Vertical Growth Phase Melanomas Research Articles

Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions

BackgroundHypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.ResultsHIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1α and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1α expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 μM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1α expression. However, a 24 h treatment with 10 μM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels.ConclusionWe speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1α expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1α in melanoma biology increases its importance as a therapeutic target.

Distinct MHC gene expression patterns during progression of melanoma

Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas.

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis

Hallmarks of malignant melanoma are its propensity to metastasize and its resistance to treatment, giving patients with advanced disease a poor prognosis. The transition of melanoma from non-invasive radial growth phase (RGP) to invasive and metastatically competent vertical growth phase (VGP) is a major step in tumor progression, yet the mechanisms governing this transformation are unknown. Matrix Metalloproteinase-1 (MMP-1) is highly expressed by VGP melanomas, and is thought to contribute to melanoma progression by degrading type I collagen within the skin to facilitate melanoma invasion. Protease activated receptor-1 (PAR-1) is activated by MMP-1, and is also expressed by VGP melanomas. However, the effects MMP-1 signaling through PAR-1 have not been examined in melanoma. Here, we demonstrate that an MMP-1/PAR-1 signaling axis exists in VGP melanoma, and is necessary for melanoma invasion. Introduction of MMP-1 into RGP melanoma cells induced gene expression associated with tumor progression and promoted invasion in vitro, and enhanced tumor growth and conferred metastatic capability in vivo. This study demonstrates that both the type I collagenase and PAR-1 activating functions of MMP-1 are required for melanoma progression, and suggests that MMP-1 may be a major contributor to the transformation of melanoma from non-invasive to malignant disease.

Clinical and histopathological risk factors to predict sentinel lymph node positivity, disease-free and overall survival in clinical stages I–II AJCC skin melanoma: Outcome analysis from a single-institution prospectively collected database

Background To investigate if the tumour infiltrating lymphocytes (TILs) are able to predict the sentinel lymph node (SLN) positivity, the disease-free survival (DFS) and overall survival (OS) in clinical stages I-II AJCC primary cutaneous melanoma (PCM). Methods The study included consecutive patients with PCM, all diagnosed, treated and followed up prospectively. Logistic regression was used to investigate the association between DFS, OS, SLN positivity and Breslow thickness, Clark level, TIL, ulceration, lesion site, gender, regression and age. Results From November 1998 to October 2008, 1251 consecutive patients with PCM were evaluated. Median age was 51 (range 15–96) with 32.2% ( N = 393) of them older than 60; 44.8% of them were males. Of the whole series, a total of 404 patients with primary vertical growth phase (VGP) melanoma and no clinical evidence of metastatic disease underwent SLN biopsy. Of these, 74 (18.8%) had a positive SLN. In a multivariate analysis, primary melanoma on the extremities versus that on the axial locations (truncal and head/neck) (OR 0.49, 95% CI 0.25–0.98, p 0.04) and TILs (TILs versus no TILs) (OR 0.47, 95%CI 0.25–0.90, p 0.02) were predictive for lower probability of SLN involvement, while thickness (>4 mm versus 0–1 mm) (OR 24, 19, 95% CI 4.91–119.13, p < .001) was predictive for higher risk of SLN positivity. A multivariate stepwise analysis confirmed these results. The histological status of the SLN was the most significant predictor of DFS and OS. Patients with a negative SLN had a 5-year DFS of 75.9%, compared with 35.2% in patients with a positive SLN ( p < .0001) and a 5-year OS of 88.7% versus 42.9%, respectively ( p < .0001). Conclusions Our study demonstrates that the absence of TILs predicts SLN metastasis, in multivariate analysis the SLN positivity predicts DFS and OS.

