Prognostic Marker In Melanoma Research Articles

TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome

Background:Clinical outcome of patients with high-risk melanoma cannot be reliably predicted on the basis of classical histopathological examination. Our study aimed to determine in melanoma metastases a gene expression profile associated with patient survival, and to identify and validate marker(s) of poor clinical outcome.Methods:Skin and lymph node metastases from melanoma patients (training population) were used to identify candidate prognostic marker(s) based on DNA microarray analysis. Additional skin metastases (validation population) were used to assess the prognostic value of the first ranked gene by real-time PCR.Results:We performed microarray analysis in the training population and generated a list of 278 probe sets associated with a shorter survival. We used the first ranked gene, tyrosinase-related protein 1 (TYRP1), further measured its expression in the validation population by real-time PCR and found it to be significantly correlated with distant metastasis-free survival (DMFS), overall survival (OS) and Breslow thickness. We also found that it was fairly well conserved in the course of the disease regardless of the delay to metastasis occurrence. Finally, although Tyrp1 protein (immunohistochemistry (IHC)) was only detected in about half of the samples, we showed that its expression also correlated with Breslow thickness.Conclusion:Our data indicate that TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma, and is particularly useful when prognostic pathology parameters at the primary lesion are lacking. Its conserved expression further supports its use as a target for therapy.

Nestin and vimentin intermediate filaments expression in cutaneous melanoma

Nestin, a class VI intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue, is considered as cancer stem cell marker of malignancies of neuroectodermal origin. It may play a role in connecting the components of the cytoskeleton and coordinate changes in cell dynamics. It is well known nestin copolymerizes with class III IF-proteins into heteromeric filaments, mostly vimentin, an intermediate filament overexpressed in various type of cancer. Moreover, nestin contributes to the disassembly of vimentin during mitosis. Vimentin is considered to be an ectodermal, neural and pancreatic progenitor cells marker, and its presence was detected in pancreatic cancer stem-like cells. The cancer stem cell hypothesis suggests that mutated melanocyte stem cells are present in skin as precursors of melanoma cells. Several investigations have provided evidence that the genetic and/or epigenic alterations occurring in the multipotent tissue-specific adult stem cells and/or their early progenies may lead to their malignant transformation into cancer progenitor cells. Melanomagenesis and tumor progression are commonly described as ‘de-differentiation’ processes of transformed, mature melanocytes; recently, it has also been demonstrated that metastatic melanoma cell lines exhibit morphological, phenotypic and functional features of stem cell population. Our recent study suggests that nestin expression in both tumoral and endothelial cells may be considered as an important early prognostic marker in melanoma; on the other hand vimentin overexpression in cancer well correlates with accelerated tumor growth, invasion, and poor prognosis. Based on these considerations, the aim of our study was to investigate the colocalization of nestin and vimentin in primary melanoma by immunofluorescence, and its association with clinico-pathological features and patients survival.

Nestin and SOX9 and SOX10 transcription factors are coexpressed in melanoma

Nestin is an intermediate filament expressed in proliferating neural progenitor cells and has been considered as a stem cell marker. Nestin is also found in melanoma and we recently demonstrated that its expression in melanoma cell lines is regulated by the transcription factors SOX9 and SOX10, but not BRN2. In this study, the expression levels of nestin, BRN2, SOX9 and SOX10 were analysed in tissues of melanoma (n = 78) and melanocytic nevi (n = 26) by immunohistochemistry. All proteins were highly expressed in primary and metastatic melanomas and, apart from BRN2, showed much lower levels in melanocytic nevi. Significant coexpression of nestin with SOX9 and SOX10 was found in primary melanoma confirming our in vitro data. Correlation analysis with clinicopathological data revealed that nestin was significantly associated with presence of ulceration in primary tumors and SOX9 with more advanced stage of disease. Our data reveal that SOX9 and SOX10 are highly expressed in melanoma and seem to have a regulatory role in nestin expression. The association with ulceration and advanced-stage tumors, respectively, suggests that nestin and SOX9 may be negative prognostic markers in melanoma.

