Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.     

Structure, Expression and Chromosome Mapping of MLZE, a Novel Gene Which Is Preferentially Expressed in Metastatic Melanoma Cells

We isolated a novel gene, termed MLZE , from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE ) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that h MLZE was expressed primarily in trachea and spleen. We mapped the h MLZE gene (by fluorescence in situ hybridization) to 8q24.1-2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6/11=55%) than in Clark levels I and II melanomas (2/15=13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.     

Co-expression of Thymidine Phosphorylase and Heme Oxygenase-1 in Macrophages in Human Malignant Vertical Growth Melanomas

Expression of thymidine phosphorylase (TP) is often associated with tumor angiogenesis and/or prognosis in patients. Further, infiltration of macrophages is closely correlated with the depth of tumor and angiogenesis in melanomas. In this study, we examined the expression of TP and an activated macrophage-specific enzyme, heme oxygenase-1 (HO-1), involved in malignancy in 22 cases with melanomas. TP was strongly expressed not only in CD68-positive macrophages in and around tumors, but also in S100 protein-positive melanoma cells, fibroblasts and keratinocytes. By contrast, HO-1 was specifically expressed in macrophages, but only slightly in melanoma cells and other cell types in the stroma of melanomas. We thus observed apparent co-expression of TP and HO-1 in macrophages infiltrating in the late stage of malignant melanomas. There appeared increasing numbers of TP-positive cells in Clark level IV and V melanoma compared with Clark level I ( in situ ) melanoma, and there was also a close correlation between numbers of TP-positive cells and HO-1-positive cells. Both TP- and HO-1-positive macrophages could be observed in the stroma in and around tumors in vertical growth melanomas.     

Co-expression of thymidine phosphorylase and heme oxygenase-1 in macrophages in human malignant vertical growth melanomas.

Expression of thymidine phosphorylase (TP) is often associated with tumor angiogenesis and / or prognosis in patients. Further, infiltration of macrophages is closely correlated with the depth of tumor and angiogenesis in melanomas. In this study, we examined the expression of TP and an activated macrophage-specific enzyme, heme oxygenase-1 (HO-1), involved in malignancy in 22 cases with melanomas. TP was strongly expressed not only in CD68-positive macrophages in and around tumors, but also in S100 protein-positive melanoma cells, fibroblasts and keratinocytes. By contrast, HO-1 was specifically expressed in macrophages, but only slightly in melanoma cells and other cell types in the stroma of melanomas. We thus observed apparent co-expression of TP and HO-1 in macrophages infiltrating in the late stage of malignant melanomas. There appeared increasing numbers of TP-positive cells in Clark level IV and V melanoma compared with Clark level I (in situ) melanoma, and there was also a close correlation between numbers of TP-positive cells and HO-1-positive cells. Both TP- and HO-1-positive macrophages could be observed in the stroma in and around tumors in vertical growth melanomas.     

A new Mr 55,000 surface protein implicated in melanoma progression: association with a metastatic phenotype

