The importance of microenvironment: the role of CCL8 in metastasis formation of melanoma.

We have attempted to characterize the changes occurring on the host side during the progression of human melanoma. To investigate the role of tumor microenvironment, we set up such an animal model, which was able to isolate the host related factors playing central role in metastasis formation. One of these 'factors', CCL12, was consequently selected and its behavior was examined alongside its human homologue (CCL8). In our animal model, metastasis forming primary melanoma in the host exhibited increased level of CCL12 mRNA expression. In clinical samples, when examining the tumor and the host together, the cumulative (tumor and host) CCL8 expression was lower in the group in which human primary melanoma formed lung metastasis compared to non-metastatic primary tumors. We could not detect significant difference in CCL8 receptor (CCR1) expression between the two groups. Increased migration of the examined tumor cell lines was observed when CCL8 was applied as a chemoattractant. The tumor cells and their interactions can be influenced the expression of CCL8 by dermal fibroblasts, as a significant change in the metastatic microenvironment. Furthermore, we examined changes in miRNA profile resulted by CCL8 and miR146a appears to be a promising prognostic marker for following this process.

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.

[Recurrent acral lentigous melanoma successfully treated with Mohs' micrographic surgery. Case report and review of the literature]. / Recidív acralis lentiginosus melanoma sikeres eltávolítása Mohs-féle mikrográfikus sebészi technikával. Esetismertetés és irodalmi áttekintés.

Mohs' micrographic surgery is an approach to selected skin cancer removal that aims to achieve the best prospect of total tumor excision simultaneously with maximal functional and cosmetic preservation. The advantage of the technique is that 100% of the surgical margin of the specimen, including the periphery and deep surface, can be examined intraoperatively by horizontally oriented frozen sections. This method offers cure rates significantly higher than conventional excision or other modalities. Mohs' micrographic surgery is the method of choice for removal of large, recurrent or incompletely excised skin cancers or for tumors located in functional and aesthetic relevant anatomic regions. The authors present a case of a 75-year-old man with a second time recurrent plantar invasive malignant melanoma successfully treated with Mohs' micrographic surgery technique and an immediate reconstruction using split-thickness skin graft.

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.

Relationship between sentinel lymph node status and regression of primary malignant melanoma.

The prognostic significance of spontaneous regression of primary melanoma is a controversial issue. Studies on sentinel lymph node status and circulating tumour cells may represent a step towards a better understanding. The clinical details of 269 melanoma patients who underwent sentinel lymph node biopsy were analysed. Correlation was sought between the parameters of the primary tumour, particularly tumours showing a partial intermediate level of regression, and sentinel lymph node status. The presence of circulating tumour cells was studied by reverse transcription-polymerase chain reaction for tyrosinase messenger RNA preoperatively in 94 patients. Of the examined tumours, 27.8% showed histological features of a partial intermediate level of regression. Regressive tumours were localized predominantly on the trunk (P=0.006), were significantly thinner (P<0.0000) and were less frequently ulcerated (P=0.003) than tumours without regression. Moreover, the majority of regressive melanomas were of the superficial spreading type (P<0.0000) and their sentinel node status was more favourable (P=0.026). We demonstrated the presence of circulating tumour cells in five of 26 (19.2%) regressive and 19 of 68 (29.4%) non-regressive tumours. The difference was not significant (P=0.32). By multivariate analysis, however, the Breslow thickness and ulceration of the primary tumour were predictors of the sentinel lymph node status, in agreement with literature data. A partial intermediate level of regression of the primary tumour did not affect unfavourably the sentinel lymph node status in our study. We failed to demonstrate a significant relationship between the presence of circulating tumour cells and either primary tumour regression or the sentinel lymph node status.

[Etiologic factors of malignant melanoma in young adults]. / Fiatalkori melanomák etiológiai tényezoi.

BACKGROUND: Malignant melanoma is the most aggressive type of skin cancers, involving the cutis and the mucosa. Its incidence keeps increasing dramatically in the last decades. It appears rarely in childhood. The main environmental risk factors are: excessive sun exposure and severe sunburns in both childhood and adolescence. Skin phototype, number of nevi, presence of congenital nevi (especially giant congenital nevi) and non-melanoma skin cancer in previous history refer to increased risk. Investigations of genetical factors have come to the front. The role of hormonal influences and traumas are recurring questions. AIM: The presence of melanoma typically concerns the middle-aged population. The purpose of this study was to determine the main risk factors, etiology factors and predisposing pediatric conditions in development of melanoma in young adulthood (under the age of 30). METHOD: A total of 70 new, histologically verified melanoma patient under 30 years were examined between 1993-2003 with a retrospective study. Results of questionnaire based survey and clinical data base about melanoma risk factors were also analysed. RESULTS: 5% of patients had giant congenital nevi, although in half of the patients (19/40) more than 20 moles were found. On the basis of patients' histories 57.5% of melanomas developed on a nevus existing from birth or childhood. 30% of melanomas developed on a pigmented brown alteration which rose on the normal skin. About 1/3 of patients had fair skin type and almost all patients (38/40) suffered from erythematous sunburn at first sunbath. Melanoma developed mostly on the trunk and lower extremities. 51.5% of patients belonged to Stage I (Breslow thickness below 1 mm in 33%). CONCLUSION: There were 70 young (under 30 years) patients were treated for malignant melanoma at the Dermatology Department of the National Institute of Oncology in Budapest from 1993 till 2003. The incidence of melanoma under the age of 30 was 3.3%. In young adulthood the main risk factors were the number of atypical nevi and repeated or severe sunburns in childhood. The skin type was also an important risk factor. 50% of the melanomas in young women developed on the trunk. Authors could not prove any relationship among hormonal factors, pregnancy and the development of the 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.

