High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV‐type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho‐S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV‐related XP nevi and melanomas were different from nevi and melanomas in the general population.     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.     

Lack of somatic alterations of MC1R in primary melanoma

Germline variation of the melanocortin 1 receptor gene (MC1R) is a risk factor for cutaneous melanoma. Recent studies have indicated that the risk is significantly higher for melanomas with somatic BRAF mutations, suggesting that MC1R variants may have a more specific role than their demonstrated effects on skin and hair pigmentation. To address the possibility that MC1R may act like a tumor suppressor gene by creating a permissive condition for melanocytes with specific somatic mutations to proliferate or survive, we analyzed 103 primary melanomas for somatic MC1R mutations and copy number alterations. This cohort included melanomas from skin with and without chronic sun-induced damage, mucosal membranes, and acral skin (palms, soles, and subungual). We did not find somatic mutations or frequent DNA copy number alterations at the MC1R locus, nor any skewed pattern of copy number alterations that would favor one allele type over the other. In conclusion, our findings indicate that MC1R is not a frequent target of somatic alterations in melanoma.     

The dysplastic nevus: recognition and management

The recognition of atypical or dysplastic nevomelanocytic nevi potentially provides clinicians with another means of identifying individuals at increased risk for cutaneous malignant melanoma. However, a great deal of controversy still surrounds these lesions, their significance, and the clinical and histologic criteria needed for their diagnosis at present. In general, dysplastic nevi tend to be asymmetrical and larger (greater than 5 mm) than ordinary acquired nevi, have a macular component, irregular and ill-defined borders, and haphazard (variegate) coloration. A clinical diagnosis of dysplastic nevi must be confirmed by histopathology, since not all clinically atypical nevi are dysplastic. While precise histopathologic criteria for dysplastic nevi are lacking, most authorities agree that an abnormal nevomelanocytic proliferative pattern as manifested by increased numbers of basilar melanocytes and/or abnormal junctional nevomelanocytic nesting in the setting of lentiginous epidermal hyperplasia, variable degrees of nevomelanocytic nuclear atypia, and a lymphocytic host response are consistent with a histologic diagnosis of dysplastic nevi. Current data for individuals with dysplastic nevi and a family history of cutaneous malignant melanoma (at least two family members with cutaneous malignant melanoma) indicate a relative risk for cutaneous malignant melanoma about 148 times that of the general population. In comparison, cutaneous malignant melanoma risk seems lower for individuals with familial dysplastic nevi (but without familial cutaneous malignant melanoma) and "sporadic" dysplastic nevi. With respect to progression to melanoma, probably the vast majority of dysplastic nevi remain stable or possibly regress. Management of individuals with histologically confirmed dysplastic nevi involves periodic skin examinations. Regional overview and life-size photography are helpful in following these patients. Patients should also be instructed in the examination of their own skin. While a definite relationship between sun exposure and dysplastic nevi remains unproved, the use of sunscreens and avoidance of unnecessary sun exposure are advised. Examination of family members for atypical melanocytic lesions is also recommended.     

Learning Reflectance Confocal Microscopy of Melanocytic Skin Lesions through Histopathologic Transversal Sections

Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM) features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane). The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods) with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features. Four melanomas and 2 nevi with different dermoscopic clues have been studied. Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections. We presented the possibility to perform transverse sections as a new approach to understand RCM features. Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections. Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion. Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.     

Apaf-1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf-1, an important mediator of mitochondrial-dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf-1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf-1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf-1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf-1 mRNA by fluorescence in situ hybridization and of Apaf-1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf-1. A significant decrease in Apaf-1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf-1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf-1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf-1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf-1 may be an important contributory factor in the development of the disease.     

Malignant transformation of melanocytes to melanoma by constitutive activation of mitogen-activated protein kinase kinase (MAPKK) signaling

Malignant melanoma is the cancer with the most rapid increase in incidence in the United States. Ultraviolet light and deficiency of the p16ink4a gene are known factors that predispose one to the development of malignant melanoma. The signal transduction pathways that underlie the progression of melanoma from their precursors, atypical nevi, are not well understood. We examined activation of the MAP kinase pathway in atypical nevi and melanoma cells and found that this pathway is activated in melanomas. To determine the functional significance of this activation, we introduced constitutively active MAP kinase kinase (MAPKK) into immortalized melanocytes. The introduction of this gene into melanocytes leads to tumorigenesis in nude mice, activation of the angiogenic switch, and increased production of the proangiogenic factor, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Activation of MAP kinase signaling may be an important pathway involved in melanoma transformation. Inhibition of MAP kinase signaling may be useful in the prevention and treatment of melanoma.     

Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions,adhesive associations,and dispersed desmoglein 2 glycoproteins

In the tissue integration of melanocytes and melanoma cells, an important role is attributed to cell adhesion molecules, notably the cadherins. In cultured melanoma cells, we have previously described a more heterogeneous repertoire of cadherins than normal, including some melanoma subtypes synthesizing the desmosomal cadherin, desmoglein 2, out of the desmosomal context. Using biochemical and immunological characterization of junctional molecules, confocal laser scanning, and electron and immunoelectron microscopy, we now demonstrate homo- and heterotypic cell-cell adhesions of normal epidermal melanocytes. In human epidermis, both in situ and in cell culture, melanocytes and keratinocytes are connected by closely aligned membranes that are interspersed by small puncta adhaerentia containing heterotypic complexes of E- and P-cadherin. Moreover, melanocytes growing in culture often begin to synthesize desmoglein 2, which is dispersed over extended areas of intimate adhesive cell-cell associations. As desmoglein 2 is not found in melanocytes in situ, we hypothesize that its synthesis is correlated with cell proliferation. Indeed, in tissue microarrays, desmoglein 2 has been demonstrated in a sizable subset of nevi and primary melanomas. The biological meanings of these cell-cell adhesion molecule arrangements, the possible diagnostic and prognostic significance of these findings, and the implications of the heterogeneity types of melanomas are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported in parts by grants from the Deutsche Forschungsgemeinschaft to W. K. Peitsch (project PE 896/1) and the Deutsche Krebshilfe to W. W. Franke (project 10-2049).     

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.     

The cell biology of human hair follicle pigmentation

Although we have made significant progress in understanding the regulation of the UVR‐exposed epidermal‐melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR‐shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis‐follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub‐populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species‐specific issues involved which the general reader of the pigmentation literature (with its substantial mouse‐based data) may not fully appreciate.     

Immunological aspects of benign melanocytic tumours and melanoma in situ

Summary Extracts were prepared from 15 malignant melanomas, 10 naevi, 8 pieces of skin containing atypical melanocytes and 4 samples of normal skin. Peripheral blood leucocytes from 52 patients with benign naevi that had altered sufficiently to cause the patient to seek attention, 57 patients with malignant melanomas, and 98 normal individuals were tested against these extracts in a capillary leucocyte migration test. Melanoma extracts preferentially inhibited the leucocytes of melanoma patients and to a lesser extent those of naevus patients. Melanoma patients' leucocytes were more frequently inhibited by naevus extracts (whether from intradermal or from compound naevi) than were the leucocytes of naevus patients and control donors. Extracts of skin containing atypical melanocytes (perimelanomatous skin and lentigenous skin) selectively inhibited melanoma patients' leucocytes.     

Localization of integrin alpha(v)beta3 and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in cutaneous and oral melanomas of dog

Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.     

Multiple primary melanoma: epidemiological and prognostic implications; analysis of 36 cases

Patients who are already diagnosed with cutaneous melanoma are at increased risk of developing another primary melanoma. The occurrence of multiple primary melanoma is a rare phenomenon, varying in frequency, with an estimated incidence ranging from 0.2% to 8.6%. The authors are presenting data on the patients with multiple primary melanoma from the Croatian Referral Melanoma Centre. The clinical, histological and epidemiological characteristics of 36 (3.6%) patients, identified from 991 patients with histologically confirmed melanoma, are analyzed in this study. Twenty-eight of the patients (78%) had two primary melanomas, six had three melanomas (16.7%) and two (5,6%) had four melanomas. Diagnosis was established synchronously in 11 patients (30%) and, in the rest of the patients, time interval between the diagnosis of the first and second melanoma varied from 1 month to the longest interval of 16 years. However, the majority of subsequent melanomas were removed within 2 years of the initial operation. The mean Breslow's thickness of the first melanoma was significantly higher than the mean Breslow's thickness of the second primary melanoma. The proportion of in situ to invasive melanomas was greater for the second melanomas compared with the first melanomas. Therefore, we emphasize the importance of regular follow-up as well as the education in regular self--skin examinations in melanoma patients in order to detect subsequent primary melanomas in the early phase.     

The Study of Cutaneous Melanomas in Camargue‐Type Gray‐Skinned Horses (2): Epidemiological Survey

An epidemiological survey was made on cutaneous melanomas occurring in Camargue‐type, gray‐skinned horses in southern France. The population investigated was composed of 264 horses, which were selected from the Camargue horse stud registry and were allowed to be examined by the owners. The presence of tumors was inspected macroscopically according to the standardized protocols of veterinary medicine specialists, and some tumors were subjected to macroscopic histopathological examination. The results indicated that: 1) the prevalence of melanomas in the overall population was 31.4%; 2) the odds ratio was obtained for an age class of 5–9 years, in which most melanomas were presumed to occur; 3) the incidence of melanomas was significantly correlated with age, giving a prevalence of 67% at ages> 15 years; 4) the size and number of tumorous lesions were significantly related to age; 5) little correlation was observed between melanoma incidence and gender; and 6) the most frequently occurring body site of these tumors was underneath the tail. Because of their frequent occurrence in shaded body regions, sun exposure was not likely to be a risk factor for melanoma formation in these horses. In view of the slightly darker skin pigmentation in these horses at younger ages, the unique characteristics of their melanocytes may be associated with melanoma formation.     