Emergence of the invasive phenotype is a key event in the progression of human melanoma from benign proliferative lesions to malignant lesions. Recently we successfully selected in vivo from a poorly metastatic M4Beu. human melanoma cell line two variants (7GP and T1P26) that generate a higher frequency of spontaneous metastases to the lungs into immune-suppressed neonatal rats. Both cell lines showed no significant differences in the integrin profile of the subunits analyzed except for beta3, which was reduced to a background level in metastatic variants. To investigate how these variant sublines of human melanomas manage to sustain growth in the absence of alpha(v)beta3, a subtractive immunization approach was used to elicit host antibody response against cell surface proteins expressed on metastatic variants. In this study, a new monoclonal antibody (MoAb), LY1, that is highly specific for the 7GP and T1P26 variants, was isolated. LY1 identifies a membrane protein of Mr 55,000 on melanoma variants with epitopes that were resistant to sugar-cleaving enzymes. Immunostaining cells from variants by LY1 showed that staining is distributed to the cell periphery with high labeling intensity at the cell-to-cell contact points. This MoAb significantly inhibited invasion of metastatic variants through a reconstituted basement membrane (Matrigel) in vitro. Moreover, tumor growth of melanoma variants was dramatically affected in vivo with this MoAb. In vitro studies indicate that the LY1 MoAb does not inhibit chemotactic migration of the metastatic variants, the adhesion of tumor cells to vitronectin, collagen IV, fibronectin, and laminin, or cell proliferation. Expression of this antigen is high in human striated muscle, heart, spleen, brain, and lung and absent in kidney, liver, and pancreas. Using 59 fixed, paraffin-embedded archival tissues of human melanomas and nevi, LY1-reactive cells were not observed in melanocytes, nevi, or radial growth phase primary melanomas. In sharp contrast, LY1 selectively stained melanocytes derived from the vertical growth phase of many primary melanomas and metastatic melanomas. These results provide evidence that the Mr 55,000 protein expressed by selected variants with increased metastatic properties in vivo plays a functionally important role in determining metastasis. This molecule may represent a new metastatic risk marker in human melanoma and may be of biological importance in the identification of fatal metastatic subpopulations that have acquired competence for metastasis production.     

Telomerase activity and expression of apoptosis and anti-apoptosis regulators in the progression pathway of human melanoma

The stages defining the progression pathway of human melanoma are atypical nevi, the precursor lesions and risk markers of melanoma, melanoma in situ and melanoma in the radical growth phase (RGP), which represent the early stages of melanoma development, and primary melanoma in the vertical growth phase (VGP) and melanoma in the metastatic growth phase (MGP), which are the advanced stages of the disease. Unlike cells obtained from VGP and MGP melanomas, which can be established as cell lines, cells derived from atypical nevi, melanoma in situ, and RGP melanoma cannot be propagated in vitro. Thus, information regarding molecular markers that may be differentially expressed in the early versus the advanced stages of this disease can only be obtained from the analysis of specimens. Since activation of telomerase and deregulation of apoptosis contribute to the pathogenesis of a significant number of human malignancies, we conducted a study, using nevus and melanoma specimens, to determine at what stage in the progression pathway of melanoma, telomerase activity can first be detected, and whether concordant with telomerase activation, one might observe a stage-specific switch from expression of promoters to inhibitors of apoptosis. The findings described here, demonstrate telomerase activity in some but not all MGP melanomas and not in any of the preceding pathological stages, and no apparent imbalance between pro- and anti-apoptotic markers in telomerase-positive MGP melanomas compared to telomerase-negative nevi and telomerase-negative VGP and MGP melanomas.     

Linear discriminant analysis of dermoscopic parameters for the differentiation of early melanomas from Clark naevi

As a first step to develop a screening system for pigmented skin lesions, we performed digital discriminant analyses between early melanomas and Clark naevi. A total of 59 cases of melanoma, including 23 melanoma in situ and 36 thin invasive melanomas (Breslow thickness < or =0.75 mm), and 188 clinically equivocal, histopathologically diagnosed Clark naevi were used in our study. After calculating 62 mathematical variables related to the colour, texture, asymmetry and circularity based on the dermoscopic findings of the pigmented skin lesions, we performed multivariate stepwise discriminant analysis using these variables to differentiate melanomas from naevi. The sensitivities and specificities of our model were 94.4 and 98.4%, respectively, for discriminating between melanomas (Breslow thickness < or =0.75 mm) and Clark naevi, and 73.9 and 85.6%, respectively, for discriminating between melanoma in situ and Clark naevi. Our algorithm accurately discriminated invasive melanomas from Clark naevi, but not melanomas in situ from Clark naevi.     

Expression of CD44s and CD44 splice variants in human melanoma.