[Report on clinical observations obtained with sentinel lymph node surgery in malignant melanoma and during their follow-up at the Department of Dermatology, National Institute of Oncology, Budapest]. / Sentinel nyirokcsomó-biopszia melanomában. Klinikai tapasztalatok az Országos Onkológiai Intézetben.

OBJECTIVES: Report on clinical observations obtained with sentinel lymph node surgery for malignant melanoma and during follow-up at the Department of Dermatology, National Institute of Oncology, Budapest. PATIENTS AND METHOD: In the period from November, 1997 to September, 2002, the above surgical intervention was made with 179 patients having primary tumour, one to two months after primary tumour removal. Staining with patent blue was combined with isotope technique. The primary melanoma and the pertaining sentinel lymph node(s) were removed. Histological evaluation of the sentinel lymph nodes was performed in serial sections. Immunohistochemical detection of S100, HMB-45, or Melan-A was used in the case of suspected micrometastases. Demonstration of positive sentinel lymph nodes was followed, preferably within 2-3 weeks, by regional block dissection. Interferon in low doses or BCG immune therapy were applied as adjuvant therapy. Bimonthly follow-up of the patients included physical examination and the use of imaging techniques as specified in the melanoma protocol. RESULTS: Sentinel lymph node surgery was successful in 177/179 cases (98%). Positive sentinel lymph node was identified in 26/177 patients (14%). In node positive patients the thickness of the primary tumour was significantly greater than that of node negative ones (p<0.0000). Patients with micrometastases had significantly poorer symptom-free and overall survival by the Mantel-Cox test than those of the other group (p=0.0001 and p=0.0007, respectively). In the discriminance analysis of our data, the discriminant function established from tumour thickness yielded 81.7% and the positivity of sentinel lymph nodes 79.9% correct classification rates. CONCLUSION: In good harmony with literature data, positive sentinel lymph node(s) were found in the case of thicker tumours. The involvement of sentinel lymph node indicated a significantly poorer prognosis.

[Angiosarcomas of the chest wall and breast after radiotherapy: The questions of radiogenic origin, diagnosis and treatment] / Mellkasfali és emlô angiosarcoma sugárkezelés után: a radiogén eredet, a diagnózis és a kezelés kérdései.

PURPOSE: To present medical history of secondary chest wall and breast angiosarcomas (AS) developed after radiotherapy, and to discuss the questions of radiogenic origin, diagnosis and treatment by the review of the literature. METHODS: Report of two cases and MEDLINE search for relevant publications. RESULTS: Secondary AS occured in a previously irradiated field after a long (6 and 8 years) latency period in both cases. Detailed histopathological and immunohistochemical examinations from the biopsy and/or surgical specimens confirmed the diagnosis as AS. The first patient with moderately differentiated AS was treated successfully with radical surgery. The second patient with irresecable AS died of rapid local progression within 4 months. The incidence of chest wall and breast AS is increased after irradiation, however, controversial data exist in the literature. The incidence of chest wall and breast AS after radiotherapy was found to be 0.39% in our patient population, which means an estimated odds ratio of 2.4 for secondary AS. Stewart-Treves syndrome is not of radiogenic origin, since postoperative lymphoedema has been considered as primary etiological factor. CONCLUSIONS: Patients treated with surgery and/or radiotherapy for primary breast cancer are at higher risk for developing secondary AS, compared to the healthy population. An etiological relationship between radiotherapy and subsequent AS of chest wall and breast is likely, but still controversial. Initial radical surgery is the only effective treatment for achieving long term survival. Further adjuvant radiotherapy is no longer feasible, due to the previous irradiation. Chemotherapy has only palliative effect. These very rare cases deserve special attention due to the atypical clinical appearance, difficulties of differential diagnosis and poor prognosis.

[Preoperative lymphoscintigraphy guided sentinel lymph node biopsy in malignant melanoma] / Preoperatív limfoszcintigráfiával végrehajtott sentinel nyirokcsomó mûtétek melanoma malignumban.

The authors present preliminary experience with preoperative sentinel lymph node biopsy carried out with lymphoscintigraphy in patients with malignant melanoma. PATIENTS AND METHODS: In the present study patients operated for primary cutaneous malignant melanoma of moderate and high severity were included. On the day of surgery isotope labelled colloid was injected intradermally around the tumor to indicate the lymphatics and to obtain basic information about the localization of the sentinel lymph node(s).During surgery the lymph node(s) previously visualized by the injection of patent-blue staining were detected with the aid of a gamma probe. Simultaneously, the excision of the primary tumor was extended. Histologically verified metastasis in the surgically removed lymph node(s) necessitated block dissection possibly within two weeks. RESULTS: The distribution of patients (19) according to tumor localisation: 2 - upper extremities; 9 - lower extremities; 2 - sacral region; 6 - trunk. Tumor thickness ranged from <1.5 mm (6 patients) to 1.5-3 mm (5 patients) and to >3 mm (8 patients). In two cases the identification of the lymph node has failed. Positive sentinel ymph nodes were detected in two patients. It is noteworthy that with one patient the sentinel lymph node was not regional but intransit. This study was aimed at the development of a suitable method. Further on we wish to try it in prospective randomized studies.