As the most potent antigen presenting cells, dendritic cells (DCs) play key roles in the immune response against tumors. Their density in the tumor tissue has been associated with prognosis in patients with various cancers. However, few studies have been aimed at the presence and maturation state of DCs in cutaneous melanoma, with regard to their potential clinical correlates. In this study, the density of DCs expressing CD1a and the maturation marker DC-LAMP was determined by immunohistochemistry in primary tumor samples from 82 patients with cutaneous malignant melanoma. Intratumoral and peritumoral cell densities were analyzed in relation to tumor thickness and the subsequent development of metastases, as well as to patients' survival. CD1a(+) DCs were found both infiltrating melanoma cell nests and in the surrounding stroma, while DC-LAMP(+) mature DCs were generally confined to the peritumoral areas, associated with lymphocytic infiltrates. DC density values significantly correlated with the number of activated (CD25(+) or OX40(+)) T lymphocytes (p < 0.001). The degree of infiltration by CD1a(+) and DC-LAMP(+) DCs showed strong inverse correlation with the thickness of melanomas (p < 0.001). High peritumoral density of mature DCs was associated with significantly longer survival (p = 0.0195), while density of CD1a(+) cells had a prognostic impact of borderline significance (p = 0.0610). Moreover, combination of high peritumoral CD1a(+) or DC-LAMP(+) cell density with high number of CD25(+) or OX40(+) lymphocytes identified patient subgroups with more favorable survival compared to other subgroups. A multivariate survival analysis involving DC and activated T-cell densities alone and in combinations, as well as traditional prognostic factors, identified high DC-LAMP(+) cell/high OX40(+) cell density and Breslow index as independent predictors of good prognosis. These results suggest that the presence of CD1a(+) DCs primarily depends on the thickness of melanomas, without direct relationship with the patients' survival. On the other hand, the density of mature DCs, especially in association with that of activated T cells, proved of prognostic importance, suggesting that these parameters could be considered as signs of a functional immune response associated with better outcome of the disease.

This study was undertaken to determine the role and the fate of the peritumoural vascular plexus during the vascularization of human malignant melanoma (hMM) and in an appropriate murine melanoma model system. The prognostic significance of the vascularity of different tumour areas was also evaluated. Despite morphometry revealing several-fold higher microvessel densities (MVDs) in the peritumoural tissue than at the centre of the tumour, the development of visceral metastases of hMM was exclusively correlated with the MVD of the tumour centre. Furthermore, the 5-year survival of the patient group with low tumour centre MVD (<30/mm(2), n=29) was 100%, compared to 1/16 patients alive with high tumour centre MVD (>30/mm(2), n=16). Morphometric analysis and three-dimensional reconstruction of vessel networks of both human and murine melanomas showed clearly that the peritumoural vascular plexus present at the melanoma base is continuously being incorporated into the growing tumour mass. Once vessels become incorporated, sprouting ceases and the proliferating endothelial cells (EC) take part only in vessel dilatation. Moreover, the immunohistochemical and ultrastructural characterization of microvessels demonstrated that the pericyte coverage of endothelial tubes was complete in all of the investigated areas, in both human and murine melanomas.

Studies on the prognostic importance of tumor-infiltrating lymphocytes have mainly focused on T cells, while little is known about the role of tumor-infiltrating B lymphocytes. We investigated the prevalence of CD20(+) B cells by immunohistochemistry in primary melanoma samples of 106 patients and analyzed in relation to clinicopathological parameters and patients' survival. The majority of samples contained a significant amount of B lymphocytes, predominantly dispersed in the stroma surrounding tumor deposits (mean peritumoral and intratumoral densities: 178.7 ± 156.1 vs. 4.9 ± 6.9 cells/mm², respectively). B cells organized in follicle-like aggregates were also observed in 26% of the samples. B-cell density correlated with that of activated (CD25(+) or OX40(+)) T lymphocytes. Infiltration by CD20(+) lymphocytes did not correlate with tumor thickness, while the presence of B-cell aggregates was observed more frequently in thick melanomas. On the other hand, B-cell infiltration was more pronounced in nonmetastatic or lymph node metastatic tumors, compared to visceral metastatic ones. Accordingly, high number of these cells provided significant survival advantage (P = 0.0391 and P = 0.0136 for intra- and peritumoral infiltration, respectively). Furthermore, combination of peritumoral B-cell density with the number of activated T lymphocytes identified patient subgroups with different disease outcome, which was most favorable in the case of high density, while very poor in the case of low density of both cell types. Multivariate survival analysis identified tumor thickness and CD20(+)/OX40(+) cell density combination as significant independent prognostic factors. Taken together, our results show correlation between low number of CD20(+) B lymphocytes and melanoma progression, indicating a possible role of tumor-infiltrating B cells in antitumoral immune response. It was also reflected in better outcome of the disease since the density of B lymphocytes alone as well as in combination with that of activated T cells proved of prognostic importance in patients with malignant melanoma.

