 010) (Table 2). BRAF mutant tumours had little evidence of univariate association with MSS (HR 14, 95% CI 07–30; P = 030). If considered separately, BRAF V600K mutant tumours tended towards an association with poorer MSS, although this was still not significant (HR 298, 95% CI 095–938; P = 006). Multivariate analysis for melanoma-specific survival
0
2
4 Time (years) BRAF/NRAS WT BRAF V600K
6
8
BRAF V600E NRAS
Fig 1. Mutation status and multivariate analysis for melanoma-specific survival (MSS). (a) Kaplan–Meier MSS graph among patients with stage I–III disease with BRAF, NRAS and BRAF/NRAS wild-type (WT) mutant tumours (adjusted for thickness and nodal status; plotted at mean log thickness and no nodal involvement). (b) Kaplan–Meier MSS graph among patients with stage I–II disease with BRAF V600E and BRAF V600K, NRAS and BRAF/NRAS WT mutant tumours (adjusted for thickness and nodal status; plotted at mean log thickness and no nodal involvement).
tumours (012 mm per month vs. 060 mm per month) (P = 005). Mutation status and other clinical characteristics Correlations between mutation status and clinical characteristics have been described previously;2 however, they are presented briefly here for this subset of 196 patients with differentiation of BRAF V600E and V600K mutations (Table 1). BRAF mutant tumours were significantly thinner at diagnosis (median thickness 09 mm) compared with NRAS mutant tumours (19 mm) and wild-type tumours (18 mm) (P < 001). BRAF mutant tumours also had a lower median MR (P = 003) and lower levels of PH3 staining (P = 001), a more sensitive marker of mitosis (Table 1). Although the number of BRAF V600K mutant tumours was small, they were thicker than BRAF V600E mutant tumours (median 28 mm vs. 08 mm; P < 001) and more mitotically © 2015 British Association of Dermatologists
The initial multivariate Cox model included mutation status together with significant covariates from the univariate analyses: nodal status, thickness, MR, ulceration and subtype (Table 2, ‘Multivariate 1’). The HRs for nodal status, thickness and mutation status were statistically significant but were not significant for ulceration, MR or subtype. Nonsignificant variables were removed (Table 2, ‘Multivariate 2’) in order to optimize the number of events per HR, and the likelihood ratio test comparing nested models was not significant. The presence of either a BRAF or NRAS mutation was strongly related to outcome for patients with stage I–III disease [BRAF: HR 260, 95% CI 120–563 (P = 002); NRAS: HR 320, 95% CI 129–730 (P = 001)] (Table 2; Fig. 1a). If BRAF V600E and V600K were considered separately, BRAF V600E mutant melanomas remained strongly associated with MSS independently of thickness and nodal status (HR 389, 95% CI 167– 909; P < 001) but BRAF V600K mutant tumours did not (HR 119, 95% CI 036–392; P = 077) (Table 2; Fig. 1b). If only stage I and II patients were considered (175 patients, 23 deaths), survival times were shorter for patients with BRAF mutant tumours than for those with wild-type tumours after adjusting for thickness (HR 294, 95% CI 107– 807; P = 004) (Table 3). There was much weaker evidence of an association between the presence of an NRAS mutation and survival in this group (HR 320, 95% CI 071–732; P = 020). As reflected in the wide CIs, this model lacked sufficient events per HR to draw strong conclusions. However, interestingly, of the four patients who died from melanomas < 1 mm in thickness, all had BRAF mutant tumours (three V600E and one V600K). In contrast to results shown in Table 2, a significant association between BRAF V600K mutant tumours and poorer survival British Journal of Dermatology (2015) 173, pp76–82
80 BRAF mutations in primary melanomas, V.J. Mar et al. Table 3 Uni- and multivariate hazard ratios (HRs) for melanoma-specific survival in patients with stage I–II disease only Multivariate 1a
Univariate BRAF/NRAS status Thickness Ulceration Mitoses Subtype SSM NM LMM Other WT BRAF V600E V600K NRAS
Multivariate 2b
HR
95% CI
P-value
HR
95% CI
P-value
HR
95% CI
P-value
152–289 105–586 134–227
< 001 004 < 001
183 069 143
106–316 025–192 095–215
003 050 009
240
169–341
< 001
8
210 248 174
11 8 1 3 7 11 9 2 5
100 058 059 409 100 128 114 291 171
104–641 008–457 114–1468
001 07 < 001
100 097 064 213
033–283 008–511 040–1138
100 070 040
050–330 042–306 060–1399 054–538
060 080 020 040
339 312 622 235
112–1022 100–973 107–3611 072–773
003 005 004 020
100 294 269 507 320
107–807 094–768 101–2539 071–732
Events
004 006 005 020
CI, confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma; LMM, lentigo maligna melanoma; WT, BRAF and NRAS wild-type. aOne hundred and seventy-five patients including 23 melanoma deaths; adjusted for thickness, mitotic rate, ulceration and subtype. bAs for multivariate 1 but adjusted for thickness only, with nonsignificant variables removed.
