BRAF Mutation Status in Primary, Recurrent, and Metastatic Malignant Melanoma and Its Relation to Histopathological Parameters

Background BRAF mutations are a common finding in malignant melanoma (MM). Nevertheless, apart from their significance as a therapeutic target in advanced melanoma, their prognostic value is still debated. Objective To assess BRAF mutation status in primary, recurrent, or metastatic MM and its correlations with histopathological findings. Methods We analyzed 203 samples from 178 consecutive patients: 129 primary cutaneous MM, 49 metastatic and recurrent MM of unknown primary site, and 25 cases of recurrences or metastases of primary MM. BRAF mutations in exon 15 were identified with real-time polymerase chain reaction and/or direct sequencing or pyrosequencing. Histopathological examination was performed according to standard procedures. Results We observed a 42.1% prevalence of BRAF mutations at codon 600 among our patients, 84% of whom harbored the V600E mutation. Mutations showed a statistically significant increase in younger patients (P = 0.011), in ulcerated tumors (P = 0.020), and in tumors lacking solar elastosis in adjacent dermis (P = 0.008). Mutations were also more common in male patients, as well as in primary MMs of the torso, and in nonvisceral metastases, however without reaching statistical significance. Logistic regression analysis identified type and ulceration as the only significant predictors of BRAF mutation. The highest frequencies of mutated BRAF were identified in superficial spreading and nodular types, and the lowest in acral lentiginous and lentigo maligna types. In situ MM and primary dermal melanoma displayed intermediate frequencies. Conclusion Frequency of mutated BRAF is type-related and correlated with ulceration, a known adverse prognostic factor.

topathological parameters associated with prognosis and survival of MMs being tumor thickness, ulceration, and, more recently, mitotic score at primary site [10].
We assessed the BRAF mutation status among 178 patients with primary, recurrent, or metastatic MM. Our aim was to study the correlations between histopathological findings in primary MM and the presence of mutated BRAF in a Greek population.

Methods Specimens
A total of 203 consecutive specimens from 178 patients referred for histopathological examination during a 12-year period (2003-2015) were analyzed (Table 1). These included 129 primary cutaneous MMs (a primary site recurrence was also included in 13 cases and a metastasis in 12 cases), 44 cases of metastatic MM of unknown primary site, and 5 cases of cutaneous recurrence without an initial tumor. The institutional review board of the hospital approved the study, while analysis was performed on anonymized data.
All specimens were fixed in a buffered 10% formalin solution, processed according to standard protocols, and initially diagnosed by the same experienced pathologist. Another, equally experienced pathologist performed a second, blind evaluation of histopathological diagnosis for the purpose of this study. In case of disagreement, slides were conjointly rereviewed.

Introduction
Malignant melanoma (MM) is one of the most common types of cancer in humans, being the fifth most common in men and the sixth in women in the United States [1]. Although MM represents less than 2% of skin cancer, it accounts for the vast majority of deaths, with increasing incidence worldwide [1].
The underlying etiopathogenetic mechanisms remain so far largely unknown.
Following discovery of BRAF oncogenic mutations in various types of malignancies, a significant pathogenetic role for mutated BRAF has been proposed for MM [2]. To date, more than 30 different mutations in the BRAF gene have been identified. The most frequent, accounting for almost 90%, concerns substitution of thymine by adenine at nucleotide c.1799, leading to valine (V) being substituted by glutamate (E) at codon 600 (p.V600E) in the activation segment of the gene promoting cell survival via the ERK or MEK signaling cascade [3]. Frequency of BRAF mutations varies widely, from more than 80% in melanomas and nevi, to 1%-3% in lung and 5% in colorectal carcinoma [2]. Of note, BRAF mutations are not exclusive to MM, having also been found in up to 80% of benign melanocytic lesions, albeit in limited study populations [4].
Oncogenic mutations of BRAF are mimicking a constantly activated state, resulting in uncontrolled cell growth and proliferation. More recently, oncogenic BRAF mutations have been connected to up-regulated cell proliferation and invasion ability [5]. It has been suggested that BRAF mutations may constitute a tumor progression event rather than an initiating event in MM tumorigenesis, and that other genetic or epigenetic factors are also involved [6,7]. The presence of a BRAF mutation in primary MM of stage III has been associated with worse prognosis and also seems to be related to progression-free interval and overall survival [8].
Mutated BRAF constitutes a therapeutic target for patients with advanced MMs. Different BRAF inhibitors have been tested, with significant benefits in terms of increased response rate, progression-free survival, and overall survival [9]. Nevertheless, the prognostic value of BRAF mutations has not been fully assessed, with the only established his-younger patients (P = 0.011), in ulcerated tumors (P = 0.020), and in tumors lacking solar elastosis in adjacent dermis (P = 0.008). Mutations were also more common in male patients, as well as in primary MMs of the torso, and in nonvisceral metastases, however without reaching statistical significance. Logistic regression analysis identified type and ulceration as the only significant predictors of BRAF mutation. The highest frequencies of mutated BRAF were identified in superficial spreading and nodular types, and the lowest in acral lentiginous and lentigo maligna types. In situ MM and primary dermal melanoma displayed intermediate frequencies.
Conclusion: Frequency of mutated BRAF is type-related and correlated with ulceration, a known adverse prognostic factor. In 12 patients, samples from both primary site and metastasis were available: in 10/12 (83.3%) cases both sites had the same genotype, whereas one primary carried a V600E mutation not identified in nodal metastasis, and another was WT in the primary with a V600E mutation in the visceral metastasis.