S100A13 is a new angiogenic marker in human melanoma

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called ‘dysplastic’ nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7±0.7) to vertical growth phase (3.6±3.1) to metastases (7.0±7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

The stem cell marker nestin predicts poor prognosis in human melanoma

The cancer stem cell hypothesis suggests that mutated melanocyte stem cells are present in skin as precursors of melanoma cells. Nestin and CD133 have been described as markers of melanocytic stem cells. The aim of this study was to establish if melanocytic stem cells could have a prognostic significance in melanoma progression. An immunohistochemical study for nestin and CD133 was performed in 130 primary tumors and 32 nodal metastasis biopsy specimens to evaluate possible differences, and to compare the results with survival data and clinicopathological variables. Nestin was expressed in cytoplasm of non-pigmented tumor cells and in endothelial cells, especially at the invading tumor front. Nestin staining in stage I and II (according to the American Joint Committee on Cancer Staging system) melanoma patients significantly predicted poor survival (log-rank test, P=0.037), with lower survival rates in cases with nestin positivity in both tumoral and endothelial cells. CD133 staining was not associated with survival. There were no significant differences in nestin or CD133 expression between primary tumors and metastases. These results suggest that nestin expression in both tumoral and endothelial cells may be considered an important early prognostic marker in melanoma.

Serum Amyloid A As a Prognostic Marker in Melanoma Identified by Proteomic Profiling

Currently known prognostic serum biomarkers of melanoma are powerful in metastatic disease, but weak in early-stage patients. This study was aimed to identify new prognostic biomarkers of melanoma by serum mass spectrometry (MS) proteomic profiling, and to validate candidates compared with established markers. Two independent sets of serum samples from 596 melanoma patients were investigated. The first set (stage I = 102; stage IV = 95) was analyzed by matrix assisted laser desorption and ionization time of flight (MALDI TOF) MS for biomarkers differentiating between stage I and IV. In the second set (stage I = 98; stage II = 91; stage III = 87; stage IV = 103), the serum concentrations of the candidate marker serum amyloid A (SAA) and the known biomarkers S100B, lactate dehydrogenase, and C reactive protein (CRP) were measured using immunoassays. MALDI TOF MS revealed a peak at m/z 11.680 differentiating between stage I and IV, which could be identified as SAA. High peak intensities at m/z 11.680 correlated with poor survival. In univariate analysis, SAA was a strong prognostic marker in stage I to III (P = .043) and stage IV (P = .000083) patients. Combination of SAA and CRP increased the prognostic impact to P = .011 in early-stage (I to III) patients. Multivariate analysis revealed sex, stage, tumor load, S100B, SAA, and CRP as independent prognostic factors, with an interaction between SAA and CRP. In stage I to III patients, SAA combined with CRP was superior to S100B in predicting patients' progression-free and overall survival. SAA combined with CRP might be used as prognostic serological biomarkers in early-stage melanoma patients, helping to discriminate low-risk patients from high-risk patients needing adjuvant treatment.

Telomerase activity in prognostic histopathologic features of melanoma

Telomerase activity (TA) is believed to play a role in the regulation of senescence and to limit the number of cell divisions. The deregulation of telomerase appears to contribute to oncogenesis and the formation of immortal cell lines. As a result, it is believed that it could be used as a prognostic marker in melanoma. TA was assayed by the polymerase chain reaction PCR-ELISA-based telomeric repeat amplification protocol (TRAP assay). One hundred and eight samples were distributed in four histological groups: 30 samples from primary cutaneous melanomas, 24 from peritumoural skin sites, 28 from benign melanocytic lesions, and 26 from normal skin sites as a control. TA was different among the four tested groups (Kruskall-Wallis test p<0.001), and increasing values of TA were observed progressing from normal skin to benign and then to malignant lesions. Among melanoma samples, there was a significant association between TA and ulceration (p=0.025), TA and vascular invasion (p=0.018) and TA and mitotic rate (p=0.029) (Mann-Whitney test). A linear regression analysis showed significant associations between the increase of TA with Breslow thickness (p=0.004) and the presence of satellites (p=0.002). We observed that TA had increased from control skin to peritumoural skin, and then to benign melanocytic lesions and finally to melanoma, suggesting tumour progression. TA showed higher values in the presence of some important histopathologic parameters related to poor prognosis in cutaneous melanoma such as ulceration, vascular invasion, satellites, high rates of mitosis, and in thicker tumours.

