Cutaneous malignancies in patients with Parkinson's disease at a dermato-oncological university centre in Hungary.

Background: The possible correlation between melanoma and Parkinson's disease (PD) has been intensively studied. In this work, we aimed to assess the coincidence of skin malignancies and PD at a dermato-oncological university centre in Central-Eastern Europe, Hungary. Methods: From 2004 to 2017, a retrospective analysis of the centre's database was performed based on International Statistical Classification of Diseases-10 codes. Results: Out of the patients who visited the clinic during the study period, 20,658 were treated for malignant skin tumours. Over the 14 years, 205 dermatological patients had PD simultaneously, 111 (54%) of whom had at least one type of skin malignancy: melanoma (n=22), basal cell carcinoma (BCC) (n=82), or squamous cell carcinoma (SCC) (n=36) (in some patients, multiple skin tumours were identified). Compared to the age- and sex-matched control group, patients with PD had a significantly lower risk for basal cell carcinoma (OR, 0.65; 95% CI, 0.47-0.89, p=0.0076) and for all skin tumours (OR, 0.74; 95% CI, 0.56-0.98, p=0.0392) but not for melanoma. Conclusions: We found a decreased risk of all skin tumours and basal cell carcinoma and an unchanged risk of melanoma among patients with PD. However, it should be kept in mind that some large-scale meta-analyses suggest a higher incidence of melanoma after a diagnosis of PD, indicating the importance of skin examination in this vulnerable population.

Correlation with lymphocyte infiltration, but lack of prognostic significance of MECA-79-positive high endothelial venules in primary malignant melanoma.

High endothelial venules (HEVs) are specialized vessels in lymphoid organs, supporting lymphocyte trafficking from the blood. As the presence of these vessels was described recently in tumors, it was proposed that they could facilitate the development of antitumor immune response, resulting in improved prognosis. The aim of our study was to analyze the correlation of the density of HEVs with that of the different immune cell types as well as with the clinicopathologic parameters and the disease outcomes in patients with cutaneous melanoma. Primary melanoma samples of 118 patients were analyzed retrospectively by immunohistochemical labeling and quantitation of vessels stained with the MECA-79 antibody, as well as a panel of eight different immune cell types (CD8 and CD45RO T cells, lymphocytes expressing the CD25, CD134, or CD137 activation markers, FOXP3 regulatory T cells, CD20 B cells, and DC-LAMP mature dendritic cells). Correlations of MECA-79 vessel density with that of the immune cells, as well as with clinicopathologic parameters and disease outcomes were evaluated. We showed that the number of MECA-79 vessels correlates strongly with the peritumoral density of B and T lymphocytes. Moreover, higher HEV numbers were detected in tumors hosting tertiary lymphoid structures as well as in those of axial location compared with the ones in the extremity and in men compared with women, whereas no association was found with patient age, tumor thickness, histologic type or ulceration, or with the survival of melanoma patients. The density of MECA-79 HEVs in primary melanomas shows a correlation with B and T-lymphocyte density and differences according to the presence of tertiary lymphoid structures, tumor site, and the sex of the patient. However, it has no prognostic value.

Retrospective Analysis of Clinicopathological Characteristics of Pregnancy Associated Melanoma.

Pregnancy associated melanoma (PAM) by definition appears during pregnancy or within 1 year after delivery. In this retrospective study we analysed the pathological characteristics and survival rate of PAM and matched the data with non-pregnant age- and stage-matched control patients. Between 2003 and 2014, 34 pregnant women (aged 32.5 ± 5.6 years) were diagnosed with melanoma at the Department of Dermatology, Venereology and Dermatooncology of the Semmelweis University. During the pathological process histologic subtype, Breslow thickness and Clark level, tumor cell type, mitotic rate, peritumoral inflammation, as well as ulceration, regression, necrosis, vascular invasion and presence of satellite were analyzed and related to clinical data. Primary tumor location and clinical staging, disease course, local recurrence and metastases, 5-year survival rate, other tumor development before or after the diagnosis of melanoma have also been documented. We found no difference in all parameters between pregnant and non-pregnant melanoma cases except peritumoral inflammation which was higher in PAM group, moreover the presence of mild inflammation was significantly higher in PAM group compared to non-pregnancy associated melanoma (NPAM) women group.

Ectopic lymphoid structures in primary cutaneous melanoma.