The ability of tumor cells to adhere and detach from extracellular matrix and endothelial cells, is a crucial step in the metastatic process and may alter the clinical prognosis of some human tumors such as melanomas. CD44, the major cell surface receptor for hyaluronate, has been implicated in cell adhesion and in tumor progression. We studied the expression of standard CD44 molecule (CD44s) and its variants v3 and v6 in 57 human primary melanoma biopsies, without previous treatment. We analyzed the association between CD44 expression and the principal clinicopathological features, including survival. Fifty-six of 57 tumors expressed CD44s, associated to the cytoplasmic membrane. No expression of CD44v3 or CD44v6 was detected. No association between CD44s expression and prognostic factors such as tumor thickness, growth type, stage or anatomic site of the lesion was found. However, a positive correlation between CD44s expression and Clark level (Spearman, p<0.001) was found. While only 33.3% of melanomas Clark I + II showed high expression of CD44s (more than 50% of positive cells), 82.6% of melanomas Clark IV + V did so. Kaplan-Meier analysis revelead that patients whose melanomas had high expression of CD44s showed a reduced relapse free survival (RFS) rate, though without statistical significance. No difference between the level of CD44 expression and overall survival (OS) was found. We conclude that melanomas only expressed CD44s, and that its level was associated with Clark's stage. CD44s seems not to be useful as a tumor marker, because it does not predict either RFS or OS.     

RaLP, a new member of the Src homology and collagen family, regulates cell migration and tumor growth of metastatic melanomas

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.     

Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF(xxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.     

Screening of N-ras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression.

PURPOSE: Mutations in the ras genes often occur during tumorigenesis. In malignant melanoma, the most common ras alterations are N-ras codon 61 mutations. This study was aimed to measure the frequency of such mutations in a large series of paired primary and metastatic melanomas to determine their role in melanoma initiation and progression. EXPERIMENTAL DESIGN: Seventy-four primary melanomas and 88 metastases originating from 54 of the primary tumors were screened for N-ras codon 61 mutations using single-strand conformation polymorphism and nucleotide sequence analyses. RESULTS: Twenty-one of the 74 primary tumors (28%) had activating N-ras codon 61 mutations. From 20 of the mutated primary tumors, a total of 34 metastases were analyzed, and all but one showed the same mutation as the primary tumor from which they originated. The remaining 53 primary tumors and corresponding metastases (n = 54) were wild-type for N-ras codon 61. Analysis of the different growth phases of the mutated primary tumors showed that the mutations were already present in the radial growth phase. Mutations were also detected in tumor-associated nevi. N-ras codon 61 mutations were associated with a higher Clark level of invasion (P = 0.012) and a lower age at diagnosis (P = 0.042) but did not affect survival (P = 0.671). CONCLUSIONS: This study shows that N-ras codon 61 mutations occur early in primary melanomas rather than in the metastatic stage and that once the mutations have occurred, they persist throughout tumor progression. This suggests that activated N-ras may be an attractive target for therapy in the subset of melanoma patients carrying such mutations.     

The adhesion molecule L1 (CD171) promotes melanoma progression

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.     

Expression of the neuroglandular antigen and analogues in melanoma. CD9 expression appears inversely related to metastatic potential of melanoma

Immunohistological methods were used to examine the relation between the metastatic potential of melanoma and expression of the neuroglandular antigen (CD63) and other members of this family of molecules, CD53, CD37, CD9 and the target of an anti-proliferative antibody (TAPA-1), as well as MHC-class-I and -II antigens. The criteria used to establish metastatic potential were their relation to thickness of the primary melanoma, and differences in expression between vertical and radial growth phases of primary melanoma and between primary and metastatic melanoma. Studies on basal-cell carcinoma (BCC) and squamous-cell carcinoma (SCC) were also included as controls for malignant skin cancers with low metastatic potential. Expression of CD9 and MHC-class-1 antigen was found to be inversely related to thickness of the primary tumor, and CD9 was expressed predominantly on primary rather than on metastatic tumors. CD9 expression correlated with MHC-class-1 expression on melanoma, and both were expressed on BCCs and SCCs having low metastatic potential, but not on compound nevi. CD63 and TAPA-1 were expressed on nevi but not on SCC and BCC. Leu 13 is a molecule associated with TAPA-1 in lymphomas, and was found to be expressed in sections from 5 out of 34 primary and 5 out of 21 metastatic melanoma. CD53 and CD37 were not detected on melanoma. Our results indicate that several members of the neuroglandular antigen are expressed in melanoma and that low expression of CD9 on primary melanomas might have prognostic significance with respect to the potential for metastasis.     

