Background: Superficial spreading melanomas (SSMs) are the most common type of melanoma and cause the majority of skin cancer deaths. More than 50% of cases harbor a mutation in the BRAF gene that activates the mitogen-activated protein kinase (MAPK) cancer signaling pathway. BRAFV600E is the most common BRAF mutation, and it represents an important biomarker that guides treatment selection. However, the relationship between the BRAFV600E gene expression and intratumoral protein distribution, on one side, and clinicopathological factors and patient outcomes, on the other, is not fully described. Additionally, whether MAPK cancer signaling activation in melanoma is due to increased biochemical activity of BRAFV600E, increased mRNA levels, or both requires further investigation. Here, we addressed these questions by examining expression patterns of BRAFV600E in primary treatment-naive melanomas and correlating them to clinicopathological factors and patient outcomes. Methods: In 166 SSM cases, we performed immunohistochemical staining to investigate the protein expression of BRAFV600E, and we measured BRAF mRNA levels using NanoString nCounter system. Results: Ninety-seven (49%) melanomas stained positive for BRAFV600E, with nearly 100% intratumoral homogeneity observed. Positive BRAFV600E expression was significantly associated with nonrecurrent disease and was found to be an independent predictor of better prognosis in univariate and multivariable analyses. Furthermore, presence of tumor-infiltrating lymphocytes, sentinel lymph node biopsy negativity, and low Breslow thickness were all independent predictors of better prognosis. We observed no difference in the BRAF mRNA levels in BRAFV600E-negative and BRAFV600E-positive melanomas, respectively. Validation in a larger publicly available cohort confirmed that there is only a weak correlation (Spearman 0.4) between BRAFV600E mRNA and protein levels and no differences in mRNA between BRAFV600E mutated and non-mutated patients. Conclusion: Our findings indicated that BRAFV600E is homogeneously present throughout the whole tumor and is associated with nonrecurrent disease and better survival in primary melanoma. We also showed that BRAFV600E mutation does not result in higher transcriptional levels, suggesting that activation of the MAPK signaling pathway in BRAFV600E mutated patients can be attributed to the increased biochemical activity caused by the mutation.

Cutaneous melanoma arises from the malignantly transformed melanocytes in the skin, and it is the leading cause of skin cancer-related mortality worldwide [1]. Genome-wide sequencing has identified a remarkable genetic complexity in melanoma, and based on the pattern of the most prevalent oncogenes, melanoma can be classified into four genomic subtypes: mutant BRAF, mutant RAS, and mutant NF1, which feature activated mitogen-activated protein kinase (MAPK) cancer signaling pathway, and triple wild-type (WT), which has no mutations in any of the three genes [2]. The presence of mutated BRAF has been documented in more than 50% of melanomas, although it is generally seen at the earlier stages of tumor evolution and considered insufficient to cause the invasive phenotype [3, 4]. Several studies have found an association between BRAF mutation and reduced survival, while other studies report no differences [5‒11]. Oncogenic BRAF mutations are more frequently found in younger patients, in anatomical regions of the skin that are not subjected to chronic sun exposure, a risk factor for melanoma [12]. Furthermore, BRAF mutation-positive melanomas very frequently display a superficial spreading or nodular morphology [13]. BRAF inhibitors have emerged as an important class of targeted therapies for malignant melanomas. Unfortunately, patients undergoing treatment with BRAF inhibitors often develop resistance, especially when BRAF inhibitors are used as a monotherapy. Previously, intertumor and intratumor heterogeneity of BRAF mutations have been shown to give rise to different cellular proliferation rates and, more importantly, responses to treatment and resistance [14, 15]. This suggests that BRAF tumor heterogeneity may give rise to rapid emergence of drug resistance. However, a recent meta-analysis suggests that BRAF mutated tumors may display relatively low overall rates of heterogeneity [16].

