New prognostic factors of cutaneous melanoma: a review of the literature (2002)

Clark et al.1 described the concept of tumor progression in malignant melanoma (MM). Subsequent studies have shown that metastasis is exclusively associated with a specific pattern of tumor growth within the dermis, which Clark et al.2 termed the vertical growth phase. Most melanomas evolve through an initial stage termed the radial growth phase (in situ or ‘microinvasive’ melanoma) in which the probability of cure approaches 100%.3 Despite recent improvements in the earlier recognition of cutaneous melanoma, by the time of diagnosis, approximately 70% of melanomas have evolved into vertical growth phase or ‘tumorigenic melanoma’. Thus, assessment of prognostic parameters is of value only in vertical growth phase lesions. However, this concept of radial and vertical growth phase is not universally accepted.4 Several clinical and pathologic factors that influence the progression and behavior of melanoma have been identified. These factors include tumor thickness, ulceration, anatomic site of the primary tumor, age, gender, level of invasion, mitotic rate, tumor-infiltrating lymphocytes (TILs), histologic regression, microscopic satellites, histologic subtype, vascular invasion, and tumor cell type.3,5–14 The first three are thought to be dominant factors that predict survival in patients with stage I and II primary melanomas.3 To date, most studies have shown that the single attribute best predictive of clinical outcome for a given patient is the depth of tumor invasion (Breslow thickness), which is the most powerful independent prognostic factor.7–14 It has become the gold standard for stratifying patients according to risk of metastatic disease. However, these studies also point out that in some cases, tumor thickness has not been as accurate an indicator of biologic behavior of MM as expected. Some patients with thick melanoma do surprisingly well and survive far longer than expected, whereas others with thin melanoma have a more aggressive course of disease.14 Distinguishing patients who truly have minimal risk of metastasis from patients at greater risk of developing progressive disease is crucial for treatment planning. Therefore, additional prognostic variables are needed with improved specificity and predictive value for melanoma patients. Rapidly expanding knowledge about mechanisms of tumor progression in melanocytic tumors and the advanced molecular biology techniques emerging in the past few years have helped in the search for such markers. So far, a number of biological markers, in addition to traditional histopathology, have been shown to serve as independent prognostic markers for patients with cutaneous melanoma. The markers with prognostic value for melanoma, especially those that can be used on routine formalin-fixed, paraffin-embedded materials by using immunohistochemistry (IHC), or in the diagnostic molecular pathology laboratory, will be reviewed in this article. Therefore, we have subgrouped these prognostic markers into three categories: (i) tissue markers, (ii) serological markers, and (iii) molecular markers. A tremendous number of melanoma markers, which provide insight into the prognosis of melanoma lesions, have been studied on tissue by immunohistochemical methods. Many of these markers can be broadly categorized according to their mechanisms of action. They are reviewed in this article as follows: (i) markers of cellular proliferation, oncogenes and tumor suppressor genes; (ii) extracellular matrix degrading enzymes; (iii) cell adhesion molecules; and (iv) other new markers. These markers are primarily experimental and have not been used on a routine basis. In several malignant neoplasms, proliferative activity is known to be related to the development of metastatic disease and to influence patient survival.15 One of the easiest and most reliable methods of assessing the degree of proliferative activity is the Ki-67 index.16 The Ki-67 antigen is a non-histone protein that can be detected in the nucleus of cells during late G1, S, G2, and M phases, which makes it a more sensitive marker of the proliferation index in tissues than the mitotic index. The prognostic significance of the Ki-67 index detected by IHC has been shown in several tumor types.16 In melanoma, most studies have shown the Ki-67 index as a useful prognostic marker for melanoma progression.17–21 An elevated Ki-67 expression was significantly and independently associated with the risk of metastatic disease or decreased overall survival in these studies. However, this association seems to occur only in thick primary MM (at least > 1.5 mm)18–22 but not in thin lesions.19,22 One study further demonstrated that for MM thicker than 4.00 mm, a new prognostic parameter, ‘proliferation-based prognostic index’ (tumor thickness × Ki-67 index/100), conveys an independent prognostic significance.22 On the contrary, some studies failed to demonstrate the prognostic value of Ki-67 in primary cutaneous MM.23,24 In addition to its prognostic value, the Ki-67 index has been used as an aid to the diagnosis of malignant of melanoma.25,26 The Ki-67 index exhibited a significant difference between benign melanocytic nevi, Spitz nevi, and melanoma. Furthermore, one study showed a significant difference in the Ki-67 index of vertical growth phase MM compared with radial growth phase melanomas