Co-Expression of Putative Cancer Stem Cell Markers,CD133 and Nestin,in Skin Tumors

Background: Cancer stem cells (CSC) are populations of cells responsible for tumor initiation, progression andtherapeutic resistance in many cancers. In the present study, we aimed to investigate the expression pattern andclinical significance of two CSC markers, CD133 and Nestin, in a series of skin tumors. Materials and Methods:One hundred and thirteen paraffin blocks from skin cancers including 16 (14%) cases of melanoma, 37 (33%)of squamous cell cancer (SCC) and 60 (53%) of basal cell cancer (BCC) were collected and assembled in a tissuemicroarray (TMA). The samples were immunohistochemically examined for the expression of CD133 and Nestin.Expression of these markers was also correlated with clinicopathological parameters. Results: A significantdifference was observed in the expression of CD133 and Nestin in melanomas, SCC and BCC (p value=0.001).Furthermore, the level of expression was significantly higher in the melanomas compared to the SCC and BCCtumors. Expression of CD133 in the melanoma was significantly associated with increased tumor invasiveness (pvalue=0.05), a higher rate of metastasis (p value=0.04) and the presence of ulceration (p value=0.02). Increasedexpression of Nestin was observed in metastatic melanoma (p value=0.04), while no statistically significantcorrelation was found with other clinicopathological parameters including Breslow thickness, Clark level andulceration. Conclusions: Elevated expression levels of CD133 and Nestin in the melanomas are associated withadvanced disease, with more aggressive and metastatic skin tumors. Therefore, these markers could be potentialtherapeutic targets for malignant tumors of the skin.     

BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma

BRAF oncogenic mutations have been identified in significant numbers of melanocytic lesions. To correlate BRAF mutation and melanoma progression, we screened BRAF mutations in 65 melanocytic lesions, including nevi, radial growth phase (RGP), vertical growth phase (VGP) melanomas, and melanoma metastases, as well as 25 melanoma cell lines. PCR and direct sequencing were used to analyze DNA samples extracted from laser capture microdissected tissues. A similar high frequency (62-72%) of BRAF oncogenic mutations was identified in melanocytic nevi, VGP, metastatic melanomas, and melanoma cell lines [H. Davies et al., Nature (Lond.), 417: 949-954, 2002; P. M. Pollock et al., Nat. Genet., 33: 19-20, 2002; and M. S. Brose et al., Cancer Res., 62: 6997-7000, 2002]. In striking contrast, we found BRAF lesions in only 10% of the earliest stage or RGP melanomas. These findings imply that BRAF mutations cannot be involved in the initiation of the great majority of melanomas but instead reflect a progression event with important prognostic implications in the transition from the great majority of RGP melanomas to VGP and/or metastatic melanoma.     

Enhanced expression of vascular endothelial growth factor in metastatic melanoma.

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.     

Differential response of primary and metastatic melanomas to neutrophils attracted by IL-8

IL-8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL-8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL-8 with IL-8 cDNA using a replication-deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque-forming units of IL-8 per cell. However, at high IL-8 transduction levels (100 ng/ml/10(5) cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL-8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL-8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL-8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL-8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.     