Furthermore, BRAF mutations in melanoma and other cancers are usually detected on DNA level, but in one study, mRNA was used to synthesize cDNA that was sequenced to detect BRAF mutation [17, 18]. Thus, it is clear that the BRAF mutation is transcribed, but there is limited information about how the mutation affects the BRAF mRNA levels. In a small thyroid cancer study, the mRNA levels of BRAFV600E exhibited surprisingly large variability [19]. In addition to the mRNA level variability, there are reports of typically poor correlation between mRNA and protein levels, leading to the question of the biological translation of these findings in melanoma [20]. Although it is well established that BRAF mutation results in higher kinase activity, it is to our knowledge yet to be reported if BRAF mRNA level is affected in any way by the mutation.

Here, we provide insights into intratumor heterogeneity in primary treatment-naive melanomas by combining immunohistochemistry (IHC), which assesses BRAFV600E mutant protein levels, with mRNA analysis. Our results show that BRAFV600E is expressed homogenously throughout the individual tumors, and the mutation does not result in an increased transcription, measured by BRAF mRNA levels. Additionally, we observed that positive BRAFV600E expression was significantly associated with nonrecurrent disease and better prognosis.

Patient Samples and Criteria for Inclusion

A search in the Danish Pathology Register in Region Zealand in the period 1995 to 2015 using search SNOMED Clinical Terms for superficial spreading melanoma identified 1709 melanomas. After applying excluding criteria (Breslow thickness <1.0 mm or >4.0 mm, the presence of regression or ulceration), 166 cases were included in the final cohort (online suppl. Fig. S1; for all online suppl. material, see We did not include thinner melanomas due to missing sentinel lymph node biopsy (SLNB) status in these patients. Melanomas thicker than 4.0 mm or with ulceration were excluded due to their known association with markedly poorer prognosis. As there is still some uncertainty regarding prognostic importance of histological regression, we excluded all cases with this phenomenon [21].

Clinical and histopathological data were retrieved and included (Table 1). From 1998, all patients in Denmark diagnosed with melanoma over 1 mm thickness had SLNB performed. All patients were staged at the time of diagnosis according to the latest edition of the American Joint Committee on Cancer (AJCC8) melanoma staging manual [22]. Treatment with BRAF/MEK inhibitors and immune checkpoint inhibitors (ICIs) was registered. In Denmark, treatment with CTLA4 inhibitors and PD1 inhibitors was available for advanced disease from 2011 to 2015, respectively. Treatment with BRAF and MEK inhibitors was approved for advanced disease in 2012 and 2013, respectively.

Table 1.

Clinicopathological data of 166 melanoma patients

 Clinicopathological data of 166 melanoma patients
 Clinicopathological data of 166 melanoma patients

Electronic and paper medical charts were searched until death or until October 01, 2020. Melanoma-specific survival (MSS) and disease-free survival (DFS) were calculated from the date of receiving the specimen to the date of death or recurrence. Patients who did not die nor had recurrence were censored on the date of the last follow-up, and patients who died of other causes were censored at the time of death. The study was approved by the Regional Ethics Committee (SJ-742) and the Danish Data Protection Agency (REG-066-2019).

IHC Analyses of BRAFV600E

Formalin-fixed paraffin-embedded (FFPE) sections (3 μm) were stained on the fully automated instrument Omnis (Dako, Carpinteria, CA, USA). Sections were deparaffinized, exposed to antigen retrieval using EnVision FLEX Target Retrieval Solution (3-in-1) pH 9 (cat # GV834; Dako), and heated for 48 minutes at 97°C. Slides were incubated with anti-BRAFV600E clone VE1 (cat # ab228461; Abcam plc, Cambridge, UK) for 30 min at 32°C and diluted at 1:1500 for brown staining or 1:3000 for red staining (2 heavily pigmented cases). Antibody was diluted in Renoir Red (cat # PD904L; Biocare Medical, Pacheco, CA, USA). After washing and blocking of endogenous peroxidase activity, the reactions were visualized using the Envision FLEX + High pH kit (cat # GV800 + GV809/GV821; Dako) with 3,3′-diaminobenzidine as a chromogen for brown color and the Envision Magenta Substrate Chromogen System (cat # GV900; Dako) for red color, according to the manufacturer’s instructions. Finally, slides were rinsed in water and counterstained with Mayer’s hematoxylin. The presence of mutated BRAFV600E protein was characterized by cytoplasmic staining of the melanoma cells. General faint diffuse staining, weak staining of single interspersed cells, and only nuclear dot staining were scored as negative. The staining intensity was recorded on a semi-quantitative scale, with 0 = negative, 1 = weakly positive, 2 = moderately positive, and 3 = strongly positive. The proportion of stained tumor cells was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). To assess interobserver variability, all cases were assessed by two pathologists (LMGR and SONA), who were blinded to the patients’ clinicopathological and mutation data (BRAF PCR test done at recurrence). In cases with disagreeing scores, the slides were viewed by both observers together, and any discrepancies were resolved by consensus.