that had a similar proliferative index to those of melanocytic nevi.26 These studies suggest that determination of the proliferative activity in melanocytic lesions may usefully complement conventional histology to recognize melanoma, but not distinguish it from benign melanocytic lesions. p53 is a tumor suppressor gene. The overexpression of p53 detected by immunostaining for p53 protein has been reported as a prognostic indicator for many tumors. Expression of p53 has been shown to correlate with Ki-67 overexpression and with disease progression in melanoma.20,26–28 In melanoma, the abnormalities of p53 expression seem to be demonstrated only in primary, thick (> 1.5 mm), invasive and metastatic melanoma,20,26–28 which is similar to that reported in Ki-67. One study, which compared thick (> 3 mm) primary melanomas with different clinical outcomes, demonstrated that overexpression of p53 was significantly associated with metastasis and death.25 However, reports regarding abnormal expression of p53 protein and p53 mutation in cutaneous MM are greatly contradictory,29–31 with some studies failing to find a link between immunohistochemical detection of p53 protein and prognosis in cutaneous melanoma. Thus, the clinical prognostic value of p53 protein expression remains uncertain. Another tumor suppressor gene, the cyclin-dependent kinase inhibitor-2 (CDKN2A) or p16 gene, which maps to chromosome 9p21, has been shown to be deleted or mutated in a high percentage of melanoma tumor cell lines.32 Mutations have also been frequently reported in the germline of familial melanoma members.32 Additionally, recent studies indicate that p16 alterations such as homozygous deletion of CDKN2A, loss of heterozygosity of 9p21 and mutations occur frequently in sporadic melanomas.33,34 Thus, the role of this gene in melanoma progression has been investigated by immunolabeling the expressed product, p16INK4a, on paraffin-embedded melanoma tissue.21,33–40 p16INK4a functions as a negative regulator of cell cycle progression and as a tumor suppressor by inhibiting the catalytic activity through the G1-S phase cell cycle checkpoint. Immunolabeling of p16INK4a is distributed to both cytoplasm and nuclei of normal and transformed melanocytes21,35,36 but is more accurately represented by nuclear staining.21 Not surprisingly, uniform immunolabeling of p16INK4a was seen in benign melanocytic nevi33,36–38 and dysplastic nevi.38 Loss of p16 protein expression by IHC was seen in primary cutaneous melanoma and has been correlated with advanced stages of melanoma progression.33,36–38 Initial studies showed that p16INK4a protein expression reduction in melanoma cells occurred only in the invasive and metastatic stages of malignant melanoma, by 38–62%,33,36,37 and was associated with significant risk of relapse in a multivariate analysis39 and with a trend to worse prognosis in a separate study.40 The data are conflicting regarding the p16 gene as an initiating event in melanoma development. Existing observations indicate that in melanoma in situ, either there are no alterations in p16 protein expression by immunolabeling or there is reduced expression of either the protein37,38 or mRNA.38 Once invasive or metastatic disease has supervened, several studies have confirmed progressive loss of p16 with increasing stage of melanoma.21,37,38 Findings in one recent study indicated that loss of nuclear p16 protein expression identified an aggressive subset of vertical growth melanoma by showing that absent or minimal nuclear staining significantly predicted decreased patient survival, with 37 and 67% estimated 10-year survival rates, respectively.21 In this study, reduced p16 expression was an independent prognosis factor, and associated with increased tumor cell proliferation (Ki-67 index). It must be noted, however, that p16INK4a protein expression was normal in some fully evolved melanomas, raising the question of whether or not the decreased p16INK4a expression in invasive melanomas will have clinical prognostic value.21 Other protein family members of cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1 and p27KIP1, have also been studied for their expression in melanoma by immunohistochemistry. Overall, p21WAF1 and p27KIP1 levels were found to be low or undetectable in the majority of benign melanocytic lesions,36 with higher expression seen in malignant melanomas.36,41 Although an association of increased expression of p21 with Clark level and Breslow thickness was found, no correlation to clinical stage or patient prognosis was demonstrated with either p21 or p27.36 Moreover, the regulatory subunits of cyclin-dependent kinase 4 (CDK4), especially the d-type cyclins (D1, D2 and D3), have been investigated for their potential value as prognostic indicators for melanoma. Limited studies revealed elevated cyclin D1 and D3 protein expression in melanomas and metastatic melanomas compared with low or absent expression in benign nevi.34,42,43 However, only increased expression of cyclin D3 expression was an indicator of early relapse and decreased overall survival for patients with superficial spreading melanoma, but not nodular melanoma.42 Thus, the prognostic value of cyclins needs to be further investigated. Over the last decade it has been appreciated that oncogenes that are involved in the transformation of normal cells into cancer cells play a pivotal role in determining the