Ectopic lymphoid structures have been described in several tumor types including metastatic lesions, but not primary tumors, of patients with melanoma. Here we present evidence of B-cell follicles in primary cutaneous melanomas, being present in 39 of 147 cases (27 %). B-cell clusters were associated with T lymphocytes, most of which belonging to CD45RO(+) memory T cells. A network of CD21(+) follicular dendritic cells was demonstrated in 8 of 22 cases studied (36 %). MECA-79(+) HEV-like venules were observed in the neighborhood of the follicles in the majority of cases, however, their presence was not confined to tumors hosting ectopic lymphoid structures. The appearance of B-cell aggregates did not show association with the outcome of the disease, although a trend for their higher prevalence was observed in thicker tumors. Our results show that neogenesis of lymphoid structures does occur in primary melanomas, albeit with lower frequency compared to that reported in metastases.

Genotype analysis in Hungarian patients with multiple primary melanoma.

Multiple primary melanoma patients (MPMps) have better prognosis and are more prone to genetic predisposition than single melanoma patients. We aimed to compare genetic background (CDKN2A, CDK4, MITF, MC1R) of 43 Hungarian MPMps with their clinicopathological data. We observed a higher rate of synchronous first and second melanoma (MM) (49%) and a higher frequency of non-melanoma tumor co-occurrence (42%) than reported previously. CDKN2A mutation frequency was 4.7% (E69G, R99P). We identified a new human MC1R variant (D117G) and reported MC1R variant distributions in Hungarian MMs for the first time. The rare R163Q was exceptionally common among Hungarian MPMps, a variant otherwise frequent in Asia, but not in Europe. MC1R 'R' carriers showed histopathological signs of a more progressive disease than 'r' carriers did; however, tumor-infiltrating lymphocytes (TILs) in their second melanomas occurred significantly more frequently. Calculating 5-year overall survival, 'R' carriers showed more unfavourable prognosis (87%) than 'r' carriers did (95%).

[Molecular classification and markers of malignant melanoma]. / A malignus melanóma molekuláris klasszifikációja és markerei.

Pathological classification of malignant melanoma did not change in the past decade, it was just completed with UV-induced skin alterations. A new feature, however, is the establishment of molecular classification of melanoma indicating that beside the most frequent genetic alterations (BRAF, NRAS, CKIT mutations) there is a wide variety of rare molecular subclasses. Unfortunately, none of these genetic alterations can be used to discriminate benign lesions from malignant ones. The frequently used "melanoma" markers are mostly melanosomal markers, therefore they are not helpful for this diagnostic purpose either. More recently, novel FISH kits have been developed analyzing characteristic copy number alterations specific for malignant melanoma. Though melanosomal markers are helpful in differencial diagnostics, the presence of normal melanocytes in various tissues (lymph nodes, intestine or brain) requires application of molecular techniques when melanoma metastasis is in question.

[Stage distribution of malignant melanomas in a Hungarian centre]. / Melanomás betegek stádium szerinti megoszlása egy hazai centrumban.

INTRODUCTION: Survival of patients with malignant melanoma primarily depends on tumor stage. Hungarian National Cancer Registry does not specify tumors according to TNM stages. AIM: The authors aimed to survey the stage distribution of melanomas at the Department of Dermatology, Dermatooncology and Venerology, Semmelweis University. METHOD: 1160 patients (558 males and 602 females, aged 60.5±16 and 57±17 years, respectively) diagnosed with cutaneous melanoma between 2004-2009 were included. RESULTS: In comparison with international studies, the case distribution was favorable in stages IA and IV, i.e. the proportion of early melanomas was relatively high (IA: 43.8%), while the incidence in stage IV was low (0.4%). In stages IB-IIA the incidence was significantly lower, while in IIC, IIIA, IIIB it was higher as compared to published data from Western-Europe, Australia and the United States. CONCLUSIONS: The study underlines the necessity of prevention and awareness campaigns that may result in increase of early diagnosis of melanomas.

Risk of subsequent primary tumor development in melanoma patients.