Gene Expression Studies Using NanoString nCounter Platform

Total RNA was isolated from five 10-µm sections of FFPE tissue using Roche Hi-Pure FFPET Kit (Roche Life Science, Mannheim, Germany) according to manufacturer’s protocol. NanoDropTM Lite spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) was used to measure total RNA quality and quantity. From each sample, 100–200 ng of RNA was used for hybridization using the nCounter PanCancer IO 360 Gene Expression Panel, according to the manufacturer’s nCounter XT protocol (NanoString Technologies, Seattle, WA, USA). The panel includes 770 genes, providing a 360-degree view of gene expression of tumor, microenvironment, and immune response. In addition, 20 genes of interest, including BRAF, were added to the panel (online suppl. Table S1). Samples were processed on nCounter sample preparation station and nCounter Digital Analyzer as previously described [23].

Statistical Analyses

Statistical analysis was performed using SPSS version 24 software (IBM Corporation, Armonk, NY, USA) and GraphPad Prism (version 8.1.1, GraphPad Software, San Diego, CA, USA). Baseline characteristics were described using means, standard deviation (SD) with ranges for the continuous variables, and numbers with percentage frequencies for the categorical variables. To evaluate the association between two variables, Pearson’s χ2 test (χ2), Fisher’s exact test, or Mann-Whitney U test were used as appropriate. MFS and DFS curves were calculated according to the Kaplan-Meier method and compared using the log-rank test. For univariate and multivariate recurrence and survival analyses, univariate and multivariate Cox proportional hazards regression models were used. A significance level of 5% (α = 0.05) was considered statistically significant.

TCGA Dataset

Skin Cutaneous Melanoma (TCGA Firehose Legacy) dataset at the cBioPortal site was used for validation [24, 25]. This cohort is based upon data generated by the TCGA Research Network: The dataset consisted of 471 patients with several available molecular profiles: methylation (HM450), mRNA expression (RNA Seq V2 RSEM), mRNA expression z-scores relative to all samples (log RNA Seq V2 RSEM), mutations, protein expression z-scores (reverse-phase protein array [RPPA]), and protein expression (RPPA). Metadata included ethnicity, sex, Breslow thickness, tumor site, Clark level, adjuvant postoperative pharmaceutical therapy administered, and staging according to the American Joint Committee on Cancer (AJCC). The Onco Query Language tool was used to query for BRAF mutation and subsequently comparison/survival and plot features were used to investigate DFS and overall survival (OS) and to correlate mRNA and protein data. Group comparison feature was used to create custom groups and compare their clinical and genomic features.

Patient Cohort Characteristics

Samples from 166 patients diagnosed with melanoma in Region Zealand between 1995 and 2015 were included in the study. Clinicopathological data of all patients are shown in Table 1. Distribution of male and female patients was almost equal, with 80 (48.2%) women and 86 (51.8%) men. The mean age was 57.7 years (range 21–84 years). The primary tumor site was predominantly on the trunk (n = 83, 50.0%) or leg (n = 41, 24.7%). The majority of patients had localized disease (N0 and M0), but 22.9% had at least one lymph node metastasis (N1-N3), and none had distance metastasis at the time of diagnosis. Information on patient survival, time, and cause of death was available in all cases. The last date of follow-up was December 1, 2020, and median follow-up time for survivors was 121 months (range 0–255 months). During the follow-up time, 40 (24.1%) patients experienced recurrence and 30 (18.1%) patients died of melanoma. Of the 40 patients with recurrence, 22.2% experienced recurrence within 1 year and 42.5 % within 2 years of diagnosis, and only 17.5 % experienced recurrent disease after 5 years (online suppl. Fig. S2). All treatment modalities were recorded for patients with recurrence, except in 10 patients (25%), due to destroyed medical charts. At recurrence, only 12 out of 40 patients had performed BRAF mutational status by PCR test, of which 6 was positive. Only 3 of these 6 patients received treatment with BRAF/MEK inhibitors alone or combined with ICIs. Of the 3 other patients, one received treatment with temozolomide (alkylating agent), one received treatment with interleukin, and one did not receive treatment because of poor physical health. In total, 3 patients received ICI monotherapy, and all of them had negative BRAFV600E expression.