Nucleolin protein expression in cutaneous melanocytic lesions

Nucleolin is a major nucleolar argyrophilic protein involved in carcinogenesis. There are only few studies on its tissue expression in human cancer and none in melanoma. We aimed at exploring this protein and its prognostic impact in cutaneous melanocytic lesions. We studied 193 cases including benign, dysplastic and malignant melanocytic lesions. Nuclear positivity was evaluated by immunohistochemistry and quantified by automated image analysis. Most dysplastic and malignant lesions showed high percentages of cells with abnormal patterns of nuclear positivity (Abn+N) consisting in multiple, irregular, positive dots (ID+) and a coarse, irregularly positive nucleoplasm (CNpl+) or both (ID+CNpl+). The patterns CNpl+ and/or ID+CNpl+ were never observed in benign lesions, in which ID+ were also virtually absent. Abn+N% was significantly lower in dysplastic nevi than in primary melanomas and metastases and in primary melanomas than in metastases (p < 0.05). Furthermore, Abn+N was the second powerful prognostic discriminator, after melanoma thickness, and a significantly lower survival was observed in vertical growth phase melanoma patients showing Abn+N in more than 50% of melanoma cells. An altered nuclear nucleolin expression seems to accompany melanoma progression. Further investigation on nucleolin functionality and subcellular trafficking could add information on its altered role in melanoma.

Ki67 as a prognostic biomarker for patients with vertical growth phase (VGP) melanomas

9043 Background: In VGP melanomas proliferation is reflected in dermal mitotic figures (“mitogenic” VGP) and/or tumor cell nests larger than any epidermal nest. An alternative to mitotic rate (MR) to characterize cell proliferation is the expression of Ki67 protein. Since Ki67 is expressed in all phases of the cell cycle except G0, it is potentially a more robust biomarker for proliferation and prognosis than mitoses. Methods: To test the hypothesis that Ki67 would replace MR as a prognostic factor, we did a retrospective cohort study of 432 patients with Stage I/II primary VGP melanomas who had at least 10 years of follow up. Tissue sections were stained using the monoclonal antibody MIB-1 to Ki67 and the % of positive melanoma cells were evaluated by two readers. ROC curves for Ki67 and MR were computed. Predicted probabilities (PP) of 10-year melanoma-specific death were computed from 3 multivariate logistic regression models, one for each biomarker (Models 1 and 2) and one with both (Model 3), controlling for established melanoma prognostic factors (thickness, gender, anatomic site, ulceration, regression and tumor infiltrating lymphocytes), and compared. Cross-validation was used to assess differences between using Ki67 and using MR including the differences in PP, Brier scores and the misclassification rates. A decision curve analysis was done to assess the clinical net benefit of the two. Results: The areas under the ROC curve (AUCs) for Ki67 and MR, both continuous factors, were 0.69 and 0.79, respectively. In the multivariate analysis, Ki67 expression was significant in Model 1 (OR=1.03, 95% CI: 1.01–1.05), mitotic rate was not significant in Model 2 (1.05, 0.99–1.1), and only Ki67 was significant in Model 3 (1.03, 1.01–1.05). The AUCs for the three models were 0.84, 0.84, and 0.85, respectively. Based on cross-validation, there was no difference between the two biomarkers in PP, Brier scores, or misclassification rates. The decision cost analysis demonstrated the same net benefit for the two. Conclusions: A prospective study needs to be conducted to confirm that Ki67 and MR are equivalent. No significant financial relationships to disclose.