is shown for patients with stage I–II disease (Table 3). Of the two deaths from BRAF V600K mutant tumours in this group, one tumour was thick with many mitoses and the other thin with few mitoses; hence, mutation status was best able to explain the deaths. However, death from BRAF V600K mutant tumours among all cases was explained by virtue of them being thicker and more mitotically active. Date of diagnosis of distant metastases was known for 33 of the 39 patients who died of melanoma. While there was no univariate association between mutation status and distant disease-free survival (DDFS), patients with BRAF mutant tumours had significantly poorer DDFS on multivariate analysis [HR 271, 95% CI 118–617 (P = 002) for patients with stage I–III disease; HR 318, 95% CI 107–947 (P = 004) for patients with stage I–II disease only]. NRAS mutant tumours were associated with poorer DDFS on multivariate analysis for patients with stage I–III disease (HR 280 95% CI 108–727; P = 004) but this association was not significant for patients with stage I–II disease alone (HR 240, 95% CI 066–864; P = 020). There was no significant association between mutation status and the time from diagnosis of distant metastatic disease to time of death on either uni- or multivariate analysis, although numbers were too small to draw strong conclusions from.
Discussion In this cohort, long-term follow-up revealed that patients with stage I–III primary melanomas harbouring BRAF mutations had approximately threefold the risk of death than patients with BRAF/NRAS wild-type tumours. There was a similar trend for patients with early stage I–II disease; however, the number of deaths in this group was too low to draw strong conclusions from. Importantly, this cohort pre-dates the use of BRAF inhibitors, the use of which does not therefore confound outcomes. British Journal of Dermatology (2015) 173, pp76–82
These findings support results from a larger cohort of 814 primary melanomas showing that BRAF mutant tumours were more likely than BRAF wild-type tumours to present with nodal metastases despite being significantly thinner.1 This study also provides an explanation for recent findings that thin melanomas are responsible for a disproportionate number of melanoma deaths,29 whereby deaths from thin tumours occurred after approximately 6 years compared with just 2 years following diagnosis of thick melanoma.29 Interestingly, melanomas harbouring a BRAF V600K mutation were associated with other poor prognosis tumour characteristics, such as increased tumour thickness, proliferative markers (MR and PH3) and nodular subtype. In accordance with this, they also tended to be more rapidly growing than BRAF V600E mutant tumours. Compared with BRAF V600E melanomas, which are associated with younger age and location on the trunk, BRAF V600K mutant melanomas have been shown to be more common in elderly patients, and on the head and neck (associated with chronic sun damage).30 As the number of tumours with this mutation was small (n = 10) in this cohort, no strong conclusions can be drawn; however, the results suggest that BRAF V600E and BRAF V600K mutant tumours differ in their clinical presentation, as well as their biological behaviour. Recent studies in metastatic disease have suggested that BRAF V600K mutant tumours have a more aggressive phenotype.30,31 The presence of a BRAF V600K mutation was not an independent predictor of outcome in this group once thickness was taken into account. Lack of distinction between these two mutations in other studies may lead to underestimation of the strength of association between the presence of BRAF V600E mutation and outcome. It is important to acknowledge that the measurement of melanoma ROG is an imperfect approximation, which relies on patient recall. However, subjective measurements of ROG have been shown to reflect objective measures of primary © 2015 British Association of Dermatologists
BRAF mutations in primary melanomas, V.J. Mar et al. 81