Protein Expression of BRAFV600E in Melanoma Patient Samples

Staining with BRAFV600E antibody was assessed in all 166 cases, with 85 (51.2%) being positive and 81 (48.8%) being negative. The staining was cytoplasmic, and the staining intensity was assessed in all positive cases, with only 2 (2.4% of positives) cases staining weakly, 35 (41.1%) cases staining moderately, and 48 (56.5%) cases staining strongly (Fig. 1b–d). In pigmented cases, it was difficult to differentiate the positive signals of IHC from melanin deposition since the 3,3′-diaminobenzidine and melanin have a similar brown color, causing possible misjudgment of IHC results. In these cases, we compared the VE1 staining with already available hematoxylin and eosin stain (HE) which in most cases enabled us to clearly distinguish VE1 staining from melanin (Fig. 1g, h). In three challenging cases, we used a red chromogen (magenta) that easily discriminated VE1 stain from the brown pigment (Fig. 1i). The VE1 staining was clearly present in tumor cells from both the radial growth phase and vertical growth phase (Fig. 1g). Difference in staining intensity between tumor areas was rarely observed and linked to the presence of necrosis, fixation artifacts, or difference in morphology.

Fig. 1.

IHC staining for BRAFV600E. Skin melanoma with negative (a), weak (b), moderate (c), and strong (d) BRAFV600E expression with magnification for all ×10. High-powered view (×25) of the rectangular area in (c) and (d) shows approximately 100% homogeneous BRAFV600E expression across tumor cells. e Melanoma with abundant melanin deposition complicating evaluation of BRAFV600E expression. f HE stain of the same area as in e confirms that the brown color observed in VE1 stain was solely representing melanin and the melanoma was considered “negative ”for BRAFV600E. g Melanoma with weak VE1 staining, melanin deposition in macrophages, and some background staining. h HE stain with high-powered view (×25) of the rectangular area in g showing abundant pigment-laden macrophages, still representing uncertainty. i VE1 stain with magenta of the same area as in g reveals a convincing BRAFV600E staining of the tumor cells, thus melanoma is regarded as “moderate positive.”

Fig. 1.

IHC staining for BRAFV600E. Skin melanoma with negative (a), weak (b), moderate (c), and strong (d) BRAFV600E expression with magnification for all ×10. High-powered view (×25) of the rectangular area in (c) and (d) shows approximately 100% homogeneous BRAFV600E expression across tumor cells. e Melanoma with abundant melanin deposition complicating evaluation of BRAFV600E expression. f HE stain of the same area as in e confirms that the brown color observed in VE1 stain was solely representing melanin and the melanoma was considered “negative ”for BRAFV600E. g Melanoma with weak VE1 staining, melanin deposition in macrophages, and some background staining. h HE stain with high-powered view (×25) of the rectangular area in g showing abundant pigment-laden macrophages, still representing uncertainty. i VE1 stain with magenta of the same area as in g reveals a convincing BRAFV600E staining of the tumor cells, thus melanoma is regarded as “moderate positive.”

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Next, we analyzed the proportion of stained tumor cells present in each VE1 positive case to examine intertumoral heterogeneity of BRAFV600E distribution. All samples were scored as 4 (76–100% of cells positive), and no notable intratumoral heterogeneity was observed. At recurrence, 12 patients had known BRAF mutational status, and correlating these findings to our VE1 staining results, we found 100% concordance (Table 1). Taken together, these results indicate that BRAFV600E molecules are homogeneously distributed within BRAFV600E-positive melanomas.

