Heterogeneous expression of immunotherapy candidate proteins gp100, MART-1, and tyrosinase in human melanoma cell lines and in human melanocytic lesions

In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predictors of the success rate of such a treatment is the extent of positive (target) tumor cells within the lesions of the patient. First, we investigated the presence of these three proteins in 18 human melanoma cell lines using RT-PCR and immunohistochemistry. In 11 cell lines, mRNA and protein of all three markers could be detected; in one cell line, only two markers were present, and six melanoma cell lines showed no evidence for these markers. Secondly, we stained frozen sections of 105 human melanocytic lesions, 13 common nevocellular nevi, 13 atypical nevi, 13 early primary melanomas (Breslow < 1.5 mm), 25 advanced primary melanomas (aPM; Breslow > or =1.5 mm), and 41 melanoma metastases (MM) with antibodies against glycoprotein 100, melanoma antigen recognized by T cells, and tyrosinase. In addition, we used the 3,4-dihydroxy-L-phenylalanine reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results in a subset of the lesions. In the benign lesions, glycoprotein 100 was more prominently expressed in epidermal melanocytes, whereas melanoma antigen recognized by T cells was encountered in all or nearly all dermal melanocytes in all nevocellular nevi and atypical nevus lesions. Tyrosinase was found in a lower percentage of melanocytes, both in the epidermis and in the dermis within these lesions. With regard to heterogeneity of staining within the malignant lesions, we found that 54% (early primary melanomas), 48% (aPMs), and 56% (MM) of the lesions stained within the same staining category for all three proteins studied. Approximately 17% of the aPM and MM lesions did not show positive tumor cells for any of the three proteins. We conclude that a subgroup of patients with high expression should be selected for immunotherapeutic treatment approaches based on the presence of these proteins.     

Expression of two morphologic parameters concerning tumor-stroma interaction in benign and malignant melanocytic skin lesions

BACKGROUND: The 13C-urea breath (13C-UBT) test value is (semi-)quantitatively related to Helicobacter pylori density in the gastric antrum, and the value correlates with the grade of gastritis. The aim of this study was to assess variation of the 13C-urea breath test value by sociodemographic factors in H. pylori-positive children. METHODS: The analysis was performed on 127 asymptomatic children (aged 5-7 years) who were identified as H. pylori-positive with the 13C-UBT test in a large population-based epidemiologic study in the city of Ulm (southern Germany). The parents of the children were asked to fill out a standardized questionnaire about sociodemographic data. RESULTS: Forty-two infected children (33.1%) were of German nationality, 47 children (37.0%) were of Turkish and 38 children (29.9%) were of another nationality. Turkish children had a significantly higher 13C-UBT value (geometric mean = 27.2%) than German children (16.7%) or children with another nationality (19.3%) (P < 0.001). Girls had a trend towards higher values than boys (P = 0.058 after adjustment for nationality). Body mass index, education of the parents, and prior use of antibiotics were unrelated to the extent of the 13C-UBT. CONCLUSIONS: This study identified significant variation in the extent of the 13C value by nationality among H. pylori-infected children. Further studies are needed to elucidate the causes and potential consequences of these variations.     

Effect of UVB 311 nm irradiation on normal human skin

OBJECTIVE: To determine the relative efficacy and morbidity of Ho:YAG versus Nd:YAG laser treatment of bullous lung disease in an animal model. SUMMARY BACKGROUND DATA: Laser coagulation procedures for treatment of emphysematous pulmonary bullae and heterogeneous emphysema continue to evolve. The role of lasers in lung volume reduction surgery remains controversial due to issues of relative efficacy and morbidity. The Nd:YAG laser is most commonly used for these procedures. We hypothesized that the shallower penetration of the Ho:YAG laser may be better suited for laser bullae coagulation and emphysema lung volume reduction with increased efficacy and reduced lung injury. METHODS: Thirty New Zealand White rabbits (15 normal rabbits; 15 with bullous lung disease) were evaluated with Ho:YAG compared to Nd:YAG laser exposures. Bullae were coagulated by either Ho:YAG or Nd:YAG treatment. In all animals (bullous-induced and normals), unaffected lung tissue in the upper lobes and contralateral lungs were treated with 5 spot exposures of Nd:YAG and Ho:YAG, each to assess depth of lung injury. Animals were sacrificed at Days 0, 7, and 21 and their lungs were examined histologically. RESULTS: Ho:YAG and Nd:YAG exposures caused equivalent lung injury to normal lung tissue. In the acute phase, parenchymal necrosis depth was similar for both Ho:YAG and Nd:YAG (850 +/- 273 microns vs. 900 +/- 270 microns respectively, p = 0.7). By Day 7, lung necrosis depth was 925 +/- 133 microns Ho:YAG vs. 1225 +/- 235 microns Nd:YAG (p = 0.33), and lung fibrosis depth was 300 +/- 134 microns Ho:YAG vs. 558 +/- 127 microns Nd:YAG (p = 0.11). By Day 21, pulmonary parenchymal necrosis was not seen. Pleural fibrosis depth was maximal at Day 21, reaching 250 +/- 102 microns for Ho:YAG vs. 300 +/- 156 microns Nd:YAG (P = 0.88). Pleural necrosis depth was 67 +/- 42 microns Ho:YAG vs 48 +/- 34 microns Nd:YAG (p = 0.42) on Day 7 and resolved by Day 21. During surgical coagulation procedures, the Ho:YAG laser was dramatically more efficient in coagulating bullae. The Ho:YAG laser required less exposure at equivalent power and resulted in immediate desiccation of bullae, in sharp contrast to the Nd:YAG laser. CONCLUSIONS: Because the Ho:YAG was more effective and did not result in more acute lung injury than the standard Nd:YAG laser in this study, Ho:YAG lasers may have improved potential for laser treatment of bullae or lung volume reduction surgery (LVRS) compared to Nd:YAG lasers.     

