Prognostic significance of ALCAM (CD166/MEMD) expression in cutaneous melanoma patients

The unique local homeostasis maintained between keratinocytes, melanocytes and the extracellular matrix that surrounds them has a key role in the regulation of melanocyte growth and proliferation [1, 2]. Each melanocyte is estimated to directly interact and regulate approximately 36 keratinocytes [3]. Melanocyte growth is regulated by the surrounding keratinocytes through various control mechanisms. The two most important involve: (1) paracrine growth factors secreted into the extracellular environment, and (2) intercellular communication of melanocytes and keratinocytes with extracellular matrix proteins via multiple adhesion molecules [4]. All disturbances of this extremely precise homeostasis may lead to abnormal expression of adhesion proteins and molecules involved in intercellular communication, which may in turn initiate melanomagenesis.

In the initial stages of melanoma growth and progression, keratinocytes lose their ability to regulate melanocytes as a result of the three main pathobiochemical processes that occur in melanoma cells. One of them involves reduced expression of receptors such as E-cadherin, P-cadherin and connexin which are crucial for melanocyte-keratinocyte communication [4, 5]. The second mechanism via which the epithelium cells lose control of melanocytes is directly connected with overexpression of receptor proteins and signaling molecules involved in cytological interactions of the melanocyte-fibroblast type. It was demonstrated that increased immunoreactivity of proteins e.g. N-cadherin, Mel-CAM and ZO-1 (zonula occludens protein-1) is related with increased invasive potential of melanoma cells [4, 6]. The last mechanism that allows melanocytes to escape from control by keratinocytes involves loss of expression of proteins anchoring melanocytes to the basement membrane [4, 7].

Maintaining intercellular adhesion is a necessary process during invasion of cancer cell nests [8]. However, adhesion deregulation must be one of the stages of metastatic foci development. It is a key moment in which single cells or nests use the changed profile of adhesion molecules expression to occupy new areas, which initiates the spread of cancer. Adhesion protein spectrum whose immunoreactivity panel would be characteristic for invasive melanoma cells has not been identified so far.

ALCAM (activated leukocyte cell adhesion molecule, CD166, MEMD) is a transmembrane protein of immunoglobulin superfamily (Ig-SF) [9]. The gene encoding ALCAM is located on the long arm of chromosome 3 and it is organized into 16 exons [10]. Ultrastructural studies show it has 27 % homology with adhesion molecule found on the surface of MUC18/Mel-CAM/CD146 melanoma cells, which promotes progression of this cancer and formation of locoregional and distant metastases [11]. The key element of ALCAM molecule that regulates its adhesive capacity is the extracellular part composed of five immunoglobulin-like domains (D1-D5) [12]. There is also a transmembrane domain and an intracellular short C-terminal tail. At first, ALCAM was identified as a ligand for CD6 receptor (heterotypic interaction, ALCAM-CD6), and its expression was observed on the surface of leukocytes, fibroblasts, epithelial and nerve cells [10, 13]. Other cytophysiological analyses revealed the existence of functional homotypic interactions of the ALCAM-ALCAM type which were identified in CD6-negative melanoma cell lines [14]. The study using melanoma cell lines showed that overexpression of ALCAM is directly related with the increase of cytoaggregation and the ability to form cell nests [14]. Additionally, it was found that the homotypic ALCAM-ALCAM interaction plays a key role during intercellular adhesion. Interestingly, Degen et al. [14] proved that ALCAM expression is correlated with increased metastatic potential of melanoma cell lines.

So far, little is known about regulating expression of ALCAM on the gene level. It was showed that the ALCAM promoter contains a sequence binding NF-κB and AP-1 (activator protein 1) which act as transcription factors [15, 16]. Studies into the potential effects of NF-κB on ALCAM expression demonstrated that its binding to the promoter sequence of ALCAM gene enhances the immunoreactivity of the resulting protein product [15]. In turn, the effect of AP-1 family, and specifically overexpression of Fra-2 (Fos-related antigen 2) is closely related with the resulting decreased production of ALCAM [16].