Association between BRAFV600E Expression and Clinicopathological Factors

To examine potential association between BRAFV600E expression and routine clinicopathological factors, we combined results of our protein expression analysis with the patient-cohort data (results of the analysis shown in Table 2). Among the factors we examined, including the presence of tumor-infiltrating lymphocytes (TILs), SLNB status, stage at diagnosis, primary tumor site, and Breslow thickness, only female sex (p = 0.0059), younger age at diagnosis (p = 0.003), and nonrecurrent disease (p = 0.002) were significantly associated with BRAFV600E expression. Surprisingly, we observed lower risk of recurrence in BRAFV600E-positive cases, and patients with BRAFV600E-positive melanoma had significantly longer MSS than those with BRAFV600E-negative melanoma (p = 0.0041, 5-year MSS 92 % and 82 %, respectively) (Fig. 2a). In addition, patients with BRAFV600E-positive melanoma had longer DFS (p = 0.0021, 5-year DFS 89 % and 69%, respectively) (Fig. 2b). Subgroup analysis comparing patients with moderate staining intensity with strong intensity showed no significant difference in MSS or DFS (online suppl. Fig. S3A, B). In addition, we investigated if patients with BRAFV600E expression had received medical treatment at higher frequency and if this could account for their better prognosis compared to BRAFV600E-negative patients. No difference was found, with 5 out of 12 patients (41.6%) with positive VE1 stain and 13 out of 28 patients (46.4%) with negative VE1 stain receiving medical treatment (p = 0.781) (Table 2). Only 3 patients received ICI monotherapy, and all of them had negative BRAFV600E staining (Table 1). As ICIs have promising clinical benefit, the advantage of the targeted therapy that was given to the 3 patients with BRAF mutation was reduced. To validate our results, we investigated the association between BRAFV600E mutational status at DNA level and prognosis in a larger cohort consisting of 471 cutaneous melanoma samples (TCGA Firehose Legacy) at cBioPortal. In line with our results, patients with a BRAFV600E mutation had a significantly better OS (p = 0.00334) and DFS (p = 0.0232) when compared to patients with no BRAFV600E mutation (online suppl. Fig. S4A, B). In the TCGA dataset, 18.3% (67/366) of the total patients with available mutation data received non-specified adjuvant postoperative pharmaceutical therapy. In comparison, 10.8% (18/166) of patients in the current cohort received adjuvant pharmaceutical therapy. Furthermore, in the TCGA dataset, we observed that a higher percentage of patients with a BRAF mutation received adjuvant postoperative pharmaceutical therapy compared to the patients with no BRAF mutation. Difference in treatment frequency could possibly explain the better OS in the dataset. There was, however, no difference in OS and DFS between the patients that received adjuvant therapy compared to those that did not (online suppl. Fig. S5A, B). Taken together, we observed no significant association between BRAFV600E and the presence of TILs, SLNB status, stage at diagnosis, primary tumor site, and Breslow thickness. However, significant association was observed between the presence of BRAFV600E and female sex, younger age at diagnosis, and nonrecurrent disease. Furthermore, BRAFV600E-positive patients exhibited better OS.

Table 2.

Clinicopathological factors associated with BRAFV600E protein positivity

 Clinicopathological factors associated with BRAFV600E protein positivity
 Clinicopathological factors associated with BRAFV600E protein positivity
Fig. 2.

a, b Kaplan-Meier curves for MSS and DFS according to BRAFV600E expression (log-rank test).

Fig. 2.

a, b Kaplan-Meier curves for MSS and DFS according to BRAFV600E expression (log-rank test).

