Discrepant alterations in main candidate genes among multiple primary melanomas

Incidence of cutaneous melanoma has increased during last decades in Western population [1, 2]. Several risk factors have been reported. A light phototype (especially when associated with excessive sun exposure and/or increased incidence of sunburns), a large number of acquired common nevi, and the occurrence of atypical nevi have been associated with a higher risk of melanoma [3, 4]. Among others, family history of melanoma (presence of two or, mainly, three or more affected relatives) confers the highest risk for the development of the disease [3, 5]. Nevertheless, patients with cutaneous melanoma present a higher incidence of second or even additional melanomas (risk seems to be highest in the first years after diagnosis of the first melanoma and decreases progressively with time) [6, 7]. However, subsequent primary melanomas have been found to be significantly thinner than index lesions [8], possibly due to increased surveillance and not to differences in tumor biology [9‐11]. In patients with multiple primary melanoma (MPM), the disease staging is based on the melanoma with the worst prognostic features [12].

From the pathogenetic point of view, the mitogen-activated protein kinase (MAPK) signal transduction pathway (including the cascade of NRAS, BRAF, MEK1/2, and ERK1/2 proteins) has been reported to play a major role in both the development and progression of melanoma [13, 14]. The increased activity of ERK1/2 proteins, which is constitutively activated in melanomas mostly as a consequence of mutations in upstream components of the pathway, has been implicated in rapid melanoma cell growth, enhanced cell survival and resistance to apoptosis [15, 16]. Oncogenic mutations of BRAF, all constituted by single amino acid substitutions, have been found in approximately 8% of all types of human cancer, including colorectal, ovarian, thyroid, and lung cancers as well as in cholangiocarcinoma and hepatocellular carcinoma [15, 17, 18], but their highest rates remain those observed in melanoma. Overall, slightly less than half of melanomas carry activating mutations in the BRAF gene [19, 20], regardless of the mutation screening approach used [21]. The affirmation of new drugs inhibiting some mediators of the MAPK pathway, including mutated BRAF and activated MEK, has led to major advances in the treatment of patients with melanoma [22].

A less common primary pathway which stimulates cell proliferation, without MAPK activation, seems to be the reduction of RB (retinoblastoma protein family) activity by CyclinD1 or CDK4 amplification or RB mutation (impaired RB activity through increased CDK4/cyclin D1 could substitute for the MAPK activation and initiate clonal expansion) [23]. Nevertheless, impairment of the p16^CDKN2A protein, which acts as an inhibitor of melanocytic proliferation by binding the CDK4/6 kinases and blocking phosphorylation of the RB protein, may also lead to uncontrolled cell growth as well as to increased aggressiveness of transformed melanocytic cells [23, 24].

It has been reported that melanomas on skin not chronically exposed to sun usually carry a mutated BRAF whereas those arising from chronically sun-damaged (CSD) skin infrequently have BRAF mutations but present an increased copy number of the proliferation-controlling CyclinD1 (CCND1) or cKIT genes, with subsequent increased expression of the correspondent proteins [25‐28]. Overexpression of the CyclinD1 gene is commonly observed in several human cancers, including breast, head and neck, and bladder cancers [29]. In melanoma, the elevated intracellular concentration of CyclinD1, related to the amplification of the gene locus at chromosomal level, has been implicated into the resistance to both BRAF and MEK inhibitors since it promotes a MAPK-independent cell proliferation [27, 30]. With no stratification for anatomical location, amplification of cKIT has been reported in about 7% of all cutaneous melanomas [25, 31]; its frequency increase up to 30% or more in acral and CSD melanomas as well as in melanomas carrying a cKIT mutation (prevalence is even higher in Chinese population [32]) [25, 31, 33].

In this study, we aimed at assessing the frequency and distribution of alterations in candidate genes (BRAF, cKIT, CyclinD1) involved in pathogenesis of melanoma in a large series of patients with synchronous or asynchronous MPM lesions.

Melanoma development and progression have been reported to occur by sequential accumulation of genetic and molecular alterations [18, 37]. Two main genetic networks have been demonstrated to play a crucial role in the control of growth, proliferation, and survival of the melanocyte cells: the CDKN2A-driven pathway and the mitogen-activated protein kinase (MAPK) signal transduction cascade [38, 39]. Genetic alterations in different members of these pathways have been associated with the pathogenesis of distinct types of primary melanomas: high frequency of BRAF or NRAS mutations (which are mutually exclusive) is mostly frequent in melanoma on skin without chronic sun-damage, whereas CyclinD1 or cKIT amplifications are prevalent in CSD or acral melanoma, respectively. In our study, we investigated the prevalence and distribution of such genetic alterations in MPM patients.