Adhesion molecule expression in normal skin and melanocytic lesions. Role of UV-irradiation and architectural characteristics in nevi

Cell adhesion between surfaces of cells and to extracellular matrices represents a fundamental mechanism in tissue organization and influences the biological behaviour and the architecture of tumors. We investigated the expression of various adhesion molecules in normal skin (n=5), nevi (n=29), and malignant melanoma (n=10) by immunohistochemistry. Special attention was paid to the correlation between adhesion molecule expression and the respective architectural features, e.g. UV-induced morphological changes, and the arrangement of melanocytes in congenital nevi. In nevi, a single erythemagenic dose of UV-light did not influence the integrin expression of melanocytes, but results in an upregulation of alpha3 beta1- and alpha6 beta1-integrin within the suprabasal layers of the epidermis. This suprabasal labelling was associated with an increased number of suprabasal melanocytes in UV-irradiated nevi which were detected with HMB-45 antibody. Nine of 10 congenital nevi demonstrated a labelling of alpha4 beta1-integrin only in melanocytes of the deeper dermis. This integrin previously has been associated with high tumor thickness and the clinical outcome in melanomas. The integrin profile observed in melanomas differed in part from that seen in nevi with expression of beta2- and beta3-integrins in some cases. The results may indicate a correlation between adhesion molecule expression and histopathological findings in melanocytic lesions.     

Mitotic cyclins and cyclin-dependent kinases in melanocytic lesions

Recent evidence has implicated cyclins and cyclin-dependent kinases in the evolution and progression of various malignancies. We studied the immunohistochemical expression of cyclin A, cyclin B, and cyclin-dependent kinase p34cdc2 in a broad spectrum of benign and malignant melanocytic lesions. Formalin-embedded, parrafin-fixed tissue sections from 66 malignant melanomas (MM) and 60 benign nevi were examined for the expression of these cell-cycle proteins. The results were compared with the standard proliferative marker Ki-67 and mitotic index. MM showed significantly higher immunoreactivity for cyclin A, cyclin B, p34cdc2, and Ki-67 compared with benign nevi. Cyclin A, p34cdc2, and Ki-67 displayed strong co-expression in MM. Overexpression of cyclin A and p34cdc2 correlated with histological type, mitotic activity, Ki-67 index, tumor thickness, Clark's level, and clinical outcome in MM. In invasive MM, increased immunostaining of cyclin A and Ki-67 were associated with decreased patient survival. These findings indicate potential roles of mitotic cyclins and cyclin-dependent kinases in the pathogenesis and progression of malignant melanoma.     

Nevus of Ota and leptomeningeal melanocytic lesions

We quantitated glomerular and cortical interstitial structures in nine type I mesangiocapillary glomerulonephritis (MCGN) patients aged 6 to 20 years whose creatinine clearance (CCr) was 10 to 129 ml/min/1.73 m,2 as compared to age-matched normal controls. Mean glomerular volume and mesangial volume fraction [Vv(mes/glom)] were increased and the percentage of the capillary endothelial circumference which was defined as filtration surface was decreased in type I MCGN patients. Vv(mes/glom) was inversely related to filtration surface area per glomerulus (r = -0.73, P < 0.05) and directly to volume density of cortical interstitium [Vv(int/cortex)] (r = +0.90; P < 0.01). Vv(mes/glom) (r = -0.87; P < 0.01), filtration surface area per glomerulus (r = +0.83; P < 0.01) and Vv(int/cortex) (r = -0.86; P < 0.01) were correlated with CCr. Thus, in type I MCGN, measures both of glomerular and of cortical interstitial structure are highly correlated with glomerular function.     