biology of malignant disease and thus clinical outcome. One particular oncogene, c-myc, is known to be deregulated in a number of human malignancies. The c-myc oncogene produces a nuclear oncoprotein, which appears to act by increasing the rate of cellular proliferation and the metastatic potential of tumor cells.44 Recent works have shown c-myc expression to be a marker of poor prognosis in head and neck cancer and cervical carcinoma.45,46 In addition, the measurement of c-myc oncoprotein by flow cytometry has recently been noted to provide prognostic stratification in primary and metastatic melanoma,47 subungual melanoma,48 acral lentiginous melanoma,49 and regional metastatic disease.50 However, the results measured by IHC were controversial.51,52 Expression of c-myc oncoprotein was higher in metastatic than in primary melanoma, but it predicted clinically poor outcome in both primary and metastatic melanomas.47,50 It was also associated with tumor thickness, ulceration and reduced disease-free survival in primary melanomas.47 c-myc, particularly, was able to discriminate prognosis in high-risk melanoma patients with thick lesions (> 3 mm).47 Most importantly, overexpression of c-myc oncoprotein was a powerful prognostic marker for patients with regional nodal metastatic melanoma,50 in which the clinicopathological parameters were no longer of prognostic significance. Conversely, Boni et al.52 failed to find significant differences in c-myc reactivity between metastasizing and non-metastasizing cutaneous primary melanoma. They also found no correlation between c-myc expression and tumor thickness. It is generally accepted that proteases like plasminogen activators (PAs), primarily by degradation of the cancer-surrounding extracellular matrix, are involved in tumor cell invasion and metastasis.53 Urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), serine proteinase inhibitors PAI-1 and PAI-2 and urokinase plasmogen activator receptor (uPAR) are members of the PA proteolytic system. High levels of uPA, PAI-1 expression and uPAR, measured by both IHC and ELISA, were repeatedly reported to be related to shorter disease-free and overall survival, whereas high tPA and PAI-2 levels were related to a favorable course of disease in some malignancies, such as breast carcinoma.54 Observations in experimental systems and on clinical material also pointed to a role for proteases in melanocytic tumor progression.55 uPA, uPAR, PAI-1 and PAI-2 appeared frequently in advanced primary and metastatic melanoma as well as atypical nevi, but not in benign nevi.56,57 Thus, uPA along with its receptor and inhibitors may be employed as markers of progression in melanocytic lesions and may provide a tool for the evaluation of atypia in nevi. In addition to the u-PA system, evidence also implicated the role of the t-PA system in melanoma progression and prognosis.54 Unlike the u-PA system, the extensive tPA expression of melanoma cells measured on paraffin tissue of clinical stage II melanoma with minimal (1.5 mm) thickness was reported to correlate with a favorable outcome.54 The best prognosis was related to lesions with 51–100% tPA tumor positivity, whereas the worst prognosis was related to lesions with intermediate (6–50%) tPA tumor cell expression.54 Moreover, tPA tumor cell positivity was an independent prognostic factor. The complex association between tPA expression and the outcome of disease in melanoma needs to be validated in further series. Another proteolytic enzyme system, matrix metalloproteinase (MMP), is also involved in the degradation of many different components of the extracellular matrix. MMPs and their specific tissue inhibitors (TIMPs) play an important role in cancer cell invasion and metastasis. Studies have shown that melanoma cells and melanoma tissue may express a number of MMP family members (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) as well as their tissue inhibitors (TIMP-1, TIMP-2, and TIMP-3).58 Expression levels of several components of MMPs and TIMPs may increase in cutaneous melanoma progression.59–62 Taken together, these studies demonstrated a significant increase in the expression of MMP-1, MMP-2, MMP-13, MT1-MMP, TIMP-1, TIMP-2 and TIMP-3 in human melanoma during progression from in situ to metastasic disease.60–62 No MMP-2 expression was found in common nevi or atypical nevi.62 These findings suggest that MMPs and their inhibitors are required for melanoma invasion and metastasis. An inverse relationship between expression of MMP-9 and melanoma progression was found,63 with MMP-9 expression being detected only in the radial growth phase and absent in the vertical growth phase of primary melanoma and melanoma metastases. These studies, in general, demonstrate that MMPs and TIMPs play an important role in melanoma progression. Additionally, expression of MMP-2 has been found to correlate with unfavorable prognoses in male patients,64 and MMP-2 positivity in primary and subcutaneous melanoma lesions has been correlated with hematogenous metastasis.60 However, comprehensive studies of the MMP profile in large defined series of patients are needed to further evaluate the prognostic significance of individual MMPs. In addition to extracellular matrix-degrading activity, invasive melanoma cells must develop the ability to migrate through the dermis for tumor progression to occur. This ability requires a variety