melanoma growth rate closely, particularly for more rapidly growing tumours.32 In the context of this study, ROG is useful for providing insight into the longitudinal clinical behaviour of different molecular subtypes in addition to proliferative markers, which are assessable only at one time point. We have previously reported an association between NRAS mutant melanomas and poorer MSS, although the presence of a BRAF mutation was not a significant prognostic factor in that study.2 The current cohort has the advantage of longer followup time (median 92 vs. 47 months) and more events (39 vs. 28 deaths), thereby extending the horizon over which these associations are assessed, and improving statistical power. In particular, Figure 1 shows separation in the survival curve for patients with BRAF mutant tumours only after the extended follow-up time. The fact that the longer follow-up period in the current study shows BRAF status to be an independent predictor of survival points towards these tumours growing and progressing more slowly, and is perhaps the reason why previous studies with shorter follow-up periods have not found any association between BRAF status and overall survival in early stage disease.2,9,12,13 Our finding that the presence of a BRAF mutation in earlystage disease is associated with poor outcome is supported by other studies of metastatic disease,5–8 and a recent study in early-stage disease that showed poorer disease-free survival , but not MSS, in 147 patients, with 17 deaths over a median followup of 48 months.9 The relative survival probability between patients with BRAF vs. BRAF/NRAS wild-type melanomas is pronounced after adjustment for thickness and nodal status, illustrating that, in early-stage disease, it is important to take other prognostic variables into account. This might explain why other studies reporting a univariate association only have not found a significant association between BRAF status and outcome.10,11,13 In our study the shorter MSS among patients with BRAF and NRAS mutant tumours was more likely to be attributable to shorter DDFS rather than a more rapid progression from stage IV disease to death. However, larger studies of stage IV cohorts have shown poorer overall survival following diagnosis of metastatic disease.5,33 Poorer survival among patients with BRAF mutant tumours is therefore likely to be attributable to both poorer DDFS and progression from stage IV disease to death. Although BRAF is a key oncogene in the mitogen-activated protein kinase pathway, which ultimately induces cell proliferation, the exact role of BRAF mutations in tumour progression remains complex. We have shown that proliferation markers are more likely to be reduced in BRAF V600E mutant tumours, and, indeed, the prognostic significance of MR is lost once mutation status is considered in this study. A few previous studies have reported a reduction in MR among BRAF mutant tumours,11,17 while others have not.5,13 It would be interesting to assess the prognostic significance of MR in larger cohorts with known mutation status. While BRAF and NRAS mutations are early initiating events in melanoma, it is likely that other molecular events are © 2015 British Association of Dermatologists
required for progression, to overcome such phenomena as melanocyte senescence (growth arrest) and suppression of apoptosis.34 Mutations in BRAF are found in benign melanocytic naevi, which proliferate after oncogene activation and then become senescent.35 Activated BRAF may induce senescence by upregulating p16 and other pathways early in melanoma formation.34,35 However, in late-stage disease, BRAF (unlike NRAS) mutations are often accompanied by loss of phosphatase and tensin homolog,36 resulting in reduced melanocyte apoptosis. This suggests that, acting alone, BRAF mutations are less efficient drivers of tumour proliferation and metastasis than NRAS.34 This may explain why the former often present as thinner lesions and are slower to progress. We have previously shown that BRAF mutant primary melanomas are more likely to show RAC1 immunoreactivity, which may be important in the early migration of these tumours.1 Our results show that the presence of a BRAF mutation increases the risk of melanoma death in patients with stage I–III disease. Evidence is growing that an assessment of mutation status along with other prognostic variables is important in assessing the risk of progression from early-stage disease, particularly as we approach an era of adjuvant therapies.
Acknowledgments We would like to acknowledge the Victorian Cancer Council for their assistance in data linkage for mortality follow-up and the contributions of Dr William Murray, Professor John Dowling, Dr Jill Magee and Dr Graham Mason, dermatopathologists involved in the reporting of cases in this cohort.
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