Incidence of subsequent malignant tumor development in 740 patients with primary cutaneous melanoma verified between 2006 and 2010 at the Semmelweis University was studied retrospectively and was compared to data of sex and age matched Hungarian population. The follow-up period was 1499 person-years for the whole group from the diagnosis of index melanoma with an average of 2 years. Standardized incidence rate (SIR) was established as the ratio of observed and expected values. The risk of all subsequent malignancies was 15- and 10-fold higher in males (SIR: 15.42) and in females (SIR: 10.55) with melanoma, than in the general population. The increased cancer risk resulted mainly from the significantly higher skin tumor development: SIR values were 160.39 and 92.64 for additional invasive melanoma and 342.28 and 77.04 for subsequent in situ melanoma in males and females, respectively. Non-melanoma skin cancers also notably contributed to the higher risk, the SIR was elevated in both genders to the same extent (males: 17.12, females: 17.55). The risk was also significantly higher for extracutaneous tumor development like chronic lymphocytic leukemia, colon and kidney cancer (both genders), non-Hodgkin's lymphoma, cervical cancer (females), and bladder carcinoma (males). These data underline the importance of patient education and the necessity of frequent medical follow up, including a close-up dermatological screening of melanoma survivors for further malignancies.

Melanoma screening in a hungarian nuclear power plant.

The industrial use of the ionizing radiation (IR) particularly stresses the safe work, regular health control is inevitable. Since previous occupational cohorts reported contradictory data on the incidence of melanoma among nuclear industry workers, and in few publications significant increase of it has been described, our clinic was requested by the industry to screen malignant skin tumours among the workers of a power plant. Within a year we have investigated 556 workers, 275 females and 281 males. Out of them 283, majorly males had been officially confirmed as to be employed at hazardous, but strictly controlled environment for an average of 18 years (1-32 years). To distinguish between IR and environmental UV (UVA+UVB) induced cutaneous malignancies we determined the sun and tanning bed exposure of the workers. One in situ melanoma developed in a woman with type I skin, bullous sunburns in the history, who had worked in safe environment for 26 years. Basal cell carcinoma was identified in two men, each of them worked for more than 20 years with IR (in hazardous environment). One had type I skin, the other had type II skin. These results didn't differ significantly (chi-squared test; p = 0, 2437 and 1, 0) from the national population data and the results of Euromelanoma screening campaign in Hungary. Our data clearly show, that 1./UV exposure and skin type should be evaluated in occupation cohort studies. 2./The melanoma incidence was not significantly higher among the employees of the power plant than in the general Hungarian population, according to the results of our study, the only Hungarian power plant is safe as far as the skin carcinogenesis is concerned.

Prognostic impact of B-cell density in cutaneous melanoma.

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.

FOXP3+ cell density in primary tumor has no prognostic impact in patients with cutaneous malignant melanoma.

Regulatory T cells (Tregs) have been implicated as inhibitors of antitumor immune reactions. However, data on the relevance of their prevalence at tumor sites in influencing disease outcome are controversial. The aim of our study was to investigate the role in tumor progression and the prognostic impact of the density of lymphocytes expressing FOXP3, a transcription factor expressed predominantly by CD4(+)CD25(+) Tregs, in primary cutaneous melanoma. We examined the infiltration of FOXP3(+) cells by immunohistochemistry in tumor samples from 97 patients and evaluated in relation to patient and tumor parameters. The degree of infiltration by FOXP3(+) cells did not show correlation with the thickness of melanomas. Moreover, no associations were found with metastasis formation during the 5-year follow-up period, patient survival, or any other clinicopathologic parameters studied. These results suggest that the presence of FOXP3(+) lymphocytes in primary tumors is not of prognostic importance in human cutaneous melanoma.

Association of microvessel density with infiltrating cells in human cutaneous malignant melanoma.

Vascularization and host response to malignant tumors may have common molecular regulators, therefore, we analyzed the relationship between microvessel density (MVD) and tumor infiltrating cells in cutaneous malignant melanoma. Density of lymphocyte subpopulations, macrophages, dendritic cells and CD34(+) microvessels was determined by immunohistochemistry in primary tumor samples from fifty-two patients with melanoma thicker than 1 mm. Intratumoral MVD did not show significant association with infiltration for any of these cell types. In the case of peritumoral reactive cell densities analyzed in the whole patient population, a positive correlation of MVD was found with CD3(+) T cell density. This association was stronger in melanomas >4.0 mm and in visceral metastatic tumors. In these subgroups similar phenomenon was observed for CD8(+) cells. We found significant correlation of MVD with CD68(+) macrophage density only in the highest thickness category, and weak associations with B-cell and dendritic cell infiltration in visceral metastatic cases. MVD did not vary significantly in tumors categorized according to thickness, localization, ulceration or histological type. However, both intratumoral MVD and macrophage infiltration were significantly higher in male patients compared to females. The correlation of immune cell density with tumor vascularization and gender differences in vascularity and macrophage infiltration of melanoma deserve further attention.