of adhesion molecules, motility factors, and homing receptors. Recently, several classes of cell adhesion molecules, including the integrins, cadherin, members of the immunoglobulin superfamily and CD44, have been shown to be key components in the metastatic cascade and to be prognostically valuable in cutaneous melanoma. Integrins are heterodimeric transmembrane glycoproteins composed of non-covalently bound α and β subunits that together form a functional cell adhesion receptor, and are involved in cell–cell and cell–extracellular matrix interactions. Because of their diverse functions, they have been implicated in tumor progression in many malignancies and also have been found to play a role in human melanoma progression and metastasis.65 In studies of human melanocytic lesions, both β1 and β3 integrin expressions have been linked to melanoma progression and metastasis.31,64–70 The collective data from these investigations suggest that the expression of β3 integrin, mainly αvβ3 integrin, as well as β1 integrin (mainly α2β1 and α3β1) progressively increases in the progression from benign nevi to malignant melanoma to metastatic melanoma. These studies emphasized that the expression of these integrins was absent or low in most benign nevi, except for Spitz nevi that strongly expressed β3 integrin.66 In primary melanoma, expression of β3 integrin was absent or low in the non-tumorigenic radial growth phase.70 In contrast, the expression of β1 and β3 integrins was higher in the vertical growth phase and in most metastatic melanomas.31,64–71 The expression of αvβ3 increased with tumor thickness and was associated with disease recurrence.70,71 However, studies of β integrin expression on clinical prognosis in melanoma are limited,31,68 and the diagnostic utility may be limited because of the strong expression of some integrins in Spitz nevi.66 In primary melanoma, high expression of both β1 and β3 integrins was associated with poor prognosis, with a worse prognosis in β3-positive patients who were more likely to die of disease31 and had significantly decreased disease-free survival.71 Moreover, individual integrin expression in primary melanomas may indicate different sites of metastasis.31β3 integrin was associated with subsequent lung metastasis,31 and β1 integrin with lymph node involvement.31,64 In contrast to primary melanoma, β1 integrin expression plays a different role for metastatic melanoma in predicting prognosis.68 Patients with β1-positive metastatic tumors had significantly longer disease-free survival and overall survival.68 In summary, αvβ3 integrin plays an important role during the transition from radial to vertical growth phase of primary melanoma; β1 and β3 are markers of poor prognosis in patients with primary melanoma; and the presence of β1 integrin in metastatic melanoma is associated with better survival. CD44 is a structurally variable and multifunctional cell surface glycoprotein found on most cell surfaces and is involved in a cell adhesion-dependent process. It forms the major cellular receptor for its ligand-hyaluronic acid (HA) of the extracellular matrix.72 The protein exists in a standard form (CD44H/CD44s) and at least 10 isoforms (v1–v10). Previous experimental data suggest that CD44 and HA enhance growth and metastatic capacity of melanoma cells,73 but the clinical significance has not been confirmed in other reports.74–78 CD44 was uniformly expressed in very high levels in benign melanocytic nevi, dysplastic nevi and melanoma in situ.74,75 In contrast, although virtually all melanomas expressed CD44, an inverse correlation in the levels of CD44s expression with increasing tumor thickness and invasiveness was found in primary melanoma74–77 and in metastatic melanomas,74,75 but no definite correlation between isoform CD44v expression and primary melanoma progression was found.74,75 Furthermore, metastases exhibited a significantly weaker or absent staining for CD44s compared with primary melanoma, although diminished expression of CD44s was observed in both.74,75 However, metastases could not be distinguished from primary lesions based on their CD44 immunostaining pattern.76 These data suggest a possible role for the downregulation of CD44s in modulating the biological behavior of malignant melanoma. Two studies addressed the prognostic value of CD44 expression in cutaneous melanoma. One study showed that patients having tumors with a high level of CD44 expression (> 2+ staining) experienced a significantly reduced 5-year survival rate compared with those having low levels of CD44 expression.78 In this study, the high level of CD44H/S predicted metastasis in patients with tumor thickness less than 0.7 mm. Conversely, another study77 demonstrated that reduced cell surface CD44 (< 90% positive tumor cells) and HA (< 70% positive tumor cells) levels within primary melanomas correlated with subsequent disease progression and short survival. In addition, this study showed reduced levels of CD44s acted as an independent unfavorable prognostic factor in clinical stage I melanoma. The conflicting results warrant further studies to confirm the usefulness of CD44s as a prognostic factor. Likewise, other cell adhesion molecules have also been implicated in tumor progression in cutaneous melanoma. The melanoma cell adhesion molecule (Mel-CAM), also known as MUC18, is a member of the immunoglobulin supergene family. Experimental