UVB radiation preferentially induces recruitment of memory CD4+ T cells in normal human skin: long-term effect after a single exposure

Acute, low-doses of ultraviolet (UV)-B radiation affect the immune competent cells of the skin immune system. In this study, we examined the time-dependent changes of the cutaneous T cell population in normal human volunteers following a single local exposure to UV. Solar-simulated UV radiation caused an initial decrease in intraepidermal T cell numbers, even leading to T cell depletion at day 4, whereupon a considerable infiltration of T cells in the epidermis occurred that peaked at day 14. In the dermis the number of T cells was markedly increased at days 2 (peak) and 4 after irradiation, and subsequently declined to the nonirradiated control values at day 10. Double-staining with several T cell markers showed that the T cells, infiltrating the (epi)dermis upon UV exposure, were almost exclusively CD4+ CD45RO+ T cells, expressing an alpha/beta type T cell receptor, but lacking the activation markers HLA-DR, VLA-1, and IL-2R. Application of UVB radiation resulted in similar dynamics of T cells, indicating that the UVB wavelengths within the solar-simulated UV radiation were responsible for the selective influx of CD4+ T cells. In conjunction with UVB-induced alterations in the type and function of antigen-presenting cells (i.e., Langerhans cells and macrophages), the changes of the cutaneous T cell population may also contribute to UVB-induced immunosuppression at skin level in man.     

Diagnosing and treating congenital melanocytic nevus simulating malignant melanoma

Many cases reported as malignant melanomas arising in benign congenital melanocytic nevi in the neonatal period have not shown evidence of metastases after several years of follow-up. These lesions were probably pathologically misdiagnosed, thus creating a controversy regarding the precise incidence. This article describes the case of an infant with a giant melanocytic nevus simulating malignant melanoma to illustrate the proper criteria for diagnosis of this condition so extensive and unnecessary therapy procedures can be avoided.     

Interobserver variability on the histopathologic diagnosis of cutaneous melanoma and other pigmented skin lesions

PURPOSE: To assess the interobserver agreement on the diagnosis and classification of cutaneous melanoma. MATERIALS AND METHODS: A set of 140 slides of cutaneous melanoma, including a small subset of benign pigmented skin lesions, were circulated to four experienced histopathologists. The kappa statistic for multiple ratings per subject was calculated using the method described by Fleiss. RESULTS: The kappa value on the diagnosis of cutaneous melanoma versus benign lesions was 0.61. There was some discordance on the diagnosis in 37 of 140 cases (26%). For the histopathologic classification of cutaneous melanoma, the highest kappa values were attained for Breslow thickness (kappa = 0.76) and presence of ulceration (kappa = 0.87). The agreement was generally poor for other histologic features, such as level of dermal invasion (kappa = 0.38), presence of regression (kappa = 0.27), and lymphocytic infiltration (kappa = 0.27). CONCLUSION: Our study suggests considerable disagreement among pathologists on the diagnosis of melanoma versus other pigmented lesions. Tumor thickness and presence of ulceration are the most reproducible histologic features of cutaneous melanoma.     

Ultraviolet B-induced squamous epithelial and melanocytic cell changes in a xenograft model of cancer development in human skin

The presence of bone morphogenetic proteins (BMPs) in 13 primary bone neoplasms was investigated with conventional bioassay method and immunohistochemically using antiserum against highly purified mixture of bovine BMPs as antibody. In conventional bioassay after implantation of lyophilized bone tumor tissues into mouse muscle pouches 9/13 samples turned out positive by radiography and 10/13 histologically. By immunohistochemical staining, using the avidin-biotin-peroxidase method, signs of BMPs could be verified in all cases investigated. Microscopical scoring showed the local concentration of BMPs to be especially high in sections from giant cell tumors when compared to other bone neoplasms.     

Risk of melanoma in medium-sized congenital melanocytic nevi: a follow-up study

BACKGROUND: The risk of the occurrence of malignant melanoma (MM) in medium-sized (1.5 to 19.9 cm in diameter) congenital melanocytic nevi (CMN) is the subject of controversy. Universally accepted recommendations regarding the management of such lesions have not been made. OBJECTIVE: Our purpose was to assess the risk of MM arising in medium-sized CMN. METHODS: The study included 230 medium-sized CMN in 227 patients, first seen in a private dermatology practice from 1955 to 1996, who were followed up for MM arising within their CMNs. Criteria for entry into the study included (1) a clinically diagnosed medium-sized CMN, (2) minimum follow-up period of 1 year, and (3) a photograph of the lesion in the patient's medical record. RESULTS: No MM occurred in a medium-sized CMN during an average follow-up of 6.7 years (median, 5.8 years) to an average age of 25.5 years (median, 19.1 years). CONCLUSION: The results of this short-term follow-up study do not support the view that there is a clinically significantly increased risk for MM arising in banal-appearing medium-sized CMN or that prophylactic excision of all such lesions is mandatory. Lifelong medical observation seems a reasonable alternative for many medium-sized CMN.     