Density of DC-LAMP(+) mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor.

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.

Molecular identification, localization and function of platelet-type 12-lipoxygenase in human melanoma progression, under experimental and clinical conditions.

As previous studies suggested the expression of a 12-LOX enzyme in murine and human melanoma cell lines, the primary aim of this project was to genetically identify the 12-LOX enzyme (platelet-, leukocyte- or epithelial form). By using reverse transcriptase-polymerase chain reaction, sequencing and various immunological techniques we have demonstrated conclusively the expression of the platelet-type 12-LOX in human melanoma cells of different origin, in their transplanted xenografts and in fresh human skin tumors. Furthermore, we found that p12-LOX is able to provide a survival signal for melanoma cells since inhibition of the enzyme by general LOX or selective 12-LOX inhibitors induced apoptosis in vitro. p12-LOX of human melanoma has been shown to be involved in the control of the metastatic phenotype, since we have detected the upregulation of the 12-LOX protein expression in spontaneously metastasizing xenografts and in thick human skin tumors (> 3.0 mm) characterized by high risk for the development of metastasis. Co-expression of two megakaryocytic genes, p12-LOX and alphaIIb integrin chains, was found to be a frequent phenomenon in human melanoma (approximately 70%) suggesting a common regulatory defect in this tumor.

Loss of vascular adhesion protein-1 expression in intratumoral microvessels of human skin melanoma.

Intratumoral vessels are different both structurally and phenotypically, and this may have clinical significance. In this study we analysed the expression of an adhesion molecule--vascular adhesion protein-1 (VAP-1)--in melanoma-associated blood vessels in 28 primary skin melanoma cases using immunocytochemistry and immunoelectron microscopy. We have found that VAP-1 protein expression is significantly decreased in intratumoral vessels compared with peritumoral ones; this difference was independent of the tumour thickness. Loss of VAP-1 protein expression occurred in both endothelial and smooth muscle cell components. Unlike in other cancer types, the VAP-1 protein expression of intratumoral vessels did not correlate with the density of cytotoxic T-lymphocytes or dendritic cells. On the other hand, the 5-year survival of melanoma patients with low VAP-1 protein expression in intratumoral blood vessels (< or =25%) was lower (26.3%) than in patients whose VAP-1 expression was higher (42.6%, P=0.0632). These results support the idea that the phenotype of intratumoral blood vessels is important in the progression of malignant melanoma.

T-cell activation marker expression on tumor-infiltrating lymphocytes as prognostic factor in cutaneous 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 alpha (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.

[Clinical characteristics of melanoma metastasis]. / A melanoma áttétképzésének klinikai jellemzoi.

Approximately one third of clinical Stage I melanoma patients will experience disease recurrence. The author reviews the main prognostic factors predicting the outcome of melanoma patients, the incidence, pattern and time of first metastases. Of all recurrences, two third will occur earlier by the lymphatic, and one third later by the hematogenous pathway. 75-80% of recurrences develop in the first 3 years, 3-4% of metastases occur after 10 years of disease-free interval. The data emphasize the value of lifelong follow-up.

Role for beta3 integrins in human melanoma growth and survival.

The role of alphaIIbbeta3 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 alphaIIbbeta3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that alphaIIbbeta3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of alphaIIbbeta3 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; alphavbeta3 was equally high all categories. To determine biological function, we stably transfected alphaIIbbeta3 into human melanoma cells that express alphavbeta3, but not alphaIIbbeta3. Surface expression of alphavbeta3 remained unaltered between alphaIIbbeta3 (+) and mock transfected counterparts. The alphaIIbbeta3 (+) 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 alphaIIbbeta3 displaced alphavbeta3 from focal contact points. When implanted subcutaneously into SCID mice, the alphaIIbbeta3 (+) cells developed approximately 4-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 beta3 integrins, alphavbeta3 and alphaIIbbeta3, in human melanoma cells enhanced cell survival and promoted growth in vivo.

Vascularization of cutaneous melanoma involves vessel co-option and has clinical significance.

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.