studies suggest that MUC18 expression confers metastatic potential and increased tumorigenicity to human melanoma cells.79 It was shown that MUC18 was expressed in all primary and metastatic melanomas studied,80,81 and the expression of MUC18 in melanoma correlated with tumor thickness and with metastatic potential.82 However, MUC18 is not specific for melanoma. It has been reported to be expressed in many malignant tumors80 and, to date, no study has been done to correlate the expression of MUC18 and prognosis of melanoma. Intercellular adhesion molecule-1 (ICAM-1) is a cell adhesion molecule that is found on many cell types, including tumor cells. Expression of ICAM-1 has been shown to be associated with melanoma progression. ICAM-1 expression is stronger and more frequent in malignant than in benign melanocytic lesions and is significantly associated with tumor thickness in primary melanoma.71,81,83 Within malignant lesions, the expression of ICAM-1 was significantly higher in metastatic lesions than in primary melanomas.84 Moreover, elevated ICAM-1 expression has been shown to be a significant marker of poor prognosis in stage I tumors and metastatic melanoma.71,80,83 Cadherin constitutes a large family of Ca2+-dependent cell adhesion molecules mediating predominantly homophilic interactions, which include E (epithelial), P (placental), and N (neural) cadherins. The most intriguing observations regarding cadherin expression and melanoma progression have come from studies of the expression of E-cadherin and to be E-cadherin is used by to with and loss of expression of E-cadherin has been linked to tumor progression in melanoma in studies have shown that E-cadherin is expressed in and cells and in metastatic melanoma However, studies of E-cadherin expression in melanocytic lesions revealed a complex the expression of E-cadherin was increased with invasive tumor growth in advanced primary and metastatic which was to the in of E-cadherin expression was observed in early primary melanomas and in radial growth whereas some expression could be observed in advanced vertical growth phase melanoma and metastatic No study has on its prognostic are the composed of of the in cell of the of the It has been that this is for cellular and a that of to the of confers to and the majority of human malignancies activity whereas most normal tissues do The evaluation of has been for diagnostic and in human In the has been shown to be low in the and in the of in and in some In cutaneous levels have been found to be high in cell and low in cell has been detected in melanoma cell and melanoma tissues in a number of by of a Taken together, these results indicate that increases from benign melanocytic nevi to atypical nevi and further to malignant melanoma and metastatic melanoma, and may play a role in tumor and Although was not detected in all its expression was demonstrated in most primary and metastatic and it showed the in Furthermore, increased significantly with melanoma level and showed an association between high and early metastatic in The significant increase of with tumor progression its potential prognostic role in cutaneous melanoma. factor the human of the gene product, is a nuclear protein for the development and of was reported to be a specific marker of melanocytic the of melanomas, all primary and metastatic cutaneous melanomas for in those that were negative for and However, in another study, only of melanomas for Although the diagnostic value of in primary melanocytic lesions and melanomas is not a recent that be a new prognostic marker in patients with melanoma. The data in this study showed that expression was associated with significantly overall survival and disease-free survival in patients with melanoma. has shown that the higher the number of melanoma cells with expression in a given primary tumor, the better the In addition, low or of expression in primary lesions was an indicator of regional node metastasis. Patients with > expression had a significantly of nodal Recently, a new cell surface found on metastatic melanoma was identified. This protein plays a role in the malignant of human The or cell surface protein was studied on tissues of human melanomas and nevi. of the benign melanocytic lesions or radial growth phase In contrast, from the vertical growth phase of many primary melanomas and metastatic Thus, this molecule may a new metastatic risk marker in human melanoma, which needs to be confirmed by additional studies. A variety of molecules or into the either by melanoma cells or as an of the system, have been studied as markers of tumor progression These include adhesion molecules and In recent new markers, and have regarding their clinical as markers to and disease progression in patients with malignant a melanoma protein is a composed of a of α first described in cells of immunohistochemical detection is used in the diagnosis of cells of melanocytic Recently, several studies have revealed the clinical significance of the of the in levels can be by an or a is more frequently used because of its and improved Although results to different level of from to it is that levels are elevated in melanoma patients in a clinical The of patients with increased levels was in stage melanoma and in stage In addition, levels were found to correlate with the of metastatic tumor increased levels have been found in and in and because it has