The prognostic significance of lymphatic invasion in primary melanoma

9050 Background: Lymphatic invasion (LI) is an under-observed phenomenon in primary malignancies that can be better detected by immunostaining and that may associate with prognosis. In this study we sought to test the hypothesis that LI was associated with melanoma-specific survival (MSS) and was an independent prognostic factor. Methods: This study included 277 patients with stage I/II melanomas in vertical growth phase (VGP) who had at least 10 years of follow up. The log-rank test was used to test the study hypothesis - 72 melanoma-specific deaths were needed for 80% power to detect an odds ratio of 2.1. Paraffin sections were stained with antibodies to podoplanin (lymphatic vessels) and S-100 (melanoma cells) to identify LI. Univariate and multivariate Cox models were used to evaluate the prognostic significance of LI. An independent cohort of 106 similar patients was used for validation of the 10-year MSS rates. Results: LI was observed in 44.5% (95% CI: 38.6% - 50.4%) of the melanomas and its presence was significantly associated with thickness, mitotic rate, gender, age, and ulceration (U). The Kaplan-Meier survival curves for those with and without LI were significantly different (log-rank test p=0.022). The final multivariate model for time to MSD identified 4 independent prognostic factors: thickness (HR=1.5, p&lt;0.001), U (HR=2.2 p=0.013), site (HR=3.9, p&lt;0.001) and LI (HR=1.9, p=0.015). These factors were used to define a prognostic tree with 5 risk groups defined by melanomas that were thin (≤1.0mm) with no LI or U; thin with LI but no U; 1–3mm with no U; 1–3mm with U; and &gt;3mm. Respectively, MSS rates were 100%, 88.6%, 77%, 48% and 42%. In the validation set, observed 10-year MSS rates in each risk group were not significantly different from those predicted from the survival curves for the tree-based risk groups. Conclusions: LI is an independent prognostic factor for MSS. Among patients with thin melanomas without U the 10-year MSS was lower for those patients with LI (89%, 95% CI=78% - 99%; n=41) compared to those without (100%, n=78). LI is an important prognostic factor that needs further validation in a population of patients from the sentinel node biopsy era. No significant financial relationships to disclose.

Maspin expression, angiogenesis, prognostic parameters, and outcome in malignant melanoma

Maspin is a serine protease inhibitor that is thought of as a tumor suppressor because of observations that loss of maspin expression in breast, prostate, and oral cancer is associated with poor prognosis. In addition, maspin may function as an inhibitor of angiogenesis. However, it has been correlated with malignant behavior in pancreatic and ovarian cancer. The role of maspin in malignant melanoma (MM) has not yet been systematically examined. We aimed to examine the immunohistochemical expression of maspin and several proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) in MM and correlate each to angiogenesis, tumor thickness, and outcome. In all, 77 formalin-fixed, paraffin-embedded MM samples were immunostained for maspin and other proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) and were correlated with angiogenesis as mean microvessel density. Three normal-appearing skin samples and 10 nevi were also immunostained for maspin. Breslow thickness, Clark level, clinical stage, and follow-up information were obtained for outcome analysis. Immunohistochemical analysis revealed strong nuclear melanocytic maspin expression in all 10 nevi (half of which were dysplastic) but none in melanocytes from 3 normal-appearing skin samples. Strong nuclear maspin staining was demonstrated in 78% of radial phase melanoma and 46% of vertical growth phase melanoma. In addition, there was a significant inverse relationship between maspin and microvessel density (P = .018) and tumor thickness greater than 0.76 mm (P = .007), indicating that maspin is expressed in thinner tumors with less angiogenesis. Conversely, vascular endothelial growth factor expression, Clark level, and Breslow thickness all significantly correlated with microvessel density (P = .047, P = .027, and P = .011, respectively). Cyclooxygenase-2 expression significantly correlated with thicker tumors (P = .006) but not with angiogenesis (P = .714). In addition, Clark level, Breslow thickness, and stage were all significant predictors of overall survival (P < .001, P = .005, and P < .001, respectively). This study represents a single institution. These results demonstrate maspin expression in nevi and radial growth phase melanoma, but this expression seems to be lost in the transition from radial growth phase to vertical growth phase melanoma. In addition, maspin is correlated with decreased angiogenesis and tumor thickness less than 0.76 mm in MM. These results indicate maspin may function as a tumor suppressor in MM.

Novel variants of muscle calpain 3 identified in human melanoma cells: cisplatin-induced changes in vitro and differential expression in melanocytic lesions

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Inducible nitric oxide synthase expression in melanoma: implications in lymphangiogenesis