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Cox Univariate and Multivariate Analysis for Patient Survival

Having noted that mutant BRAFV600E protein expression is associated with MSS and DFS, we wanted to examine if it was an independent prognostic factor for MSS and DFS (Tables 3, 4). The presence of TILs, BRAFV600E positivity, SLNB negativity, and low Breslow thickness were significantly associated with better survival in univariate analysis of MSS, and all variables except Breslow thickness remained significant in final multivariable analysis. In the univariate analysis, we also included TILs and VE1 staining intensity as categories and observed significant association only when comparing non-brisk TILs with total absence of TILs and when comparing strong VE1 staining intensity with no staining. BRAFV600E positivity, SLNB negativity, and low Breslow thickness were significantly associated with improved DSF in univariate analysis and remained significant and independent predictors of better prognosis in the multivariable analysis. Therefore, BRAFV600E status represents an independent predictor of patient’s survival.

Table 3.

Cox proportional hazards regression models of variables associated with MSS

 Cox proportional hazards regression models of variables associated with MSS
 Cox proportional hazards regression models of variables associated with MSS
Table 4.

Cox proportional hazards regression models of variables associated with disease free survival

 Cox proportional hazards regression models of variables associated with disease free survival
 Cox proportional hazards regression models of variables associated with disease free survival

BRAFV600E Protein Expression and Correlation to mRNA Levels

We next examined whether BRAFV600E-positive and -negative tumors exhibit significantly different BRAF mRNA levels. As shown in Figure 3, no difference between mRNA levels of the two groups was observed (p = 0.352). We next correlated the mRNA levels to BRAFV600E protein expression intensity, finding no significant changes (online suppl. Table S2). We compared our results to those of a larger melanoma cohort (TCGA Firehose Legacy), comparing the mRNA levels across samples with and without BRAFV600E mutation. We could not identify substantial changes between these two groups, p = 0.052 (online suppl. Fig. S6A). A weak correlation was found comparing the protein levels of BRAF measured by RPPA to mRNA levels in the same cohort (Spearman correlation 0.38, p = 9.83e−14) (online suppl. Fig. S6B).

Fig. 3.

Box plot of BRAF mRNA levels across samples with positive and negative expression of BRAFV600E, Mann-Whitney U test, p= 0.352.

Fig. 3.

Box plot of BRAF mRNA levels across samples with positive and negative expression of BRAFV600E, Mann-Whitney U test, p= 0.352.

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Taken together, these analyses suggest that melanoma tumors express similar levels of BRAF mRNA, regardless of the mutational status. Therefore, any changes in MAPK signaling pathway activity are likely due to well-documented increased enzymatic activity of BRAFV600E protein and not increased mRNA levels.

Here we describe results of a regional population-based study aimed at characterizing the interpatient intratumor heterogeneity with respect to the most frequent BRAF mutation (V600E) in melanoma. We employed IHC staining and correlated the results to clinicopathological factors, prognosis, and BRAF mRNA levels. We observed that BRAFV600E displayed a 100% homogenous staining pattern throughout the tumor when present and that these patients did not have higher corresponding mRNA levels when compared to those with no BRAFV600E expression. Furthermore, the presence of BRAFV600E predicted an improved MSS and DFS after adjustments for other prognostic factors. Our results contribute new knowledge to melanoma biology and may have important implications for melanoma treatment and management, as we discuss below.

It is well known that melanomas harbor heterogeneous molecular profiles, and BRAF intratumor heterogeneity has been suggested as one of the several causes of drug resistance [26]. However, numerous BRAF testing methods, including PCR DNA based tests, next-generation sequencing, and qPCR, do not create clear in situ visualization of tumor heterogeneity. Therefore, we chose to employ IHC as it provides a very fast indication of mutational status, is less expensive, and is readily available in most pathology departments. Furthermore, we chose to focus on visualizing BRAFV600E because (1) this mutation is the most frequently reported mutation in melanomas and (2) a mutation-specific, highly specific (1.00) and highly sensitive (0.96) antibody (VE1) is readily available [27, 28], thus ensuring highly reliable IHC analysis. In this study, we used VE1 antibody to characterize the BRAFV600E expression in 166 patients with primary superficial spreading melanomas. The staining was positive in just over half (51.2%) of the cases, in accordance with other reports [2]. We found a homogenous BRAFV600E expression in all cases, similar to some previous studies [29, 30] and contrary to other studies [31, 32]. It is known that BRAFV600E arises early during melanoma development, and this could help to explain the homogenous immunolabelling of the majority of tumor cells.