Interesting conclusions concerning new biological functions and role of melanoma progression were drawn by Luntner et al. [17] based on their experiment. ALCAM is instrumental for the formation of a triple complex on the surface of melanoma cells MT1-MMP/TIMP-2/pro-MMP-2 and the subsequent conversion of inactive form of metalloproteinase 2 (pro-MMP-2) into an active form responsible for the degradation of extracellular matrix and contributes to the formation of nodal and distant metastases [17]. Inhibition of ALCAM expression resulted in significantly decreased MMP-2 activity, and thus in decreased metastatic potential. Pooled analysis of results has allowed establishing a hypothesis that ALCAM is involved in regulating proteolysis via some still unknown mechanisms and seems to act as a cell-surface sensor for invasive growth [15, 17].

The aim of the study was to assess the expression and intracellular localization of ALCAM in 104 primary skin melanomas and 16 metastatic lesions from regional lymph nodes. Also, prognostic significance of ALCAM expression in primary tumor cells and metastatic lesion cells was evaluated in the context of 5-year observation. Correlations were also analyzed between ALCAM immunoreactivity parameters and detailed clinicopathological parameters such as Breslow thickness and Clark level, presence of nodal and distant metastases, recurrence of primary tumor, mitotic rate, ulceration, lymphocytic inflammatory infiltration, regression and microsatellitosis.

Our study is the first one to evaluate the effect of increased ALCAM expression on long-term survival in melanoma patients. We demonstrate that high ALCAM expression in primary tumor cancer cells (IRS ≥8) is strongly correlated with unfavorable prognosis as compared with patients with lower ALCAM immunoreactivity in tumor compartment as regards cancer specific overall survival (CSOS) (P = 0.001) and disease free survival (DFS) (P < 0.001). Furthermore, the results were similar for a high percentage (>75 %) of ALCAM-positive melanoma cells in primary tumor (P = 0.007 and P = 0.025 for CSOS and DFS, respectively). An important part of our study was to evaluate the correlation between ALCAM expression in metastatic foci in regional lymph nodes and detailed clinicopathological parameters. It was found that decreased ALCAM expression (IRS <8) in nodal metastases shows a trend related with a correlation with shorter cancer specific overall survival (P = 0.083). Additionally lower ALCAM immunoreactivity in nodal metastatic foci was significantly statistically correlated with deeper melanoma invasion in the primary tumor according to Clark scale (P = 0.032).

A review of world literature (PubMed; 1970–2014; key words: ALCAM, malignant melanoma) identified only 2 reports that were concerned with analysis of ALCAM expression in tissue material obtained from cutaneous melanoma patients using immunohistochemistry [9, 19].

Van Kempen et al. [9] performed a thorough immunohistochemical analysis of ALCAM expression in 38 benign melanocytic lesions, 55 primary melanomas and 28 metastases (11 originating from the skin, 17 as nodal metastatic foci). It was demonstrated that in the majority of benign lesions (34/38) no ALCAM expression was observed in melanocytes. Interestingly, no ALCAM immunoreactivity was observed in any of the early stage melanomas (Clark I and II). As regards Breslow thickness it was shown that over 70 % of over 1.5 mm thick melanomas had increased ALCAM expression in cancer cells [9]. These results are similar to ours in that increased ALCAM expression in melanoma cells in the primary tumor (defined as higher IRS and a high percentage of positive cells) was strongly correlated with deeper thickness in Breslow scale and higher level in Clark scale. Analysis of ALCAM immunoreactivity in cancer cells from metastases brought interesting results that were similar to ours because van Kempen et al. [9] identified ALCAM expression in 42 % (7/17) of the analyzed nodal metastatic foci while in our study we observed high expression (defined as IRS ≥8) in 5 out of 16 nodal metastatic foci (32 %). Van Kempen et al. [9], due to a different nature of the study (the article was only to present ALCAM expression pattern in benign and malignant melanocytic lesions), did not discuss any correlations with clinicopathological data or their bearing on prognosis.