The ABCD rule in dermatoscopy: analysis of 500 melanocytic lesions

500 melanocytic lesions were examined by dermatoscopy using the ABCD rule prior to excision and histologic diagnosis. Regular nevi (n = 272) exhibited a score of 3.55 +/- 0.87, nevi with histologic signs of dysplasia (n = 190) a score of 4.0 +/- 0.68 and melanomas (n = 30) a score of 5.08 +/- 1.24. This study suggests that the ABCD rule of dermatoscopy greatly facilitates the evaluation of melanocytic lesions. When the dermatoscopic score is higher than 4.2, melanoma should be considered.     

Effect of low dose UVB irradiation on the migratory properties and functional capacities of human skin dendritic cells

We recently described the 'spontaneous' migration of skin dendritic cells out of human split skin during culture. Since newly infiltrating cells from the circulation are excluded, this in vitro model is very suitable for studying the effect of UVB irradiation on the migratory properties, phenotype and functional capacities of skin cells. In the present study, we show that UVB irradiation of the skin before the culture period results in a significantly lower number of migrated cells that could be obtained compared with untreated skin. Relatively more dendritic cells of the population that migrated from UVB-irradiated skin were of dermal origin, as indicated by a higher percentage of CD1b+ cells. These data imply that UVB irradiation inhibits migration, especially of the epidermal Langerhans cells. Ultrastructural analysis of the irradiated skin revealed that the UVB dose used did not cause any directly visible damage to the cells. However, the cell population that had migrated from UVB-irradiated skin showed a significantly lower capacity to stimulate allogeneic T cells. This was not due to a lower expression of MHC class II on these cells. The percentage of cells expressing B7.1, B7.2 and LFA-3 was decreased in the population migrated from irradiated skin. The possible mechanism underlying the UVB-induced suppression is discussed.     

Simulants of malignant melanoma: a rogue's gallery of melanocytic and nonmelanocytic imposters

Although overdiagnosis of malignant melanoma involves no potential loss of life, it affects the patient profoundly and therefore should be addressed conscientiously. This review of both benign and malignant, melanocytic and nonmelanocytic simulants of malignant melanoma emphasizes the pitfalls and repercussions of misdiagnosis. For more accurate differentiation, simulants are classified first as superficial (intraepidermal) or deep (dermal), then as melanocytic or nonmelanocytic.     

Methacholine induces wheal-and-flare reactions in human skin but does not release histamine in vivo as assessed by the skin microdialysis technique

A number of investigations have indicated that cholinergic agonists release histamine from isolated mast cells and suggested that cholinergic stimulation releases histamine in vivo. The purpose of this study was to investigate whether the cutaneous wheal-and-flare reaction induced by methacholine challenge in human skin involves histamine release as measured by the skin microdialysis technique. Five hollow dialysis fibers were inserted intradermally in forearm skin in eight healthy subjects. Each fiber was perfused with Kreb's-Ringer bicarbonate at a rate of 3 microliters/min. Dialysates were collected in 2-min fractions before skin challenge and for 20 min after intradermal injection of methacholine 10(-3)-10(-1) M, the vehicle, and a positive control, codeine phosphate 0.3 mg/ml. Histamine was assayed spectrofluorometrically. Methacholine caused a statistically significant dose-related wheal-and-flare reaction, the flare reaction to methacholine 10(-1) M being comparable with that seen with codeine 0.3 mg/ml. No significant histamine release was observed with methacholine, cumulative histamine release of 16 +/- 8 nM by methacholine 10(-1) M being similar to vehicle responses of 15 +/- 9 nM. Histamine release by codeine was 2524 +/- 435 nM. In conclusion, methacholine-induced wheal-and-flare reactions in human skin appeared not to involve histamine release from skin mast cells.     

Naevi spili, Café-au-lait spots and melanocytic naevi aggregated alongside Blaschko''s lines, with a review of segmental melanocytic lesions

Lipofibromatous hamartoma of the nerve is a very uncommon congenital tumor. An association between this condition and vascular malformations is not well known. We present an 11-year-old girl with a lipofibromatous hamartoma of the right median nerve with macrodactyly. She had small red macules on her right neck, chest, and arm, which were diagnosed clinically as port-wine stains. The specimens of the enlarged nerves showed fibrous and fatty growth surrounding the nerve bundles and proliferation of the small veins. We suggest that this disorder can be accompanied by a vascular malformation.