Cutaneous melanoma preferentially metastasizes via the lymphatic route. However, the mechanisms of lymphatic invasion and metastasis to regional lymph nodes are poorly understood. Nitric oxide is a free radical molecule synthesized from L-arginine by nitric oxide synthases that plays a critical role in various physiological and pathological processes, including tumor growth and angiogenesis. We have tested whether inducible nitric oxide synthase expression correlates with lymphatic vessel density identified with D2-40 antibody and/or blood microvessel density identified with CD105/endoglin in a series of melanocytic nevi (n=28) and cutaneous melanomas (n=38), representative of various pT. Inducible nitric oxide synthase expression was significantly lower in melanocytic nevi in comparison with primary and metastatic melanomas (P<0.001). Mean microvessel density was significantly higher in primary and metastatic melanomas in comparison with melanocytic nevi (P<0.001 for intratumoral and P=0.001 for peritumoral vessels). Vertical growth phase melanomas showed a higher intratumoral microvessel density in comparison with radial growth phase melanomas (P=0.02). The number of peritumoral lymphatics was significantly lower in nevi as compared with primary and metastatic melanomas (P=0.01). No correlation between microvessel or lymphatic vessel and clinical outcome was found in melanomas. A significant direct correlation was observed between inducible nitric oxide synthase immunostaining in melanocytic tumor cells and the density of lymphatic vessels (peritumoral: P=0.001; intratumoral: P=0.08), and the density of peritumoral blood microvessel (P=0.02). Our findings support the hypothesis that inducible nitric oxide synthase is implicated not only in blood, but also in lymphatic vascular neoformation in melanoma. Mechanistic studies are needed to address the possibility that inducible nitric oxide synthase controls lymphangiogenesis, dissemination and lymphatic borne metastases.

In vivo characterization of melanin in melanocytic lesions: spectroscopic study on 1671 pigmented skin lesions

The purpose of this study is to determine the role of melanin in the various steps of progression of melanocytic neoplasia. To this aim, we perform a retrospective analysis on 1671 multispectral images of in vivo pigmented skin lesions previously recruited in the framework of a study focused on the computer-assisted diagnosis of melanoma. The series included 288 melanomas in different phases of progression, i.e., in situ, horizontal and vertical growth phase invasive melanomas, 424 dysplastic nevi, and other 957 melanocytic lesions. Analysis of the absorbance spectra in the different groups shows that the levels of eumelanin and pheomelanin increase and decrease, respectively, from dysplastic nevi to invasive melanomas. In both cases, the trend of melanin levels is associated to the progression from dysplastic nevi to vertical growth phase melanomas, reflecting a possible hierarchy in the natural history of the early phases of the disease. Our results suggest that diffuse reflectance spectroscopy used to differentiate eumelanin and pheomelanin in in vivo lesions is a promising technique useful to develop better strategies for the characterization of various melanocytic lesions, for instance, by monitoring melanin in a time-lapse study of a lesion that was supposed to be benign.

Role of nodal signaling and the microenvironment underlying melanoma plasticity

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.

Phospho-ERK (pERK) and Ki67 expression as compared to thickness and mitotic rate (MR) as prognostic factors in patients with stage I/II melanomas

9020 Background: Tumor thickness and MR in the vertical growth phase (VGP) are key prognostic factors for melanoma patients. We hypothesized that the expression of two biomarkers associated with proliferation, Ki67, a measure of cell division, and pERK, a consequence of melanoma cells signaling through the MAP kinase pathway, would be associated with poor prognosis and more accurately predict those who would have a metastasis within ten years (MET). Methods: We evaluated Ki67 and pERK expression by immunohistochemistry in a nested cohort study of 50 patients with stage I/II VGP melanomas seen at Penn's Pigmented Lesion Clinic (1972–91). Ki67 expression was determined by the % of melanoma cells positive in VGP hot spots. Nuclear pERK expression by melanoma cells in the tumor's invasive edge (pERK/IE) was quantified using a modified H-score. Cut-points for both were established by maximizing the difference between survival distributions. Fisher's exact test (0.05, one-sided) had 75% power to detect an odds ratio (OR) of 4.3 with 19 and 42 patients with and without MET. Results: The odds of MET were 10.6-fold and 4.3-fold higher in patients having high Ki67 (p=0.013) and high pERK/IE (p<0.01) expression, respectively, compared to those with low expression. Thickness (OR=1.7, p=0.004) and MR (OR=1.3, p=0.002) were also each associated with MET. In the multivariate model the adjusted odds ratios for Ki67 and pERK/IE remained strong [4.0 (p=0.061) and 5.9 (p=0.030)], but thickness and MR were no longer significant. Conclusions: New biomarkers strongly associated with clinical outcome are needed. Both Ki67 and pERK/IE expression were strongly associated with prognosis, were notably more informative than thickness and MR in predicting MET, and are therefore candidates for validation in larger datasets. No significant financial relationships to disclose.