A high prevalence of somatic mutations in BRAF gene was detected in incident and subsequent melanomas. The frequency of BRAF mutations in primary melanomas (47%) was consistent with that observed in our previous study on 451 Italian patients with single melanoma (49%) [40] and slightly higher than that reported in a meta-analysis on 2521 patients with cutaneous melanomas (41%) [41]. In our series, two BRAF^V600 mutation subtypes were detected: V600E and V600K (in 41% and 7% of cases, respectively). Such two variants represent the most prevalent BRAF mutations (our frequencies were consistent with most of those reported in literature [41]) and are able to constitutively activate BRAF kinase [21]. Amplification of CyclinD1 and cKIT genes, as determined by FISH analysis, was found in about 14% and 5% of melanoma tissues from our series, respectively (see Table 3). Again, such frequencies were consistent with those reported in literature (ranging from 12% to 19% for CyclinD1 amplification [27, 42‐44] and calculated in about 7% of all cutaneous melanomas for cKIT amplification [25, 31]). One (0.4%) out of 229 melanoma samples presented a coexistence of BRAF mutation and cKIT amplification (see Table 4), confirming that aberrations in these two genes can be considered as mutually exclusive [26].

A markedly higher rate of either BRAF mutations (59%) or CyclinD1 (38%) or cKIT (13%) amplifications was previously observed in 32 melanoma cell lines as controls by our group ([45] and unpublished data). As reported [45], these control cell lines were established as primary cell cultures from tumor samples obtained from donor patients with documented diagnosis of melanoma. Since cultured melanomas are thought to represent cells with the most malignant phenotype, one could speculate that genetic alterations in these three candidate genes play a role in tumor progression.

Sixty-two paired samples from 54 (51%) patients showed discrepancies in BRAF/cKIT/CyclinD1 mutation patterns between first and subsequent primary melanomas (see Table 5). In the discrepant cases, we observed 20 (37%) patients with a wild-type first tumor and a mutated subsequent tumor, 14 (26%) with a mutated first tumor and a wild-type subsequent tumor, 8 (15%) with change in alteration variants between the two tumor lesions, and 12 (22%) with an additional gene amplification in the two BRAF-mutated tumors (3 cases in first but not in subsequent tumors and 9 with an opposite condition). In majority of cases (29/54; 53%), gene alterations seem to be acquired in subsequent melanomas. Moreover, while BRAF mutations were equally distributed among discrepant multiple melanomas (47.5% wild-type first tumors and mutated subsequent tumors, 47.5% mutated first tumor and wild-type subsequent tumors), rates of cKIT and CyclinD1 amplification were found to significantly increase moving from incident to subsequent primary melanomas (p values, <0.001 and 0.002, respectively). Such discrepancies were also confirmed among paired primary melanomas located at the same anatomical site as well as in synchronous primary melanomas (see Table 5). Overall, these observations provide evidence about the heterogeneity of the molecular mechanisms underlying the development of MPM in the same patients. The knowledge that molecularly heterogeneous cell types may coexist in primary melanomas [45, 46] is a further confirmation that complex pathogenetic scenarios exist in melanomagenesis.

About one third of patients presented a discrepant pattern of BRAF mutations between incident and subsequent primary melanomas (overall, 40/122; 32.8%). The introduction into the clinical practice of vemurafenib and dabrafenib, potent inhibitors of BRAF^V600 mutants, makes the assessment of BRAF mutations as a crucial step toward the appropriate use of a targeted melanoma treatment. The low consistency in BRAF mutation patterns among MPM lesions from the same patients arises the practical question on how cases with coexistence of BRAF ^wild-type and BRAF ^mutant primary melanomas (and, to a less extent, those carrying different BRAF variants - which may present a different degree of responsiveness to BRAF inhibitors) should be molecularly classified. Nevertheless, progression of disease in patients with such discrepancies in primary melanomas may suggest taking into consideration all developing metastases for BRAF mutation analysis cucaccording to the recent indications provided by the National Comprehensive Cancer Network (NCCN; at http://​www.​nccn.​org/​professionals/​physician_​gls/​f_​guidelines.​asp) guidelines, most recent melanoma tissue samples should be considered as adequate for BRAF mutation screening].