The central role of T cells in antitumor immunity is well established. However, tumor progression, often seen in the presence of substantial lymphocytic infiltration, suggests that these T cells are not capable of mounting an effective immune response to control tumor growth. Evidence has accumulated that T lymphocytes infiltrating human neoplasms are functionally defective, incompletely activated, or anergic. Therefore, when characterizing the immune competent cells within lymphoid infiltrates of tumors, it is important to assess their activation state. We investigated the expression of two T-cell activation markers, interleukin 2 receptor ␣ (CD25) and OX40 (CD134), by immunohistochemistry in primary cutaneous melanoma samples of 76 patients and analyzed it in relation to tumor stage and tumor progression (>5 years follow-up), as well as to patients' survival. We found that the degree of infiltration by CD25 ؉ and intratumoral OX40 ؉ lymphocytes showed a tendency to decrease in thicker melanomas. The frequency of samples with high numbers of peritumoral CD25 ؉ and OX40 ؉ cells was significantly lower (P ‫؍‬ 0.0009 and P ‫؍‬ 0.0087, respectively) in melanomas developing distant visceral metastases, compared with nonmetastatic or lymph node metastatic tumors. For both activation markers studied, high peritumoral densities were associated with longer survival by univariate analysis (P ‫؍‬ 0.0028 and P ‫؍‬ 0.0255 for CD25 and OX40, respectively), whereas peritumoral OX40 ؉ lymphocyte infiltration had an impact on survival also in multivariate analysis (P ‫؍‬ 0.035). The results suggest that the presence of lymphocytes expressing the T-cell activation markers CD25 or OX40 shows correlation with tumor progression as well as with patients' survival in cutaneous malignant melanoma.

The role of ␣IIb␤3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of ␣IIb␤3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n ‫؍‬ 24) showed that ␣IIb␤3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of ␣IIb␤3 was 8% (؎1.8), 33% (؎10.4) and 62% (؎5) in melanomas ranging in thickness from 0 -1.5 mm, 1.5-4.0 mm and >4 mm, respectively; ␣v␤3 was equally high all categories. To determine biological function, we stably transfected ␣IIb␤3 into human melanoma cells that express ␣v␤3, but not ␣IIb␤3. Surface expression of ␣v␤3 remained unaltered between ␣IIb␤3 (؉) and mock transfected counterparts. The ␣IIb␤3 (؉) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of ␣IIb␤3 displaced ␣v␤3 from focal contact points. When implanted subcutaneously into SCID mice, the ␣IIb␤3 (؉) cells developed ϳ4-fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co-expression of the 2 ␤3 integrins, ␣v␤3 and ␣IIb␤3, in human melanoma cells enhanced cell survival and promoted growth in vivo. © 2002 Wiley-Liss, Inc. GPIIb/IIIa; ␣v␤3; integrin; tumor; growth Integrins are heterotypic adhesion receptors composed of noncovalently associated ␣ and ␤ subunits. There are a variety of integrin ␣ and ␤ subunits that pair differently to form at least 20 receptors. 1 Even though integrins were originally thought to function purely as anchor molecules, today they are well appreciated as signaling receptors. Integrins do not have intrinsic enzymatic activity, however by recruiting and activating tyrosine (pp125FAK, pp60src), serine (PKC␣, ERK, JNK, ILK) or lipid (cPLA2, PI3K, PI4P5K) kinases they can simultaneously control multiple signaling pathways, including the MAP kinase, JAK-STAT pathways, modify intracellular concentration of ions (i.e., H ϩ and Ca 2ϩ ), influence lipid synthesis and synthesis of cyclins. 2-5 These integrin-mediated signaling pathways are involved in diverse biological processes such as cell proliferation, apoptosis, angiogenesis, adhesion, spreading, motility and invasion. 6 Different integrins may crosstalk with each other, thereby regulating each other's function. For example ␣IIb␤3-mediated signaling in CHO cells influences ␤1integrin-mediated cell adhesion and spreading. 7 This phenomenon of transdominant signaling appears to involve MAPK activity. Activation of myosin light chain and lipid kinases by the MAP kinase pathway and the Rho family of GTPase is modulated by integrin activity, which provides a link between the extracellular matrix and effect on cell shape and motility that ultimately influences tumor cell invasion. Although the relationship be...