The study by Klein et al. [19] is the second report on immunohistochemical analysis of ALCAM in melanoma. It evaluates ALCAM reactivity in 71 benign melanocytic lesions, 71 melanomas and 84 metastases. The authors did not specify the type of metastatic foci they examined (nodal or in parenchymal organs). Klein et al. [19] confirmed ALCAM expression in 11/71 of benign lesions, 37/70 of melanomas and 58/84 of metastases. At the same time they observed that in melanomas and metastatic foci ALCAM immunoreactivity was most intense. Klein et al. [19] did not examine correlations with any clinicopathological parameters, nor did they evaluate the effect of ALCAM immunoexpression on long-term survival of cutaneous melanoma patients. The article focuses only on the description of ALCAM expression in a relatively wide spectrum of melanocytic lesions.

Most reports say that homophilic ALCAM-ALCAM interactions are more important in cytophysiology than heterophilic CD6-ALCAM interactions. Interestingly, the study by Swart et al. [20, 21] suggested, based on the current knowledge of intercellular connections, that homophilic ALCAM-ALCAM interactions from a theoretical point of view are generally not necessary for the maintenance of cell adhesion if the proteins of cadherin family and other adhesive glycoproteins work efficiently. What is more, as shown by numerous reports, it is the loss of expression of adhesion molecules that is strongly correlated with cancer progression and formation of nodal and distant metastases. Paradoxically, an opposite situation occurs in the case of ALCAM adhesion protein, where it is the upregulation and increase of homophilic interactions is strongly correlated with melanoma progression (ALCAM paradox). It was showed in in vitro models [12, 14, 15], and our results demonstrating that the upregulation of ALCAM in primary tumor and decreased expression of ALCAM in melanoma cells from nodal metastatic foci as the bad diagnostic indicators is the first report of this kind and is based on the analysis of well-documented clinical material and confirm experimental considerations conducted during studies in cell lines and using laboratory animals [12, 15].

In our opinion the different prognostic significance of ALCAM expression in primary tumor and lymph node metastasis should not be viewed as ALCAM paradox. The involvement of ALCAM in melanoma progression should be seen as composed of two stages [20, 21]. The first stage involves ALCAM overexpression during invasion of the dermis layers (depth of infiltration) by nests of cancer cells (which may be formed as a result of homophilic ALCAM-ALCAM interactions) (local growth stage), while the second stage involves decreased ALCAM expression which facilitates invasion of single cells and formation of metastatic foci in regional lymph nodes, which greatly worsens prognosis. In our study in cancer cells from the primary tumor we observed ALCAM expression in membranous-cytoplasmic and cytoplasmic location, dominantly in cell nests forming specific cytoaggregates, while in cancer cells from nodal metastatic foci the only pattern of intracellular distribution was cytoplasmic expression, which is in line with the considerations of the structural-functional ALCAM analysis (two-stage model of ALCAM expression). It must be stressed that it is a hypothesis that needs to be supplemented and validated empirically with a much larger study group and animal models of metastasis, however, it is a voice in the discussion and confirms the hypotheses concerning the role of ALCAM in melanoma progression.

In spite of large scale multicenter research on cytobiochemistry and oncobiology of ALCAM, numerous questions remain that if answered could help to develop new, promising treatment options and implement a more personalized treatment of melanoma patients.

Our study is the first one to evaluate the effect of increased ALCAM expression on long-term survival in melanoma patients. High ALCAM expression in melanoma cells of the primary tumor can be used as a marker of negative outcome and may indicate a more invasive phenotype of cancer cells, which would require a more intensive therapeutic strategy. Low expression of ALCAM in regional lymph node metastases is a feature associated with unfavorable prognosis in patients with cutaneous melanoma.