Regional Nodal Metastatic Disease Is the Strongest Predictor of Survival in Patients with Thin Vertical Growth Phase Melanomas: A Case for SLN Staging Biopsy in These Patients

Regional Nodal Metastatic Disease Is the Strongest Predictor of Survival in Patients with Thin Vertical Growth Phase Melanomas: A Case for SLN Staging Biopsy in These Patients

Ataxia Telangiectasia-Mutated Gene Is a Possible Biomarker for Discrimination of Infiltrative Deep Penetrating Nevi and Metastatic Vertical Growth Phase Melanoma

The deep penetrating nevus (DPN) is a variant of benign melanocytic nevus with clinical and histologic features mimicking vertical growth phase, nodular malignant melanoma (NMM). Because fatal misdiagnosis such as NMM occurs in 29% to 40% of the DPN, molecular differentiation markers are highly desirable. Beyond the clinical demand for precise diagnosis and diagnosis-adapted, preventive therapeutic strategies, the DPN represents a valuable natural model for melanocytic invasion without metastatic potential that per se deserves further investigations. In the present study, at first, we used a genome-wide, microarray-based approach to systematically prescreen for possible molecular markers differentially expressed between selected cases of typical DPN (n=4) and metastatic NMM controls (n=4). Gene expression profiling was done on Affymetrix Human X3P microarrays. Of the 47,000 genes spotted, we identified a list of 227 transcripts, which remained significantly regulated at a false discovery rate of 5%. Subsequently, we verified the expression of a subset of the most interesting transcripts in a larger immunohistochemical series (DPN, n=17; NMM, n=16). Of these transcripts, three were selected for immunohistochemical confirmation: tissue inhibitor of metalloproteinase-2, tumor protein D52, and ataxia telangiectasia-mutated gene (ATM). Additional criteria for selection from the list of 227 significantly regulated transcripts were grouping into functional Ingenuity networks and a known melanoma- or cancer-relevant function. Following these criteria, we detected a highly significant up-regulation of ATM transcription in NMM, which was also mirrored by ATM protein up-regulation. In contrast to the other markers, ATM particularly might serve as a suitable diagnostic and reliable discriminator of DPN/NMM because ATM immunoreactivity also showed a reliable staining consistency within all samples of both entities.

Evaluation of tumour-infiltrating CD4+CD25+FOXP3+ regulatory T cells in human cutaneous benign and atypical naevi, melanomas and melanoma metastases

CD4+CD25+FOXP3+ regulatory T cells (Tregs) are thought to induce immunotolerance in melanoma. They have not yet been investigated in the entire spectrum of melanocytic cutaneous lesions within a tumour site. To evaluate CD4+CD25+FOXP3+ Tregs among tumour-infiltrating lymphocytes in cutaneous melanocytic lesions. We analysed 128 lesions (10 benign junctional common naevi, 10 benign compound common naevi, 10 compound Spitz naevi, 10 junctional atypical naevi, 20 compound atypical naevi, 20 radial growth phase melanomas, 30 vertical growth phase melanomas and 18 melanoma metastases). Tregs were identified by CD25-FOXP3 double immunostains. This study indicates that CD4+/CD25+FOXP3+ Tregs are present in all groups of lesions. Junctional atypical naevi, compound atypical naevi and radial growth phase melanomas showed the highest percentages of CD4+CD25+FOXP3+ Tregs (junctional atypical naevi vs. junctional common naevi, compound common naevi, compound Spitz naevi, melanoma metastases: P < 0.0001; junctional atypical naevi vs. vertical growth phase melanomas: P = 0.001; compound atypical naevi vs. junctional common naevi, compound common naevi: P < 0.0001; compound atypical naevi vs. compound Spitz naevi, melanoma metastases: P = 0.002; compound atypical naevi vs. vertical growth phase melanomas: P = 0.02; radial growth phase melanomas vs. junctional common naevi, compound common naevi, compound Spitz naevi, melanoma metastases: P < 0.0001; radial growth phase melanomas vs. vertical growth phase melanomas: P = 0.008). The strong prevalence of CD25+FOXP3+ Tregs both in junctional and compound atypical naevi and radial growth phase melanomas, suggests that they induce immunotolerance early during melanoma genesis, favouring melanoma growth. Their evaluation within a tumour site could be useful for prognostic and therapeutic purposes.