Correlation of BRAF Status to Epidemiological and Clinical Parameters
Correlation of BRAF mutation status with epidemiological and clinical parameters is shown in Table 1. Results for sequenced in-house using a commercially available kit (Therascreen BRAF pyro kit, Qiagen, Düsseldorf, Germany) [11].

Statistical Analysis
Statistical analysis was performed using IBM Statistics SPSS 24 (IBM Corporation, New York, NY) and involved the Fisher exact test for 2 × 2 tables, χ 2 for trend for 2 × Y tables, Mann-Whitney test for differences of mean of categories, and Spearman correlation analysis for scale correlations. Binary logistic regression was used for validation of observed differences of BRAF status with all mutations grouped in a single category.

Demographic and Clinical Data
One hundred seventy-eight melanoma patients aged 21-95 years (mean 62.1 years) were included in the study. There

Association of BRAF Status to Histopathological Parameters
In primary MMs (  significance, as some of them did in previous studies [12,13].
Almost all pairs of primary and metastatic sites had the same genotype, except 2 patients, in whom a discordance of BRAF status between primary site and metastasis was found [7,14].
In a recent meta-analysis, BRAF and NRAS mutations were associated with histological subtype and tumor site, but not with patient age or sex [15]. BRAF mutations were frequently detected in patients with SSM and in MMs arising in non-chronically sun-damaged skin. In contrast, NRAS mutations were more frequent in patients with NM and in MMs arising in chronically sun-damaged skin [15]. Another meta-analysis of BRAF mutations and their associations with the clinicopathological characteristics of primary MM showed an association of BRAF mutations with younger age, location at the torso, non-chronically sun-damaged skin, SSM type, and advanced melanoma stage [16]. Similar results were identified in our sample set, but after using a logistic regression model, only ulceration and melanoma type were significant predictors.
In agreement with previous studies, BRAF mutations were more commonly found in SSM, and with decreasing frequency in NM, ALM, and LMM [12,13,16,17] growing ex-SSMs, whose adjacent intraepidermal component was phased out [18].
Another histopathological parameter significantly related to BRAF mutations was ulceration. Some studies have also reported increased BRAF mutation percentage in ulcerated samples [12,17,[19][20][21], while others, including a meta-analysis, did not [13,16]. The 2 studies that included only 1 type from a slight increase in BRAF mutations among cases with  We then used a backward step-wise Wald binary logistic regression analysis including all borderline and statistically significant parameters. Topology and age were removed during model creation, while ulceration (P = 0.008, odds ratio = 3.509) and type (P = 0.024), specifically SSM type (P = 0.020, odds ratio = 7.626), were the only predictors.

Discussion
Per our results, a 42.1% prevalence of BRAF mutations at codon 600 among our patients with MM was observed. These mutations were statistically more common in younger patients, in ulcerated MMs, and in MMs lacking solar elastosis in adjacent papillary dermis. These findings are consistent with previous experience [7,12]. A trend for increased percentage of BRAF mutations was documented in men with MMs of the torso, in MMs more than 1 mm Breslow thickness, higher mitotic rate, reported metastasis, and earlier reported recurrences. However, these findings did not reach statistical  BRAF mutations have been previously linked to transition from radial to vertical growth phase [6,23]; to shorter recurrence-free, disease-specific survival [8]; and to overall survival with a more protracted growth rate [24]. However, the exact role and the pathways mirrored in specific morphological features such as ulceration and pathology types is elusive.

Conclusions
Our results suggest that BRAF mutations are more frequent in SSM, ulcerated MMs independent of histological type, MMs may contribute to distinguishing melanoma types and estimating prognosis more accurately [25].