In addition to the high level of quality control that went into this work, the strength of the current study is a large and well-characterized patient cohort. This allowed us to perform in-depth analysis of association between BRAFV600E positivity and clinicopathological factors. We observed no association between BRAFV600E status and important clinicopathological parameters that could potentially effect survival, such as presence of TILs, SLNB status, stage at diagnosis, primary tumor site, and Breslow thickness. We did observe a higher frequency of positive BRAFV600E staining in women, although previous reports have shown no gender differences [9, 33]. Biological differences (e.g., hormonal) or different behaviors between men and women (e.g., level of sun exposure) could explain our results. Additionally, we also noted higher frequency in younger patients in line with other studies [12, 13].

Generally, patients with BRAF mutated tumors have been reported to have a higher risk of recurrence [34, 35], but surprisingly, we report a lower risk of recurrence in patients with BRAFV600E expression. Our survival analysis also supported the positive relationship between BRAFV600E positivity and MSS and DFS. Overall, our study indicates that BRAFV600E positivity, SLNB negativity, and low Breslow thickness represent independent predictors for better survival considering both MSS and DFS. Furthermore, we report that the presence of TILs is an independent predictor for longer MSS. Our results are consistent with a large body of research concluding that TILs can predict melanoma survival and identify patients at risk for metastatic recurrence using both IHC and molecular analyses [36, 37]. However, in our cohort, TILs were not associated with DFS, consistent with the results from a larger meta-analysis that failed to show a prognostic effect of TILs of any grade on DFS [38].

Nevertheless, given that the current literature report contradictory results on this point, including a meta-analysis assessing 52 studies (7,519 patients) revealing poorer prognosis and OS for patients with mutant BRAF versus WT patients and several studies that reported no difference in survival between the different mutation profiles [39‒43], at this point, we argue that the question regarding correlation of BRAF mutational status and clinical behavior remains open.

Of note, in the meta-analysis by Ny et al. [39], studies with lower stage melanomas either reported no difference in OS or better but not statistically significant OS in BRAF mutated melanomas. The majority of included studies, where BRAF mutation was associated with poor survival, involved patient samples from stage III and IV melanomas. However, the meta-analysis did not adjust for Breslow thickness, the presence of TILs, or ulceration status – factors that are known to affect prognosis in early-stage melanoma [44]. For advanced melanoma, on the other hand, the survival is associated with factors such as the number of positive regional lymph nodes and site of metastases [22]. In our cohort, 77.1% (128/166) of the patients had stage I-II disease at time of diagnosis. Our prediction of the clinical impact of BRAF mutation is based on a multivariate survival analyses, thereby adjusting for Breslow thickness and presence of TILs.

Another challenge in interpreting the previous results from Ny et al. [39] is that there is no consensus in regards to WT melanomas. In the aforementioned meta-analysis, WT was defined as dual BRAF/NRAS WT in 19/52 studies and, in the remaining 33 studies, WT included solely BRAF WT tumors. According to the TCGA research network, WT is defined as triple BRAF/NRAS/NF1 WT, as all of them exert their effect in the MAPK pathway [2]. Furthermore, differences in the level of BRAF subtyping also represent limitations, as some of the studies measure all the known BRAF mutations and while others map the most prevalent, complicating direct comparisons between studies.

A potential limitation to the current study could also have been different treatment regimens depending on mutational status. We therefore investigated if patients with positive BRAFV600E had the advantage of better treatment at recurrence and found no differences compared to patients with negative BRAFV600E. We also validated our results in online melanoma cohort with known BRAFV600E DNA mutational status, showing similar results. The validation cohort is larger than our cohort, broadly inclusive and comprises extensive clinical and molecular data. In the TCGA dataset, patients with a BRAF mutation received adjuvant therapy at a higher frequency, but the adjuvant therapy did not seem to affect OS and DFS. Another issue with interpreting prognosis from various studies is the different populations, patient selection, and percentage of histologic subtypes, genetic variation, level of UV exposure, skin pigmentation, and other unknown factors effecting the progression of melanoma [45]. Therefore, going forward, studies correlating BRAF mutational status to survival in large cohorts including different regions in Denmark or countries in Europe should be conducted to clarify whether correlations identified by us and others are influenced by the variance of different populations.