Protective effect of a brisk tumor infiltrating lymphocyte infiltrate in melanoma: An EORTC melanoma group study

8519 Background: Tumor-infiltrating lymphocytes (TILs) in melanoma are responsible for tumor killing and may induce spontaneous regression. Brisk TILs in the melanoma vertical growth phase (VGP) is a strong, albeit not independent, prognostic factor associated with superior survival. This study investigated whether more detailed classes of TIL patterns, based on topography and intensity, are independent prognostic factors and identify patients with good prognosis. Methods: The study included 1,171 patients with cutaneous VGP melanoma with at least 3 years of follow up for whom slides were available for review. TIL infiltrate was assessed for pattern (absent, non brisk, brisk), intensity (scanty, moderate, dense), and topography (peripheral, central, both). Pattern was assessed qualitatively: brisk TIL infiltrate was defined as a continuous band of lymphocytes at the base of the melanoma, or throughout the tumor. Other studied variables were: thickness, mitotic count (MC), ulceration, gender, age, anatomic site, melanoma-related death (MRD). Subsequently, Chi-square tests, Kaplan-Meier curves (KM), logrank tests, and Cox models were applied to examine the relation between MRD, TIL categories and interaction with other variables. Results: A brisk infiltrate was observed in 21.2% of the cases. Adjusted hazard ratio for MRD as compared to the absent category was 0.82 (95% CI=0.64–1.06) in the non-brisk category, and 0.43 (95% CI=0.28–0.68) in the brisk category. Based on the Cox model, brisk TIL was an independent prognostic factor for MRD (p&lt;0.001) controlling for thickness, MC, ulceration, gender, age and site. Intensity was significantly associated with MRD for melanomas with peripheral and central brisk TILs (p=0.027) but not for other melanomas. Remarkably, no death was observed in the 5.5% of melanomas with dense, brisk TIL infiltrate at ten years. Conclusions: A brisk TIL pattern was shown to have prognostic value for MRD underscoring the importance of TILs in the outcome of VGP melanoma patients. Furthermore, we identified a small population of “super-responder” patients whose tumor was characterised by a continuous and dense TIL infiltrate and who did not display signs of tumor progression. No significant financial relationships to disclose.

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.

A High-Throughput Study in Melanoma Identifies Epithelial-Mesenchymal Transition as a Major Determinant of Metastasis

Metastatic disease is the primary cause of death in cutaneous malignant melanoma (CMM) patients. To understand the mechanisms of CMM metastasis and identify potential predictive markers, we analyzed gene-expression profiles of 34 vertical growth phase melanoma cases using cDNA microarrays. All patients had a minimum follow-up of 36 months. Twenty-one cases developed nodal metastatic disease and 13 did not. Comparison of gene expression profiling of metastatic and nonmetastatic melanoma cases identified 243 genes with a >2-fold differential expression ratio and a false discovery rate of <0.2 (206 up-regulated and 37 down-regulated). This set of genes included molecules involved in cell cycle and apoptosis regulation, epithelial-mesenchymal transition (EMT), signal transduction, nucleic acid binding and transcription, protein synthesis and degradation, metabolism, and a specific group of melanoma- and neural-related proteins. Validation of these expression data in an independent series of melanomas using tissue microarrays confirmed that the expression of a set of proteins included in the EMT group (N-cadherin, osteopontin, and SPARC/osteonectin) were significantly associated with metastasis development. Our results suggest that EMT-related genes contribute to the promotion of the metastatic phenotype in primary CMM by supporting specific adhesive, invasive, and migratory properties. These data give a better understanding of the biology of this aggressive tumor and may provide new prognostic and patient stratification markers in addition to potential therapeutic targets.