Differences of ploidy status in progression of head and neck melanomas

Thirty-three common naevi, 26 dysplastic naevi, 58 primary melanomas of facial skin, 24 oral melanomas, 32 lymph nodes and 12 distant melanoma metastases were stained using Feulgen method to evaluate the ploidy status by image analysis GAS-200 system. Eight percent of common naevi, 22% of dysplastic naevi, 43% of facial melanomas, 65% of oral melanomas, 40% of lymph nodes with melanoma metastases and 66% of distant melanoma metastases were classified as aneuploid. In facial melanomas the percentage of aneuploid cases increased with Clark level. Survival time of patients was significantly shorter for aneuploid cases as compared to euploid ones (p <0.01).     

Apolipoprotein D expression in cutaneous malignant melanoma

BACKGROUND AND OBJECTIVES: Apolipoprotein D (Apo D) is a protein component of the human plasma lipid transport system, and an androgen-regulated protein in both breast and prostate cancer cell lines. Our goal was to evaluate the expression of Apo D in malignant cutaneous melanomas, as well as to assess its possible relationship to clinical and pathological parameters. METHODS: Apo D expression was analyzed in 32 paraffin-embedded tissues from patients with invasive cutaneous malignant melanomas, in 8 samples from in situ melanoma, and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi, and 2 dysplastic melanocytic nevi), using immunohistochemical techniques. RESULTS: The benign lesions were consistently negative for Apo D, whereas 3 of the 8 "in situ" melanomas (37.5%) and 12 of the 32 invasive melanomas (37.5%) showed positive immunostaining for Apo D. The percentage of Apo D-positive tumors was significantly higher in nodular than in superficial spreading melanomas (P = 0.011) and in melanomas with vertical growth phase than in melanomas with radial growth phase (P = 0.02). In addition, the percentage of Apo D-positive tumors was positively and significantly correlated with Clark's level of invasion (P = 0.046). CONCLUSIONS: Apo D may be a new prognostic factor of unfavorable evolution in cutaneous malignant melanoma.     

Identification of higher risk thin melanomas should be based on Breslow depth not Clark level IV

BACKGROUND: There is good prognostic correlation for the two microstaging systems, Breslow depth and Clark level, commonly used to stage melanomas. Many investigators have reported that Breslow depth is the superior microstaging method. Although Clark level has been dropped from most of the proposed American Joint Committee on Cancer (AJCC) melanoma staging system, the AJCC system still includes Clark Level IV as a criterion for upstaging thin melanomas. The authors sought to determine whether this is appropriate, based on melanoma patient data in the Duke Comprehensive Cancer Center database. METHODS: Of the 8833 patients registered between January 1, 1970 and December 31, 1995, complete data on Breslow depth and Clark level was available for 4560 patients who were without nodal or metastatic disease at presentation. Ten-year survival was measured from the date of excision of the primary tumor until death from melanoma and analyzed using Kaplan-Meier and Cox proportional hazard methodologies. RESULTS: When analyzed separately, both increased Breslow thickness and Clark level correlated with shorter survival times. During subgroup analysis, Breslow thickness remained a significant prognostic indicator of survival at Clark Levels III and IV. Conversely, at narrow levels of Breslow thickness (i.e., 0-0.75 mm, > 0.75 -1.0 mm, > 1.0-1.5 mm) survival times were indistinguishable between Clark Levels III and IV. For the broader Breslow thickness interval of 0-1.0 mm, a barely significant difference between Clark Levels III and IV could be obtained. However, for this thickness range, even greater differences in survival could be obtained by merely comparing Breslow subgroups (i.e., < or = 0.8 mm vs. > 0.8-1.0 mm, < or = 0.9 mm vs. > 0.9-1.0 mm). CONCLUSION: The authors' data suggested that, after controlling for Breslow depth, Clark level was not a good prognostic indicator for survival. If the AJCC's objective is to design a classification system that will reliably predict the higher risk melanomas, then the system should be based on tumor thickness, which is clearly a better prognostic indicator, and should not be modified because of Clark level.