High sensitivity and specificity of VE1 allowed us to bypass DNA testing and to, for the first time, correlate BRAFV600E results to mRNA levels. Positive VE1 staining in our cohort, which is representative of BRAFV600E mutation, was not associated with higher mRNA levels. Moreover, subsequent analysis in a larger online melanoma cohort also showed lack of association between mRNA levels and BRAFV600E mutation. Many factors are involved in regulation of mRNA levels and protein levels, such as translation and stability of mRNA and protein half-life. In addition, the majority of BRAF mutations constitute missense mutations that change the amino acid sequences of the protein and not necessarily result in a higher mRNA transcription and protein levels. Although BRAF mRNA may have no current clinical application value, it gives important knowledge about biology and may be useful for researchers studying gene expression in melanoma by targeted gene expression profiling, RNA seq., etc. Our results indicate that upregulation of MAPK kinase signaling pathway in BRAFV600E melanoma is not due to increased levels of BRAFV600E mRNA but to increased enzymatic activity of the mutant BRAF, which is known to be 500-fold more active than the WT in vitro [46]. This theory is also supported by the fact that no association was found when correlating mRNA to BRAFV600E protein expression intensity in our cohort.

In conclusion, we have shown that BRAFV600E is expressed homogenously throughout the individual SMM tumors and the mutation does not result in an increased transcription, as measured by mRNA levels. We also identified associations between several important clinicopathological factors and BRAFV600E positivity, including a surprising positive association between BRAFV600E levels and MSS and DFS. The common view that BRAF mutation promotes poor prognosis is based on studies of advanced stage melanomas. Surprisingly, in our cohort of primarily low stage melanomas, we found a better prognosis in patients with BRAFV600E mutation. By controlling for other factors known to affect survival, including Breslow thickness and presence of TILs, we strengthen the power of BRAFV600E as an independent predictor for better survival. However, gene expression studies in melanoma have also indicated that additional factors act in concert with the BRAF mutation to produce a prognostic phenotype [47, 48]. These observations are in accordance with the fact that the progression of melanoma is a complex process, involving several interconnected pathways and multiple oncogenes [3]. Although BRAF is arguably an important driver of tumor growth, underlined by the efficacy of targeted treatments, there is still a need for a better understanding of the biological translation of these mutations in melanoma and clinical biomarkers to predict and monitor disease progression and treatment efficacy.

BRAFV600E expression in melanoma predicts better prognosis.

The study protocol was reviewed and approved by the data protection agency in Region Zealand, Denmark, approval number (REG-066-2019) and by the Ethics Committee in Region Zealand, Denmark, approval number (SJ-742). The approval from the Ethics Committee in Region Zealand, also included exemption from informed consent.

Lise Mette Rahbek Gjerdrum has received funding from NanoString Technologies. The remaining authors state no conflict of interest.

This work was supported by grants from the Novo Nordisk Foundation (NNF21OC0066694, NNF15OC0014092), Lundbeck Foundation (R182-2014-3641), the Aage Bangs Foundation, Region Sjaellands forskningsfond, and the Harboefonden.

Lise Mette Rahbek Gjerdrum and Beatrice Dyring-Andersen designed the research; Soraya Naimy, Michael Bzorek, and Jens Ole Eriksen performed experiments; Soraya Naimy, Lise Mette Rahbek Gjerdrum, Beatrice Dyring-Andersen, and Jens Ole Eriksen analyzed the data; Soraya Naimy, Lise Mette Rahbek Gjerdrum, Beatrice Dyring-Andersen, and Michael Bzorek wrote the paper. All authors read and approved the manuscript.

Dataset related to this article have been deposited in the Gene Expression Omnibus (GEO) with accession number GSE193802 and were made publicly available on August 31, 2022. Further inquiries can be directed to the corresponding author.

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