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.

Prognostic signs in melanoma: state of the art.

Prognoses for melanoma patients are currently based on statistically confirmed parameters, above all the Breslow thickness and number of lymph node and/or distant metastases. However, metastases can develop even with "thin" melanomas (< 0.7 mm), while survival has been recorded in patients with tumours classified as "thick" (> 4 mm). This review of the literature examines the most recent advances in prognostic markers for melanoma (serological, immunohistochemical, histological, genetic and surgical). These markers offer interesting possibilities in terms of diagnostic certainty, identification of early growth phases and estimation of the tumour's potential for progression and metastasis. It is reasonable to assume that their combined use can provide useful information for formulating prognoses that are not only statistically valid but also individualized.

TAP1 down-regulation in primary melanoma lesions: an independent marker of poor prognosis.

Melanoma tumor thickness is a major prognostic factor. Thin lesions, however, may metastasize, and sometimes thick tumors may not. To investigate the role of HLA class I-mediated antigen presentation, we correlated the expression of components of the antigen-processing machinery in primary melanoma lesions with their thickness and with the development of metastases. Seventeen formalin-fixed, paraffin-embedded primary melanomas thinner than 0.76 mm and 21 thicker than 1.50 mm were stained with anti-LMP2, -LMP7, -TAP1, -TAP2, -HLA class I and -beta2-microglobulin monoclonal antibodies. Twenty patients remained tumor-free in the follow-up period (10.5 +/- 1.8 years). Eighteen patients relapsed within a median period of 15.0 months following tumor excision. Expression of all markers in the tested lesions was down-regulated, the frequency ranging from about 40% for LMP and TAP subunits to about 70% for HLA class I antigens. Expression of all markers was not correlated with tumor thickness. Only TAP1 and TAP2 down-regulation was significantly (p = 0.026 and 0.042, respectively) correlated with the development of metastases. This correlation was independent of tumor thickness for TAP1. We suggest that TAP1 and probably TAP2 expression in primary lesions represents an independent prognostic marker in melanoma. Abnormalities in antigen presentation may account for the lack of absolute correlation between tumor thickness and prognosis.

Prognostic value of MMP‐2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (≥34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P<0·05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P<0·05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4·5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis. © 1998 John Wiley & Sons, Ltd.

Prognostic value of MMP-2 immunoreactive protein (72 kD type IV collagenase) in primary skin melanoma.

The penetration of the subepithelial basement membrane is the first critical step in the dissemination of melanoma. In vitro studies have suggested that the 72 kD type IV collagenase (MMP-2) may be important in melanoma invasion. It has recently been demonstrated that the expression of MMP-2 immunoreactive protein increased with increasing atypia in melanocytic tumours and was associated with later haematogenous metastases in melanoma. This paper investigates the value of MMP-2 as a possible prognostic marker in melanoma. The expression of MMP-2 immunoreactive protein was studied with immunoperoxidase staining in paraffin-embedded sections of 50 cases of primary skin melanoma by using specific, affinity purified antibodies. Positive immunostaining was quantified by counting the percentage of positive cancer cells and was compared with clinical patient characteristics and survival. Sixty-four per cent of the primary melanoma cases displayed positive cytoplasmic immunostaining for MMP-2 in tumour cells. Marked overexpression of MMP-2 protein (> or = 34 per cent of melanoma cells positive) correlated with the 5-year survival of the patients when compared with patients with lower MMP-2 positivity, 55 per cent vs. 85 per cent, respectively (P < 0.05). Male patients displayed positive staining more often than females (75 per cent vs. 54 per cent, respectively). There was no correlation between MMP-2 positivity and Clark level or Breslow classification. A distinct group with unfavourable prognosis was identified. The 10-year survival for MMP-2-positive male melanoma patients was 39 per cent as opposed to 79 per cent with the other melanoma patients (P < 0.05). In the hierarchic Cox regression model for survival, MMP-2 immunoreactive protein was found to be independent of Clark level and Breslow classification. Overexpression of MMP-2 protein indicated a 4.5-fold relative risk of dying from melanoma. It is concluded that MMP-2 immunoreactive protein in melanoma cells is an independent prognostic factor for survival. High MMP-2 expression in male melanoma patients indicates an unfavourable prognosis.