A receptor-type protein tyrosine phosphatase PTP zeta is expressed in human cutaneous melanomas

We describe the expression of a receptor-type protein tyrosine phosphatase PTP zeta (or RPTP beta) in human cutaneous melanomas as detected by means of immunohistochemistry. The expression of PTP zeta has been described to be restricted to the central nervous system. In developing mice brain high levels of PTP zeta have been detected indicating its developmental importance; PTP zeta is also expressed in glioblastoma and neuroblastoma. By the use of immunohistochemistry we detected PTP zeta in human primary and metastatic melanomas. The melanocytes of healthy skin remained negative. Due to the developmental origin of the melanocytes from neural crest, this represents a further example for transformed cells switching back to express molecules related to their ontogenetic history. These promising initial results have to be verified in larger scaled studies; the inclusion of nevi will be necessary to further elucidate the role of PTP zeta in melanocyte transformation and melanoma development.     

Purification of human melanocytes by monoclonal antibody combined with Percoll gradients

Cultures of human melanocytes obtained by differential plating of human epidermal cells in 12-myristate 13-acetate (PMA) were purified using a monoclonal antibody R₂₄ which detects a restricted glycolipid antigen present on melanoma cells and melanocytes. Melanocytes were rosetted using protein A-conjugated human red blood cells and separated from non-rosetted ftbroblasts on discontinuous Percoll^™ gradients. 100% pure cultures of melanocytes obtained in this fashion were then successfully grown in tissue culture in the presence of PMA and cholera toxin for at least thirty passages (corresponding to approx. sixty cell doublings). We conclude that:

1. 1. Pure cultures of melanocytes are a prerequisite for the establishment of long-term cultures.

2. 2. Since most human melanomas express substantial levels of R₂₁ antigen, this method can be applied to purification of melanomas and can be easily adapted to separation of subpopulations of melanocytes and melanoma cells recognized by specific monoclonal antibodies.

     

Melanoma,Growth Factors,and Cutaneous Paraneoplastic Syndromes

Paraneoplastic syndromes are systemic reactions in patients with cancers that are unrelated to tumor size or location. Cutaneous paraneoplastic syndromes include proliferative, metabolic, and inflammatory skin disorders. Both systemic and cutaneous paraneoplastic reactions may occur in patients with malignant melanoma. Cancers, including melanoma, may produce growth factors, which may be responsible for proliferative cutaneous paraneoplastic syndromes. A patient with malignant melanoma we previously reported, who had the sudden onset of acanthosis nigricans, skin tags (acrochordons), and seborrheic keratoses provides a model for proliferative cutaneous paraneoplastic syndromes. High levels of α-TGF were found in the patient's urine prior to melanoma removal. The increased level of α-TGF declined after the melanoma was removed, and a corresponding clinical improvement in his acanthosis nigricans, skin tags, and seborrheic keratoses occurred. In the skin lesions, EGF receptors were abnormally present throughout all epidermal layers prior to melanoma removal, and returned to their normal distribution in the basal layers after surgery. Ectopic growth factor production by malignant melanomas and other epithelial neoplasms may cause rare, but distinctive cutaneous paraneoplastic lesions. The model of melanoma, cutaneous paraneoplastic syndromes, and growth factors may provide understanding of both cutaneous lesions associated with neoplasia, and benign cutaneous lesions.     

BRAF mutations in cutaneous melanoma are independently associated with age, anatomic site of the primary tumor, and the degree of solar elastosis at the primary tumor site

Oncogenic BRAF mutations are more frequent in cutaneous melanoma occurring at sites with little or moderate sun-induced damage than at sites with severe cumulative solar ultraviolet (UV) damage. We studied cutaneous melanomas from geographic regions with different levels of ambient UV radiation to delineate the relative effects of cumulative UV damage, age, and anatomic site on the frequency of BRAF mutations. We show that BRAF-mutated melanomas occur in a younger age group on skin without marked solar elastosis and less frequently affect the head and neck area, compared to melanomas without BRAF mutations. The findings indicate that BRAF-mutated melanomas arise early in life at low cumulative UV doses, whereas melanomas without BRAF mutations require accumulation of high UV doses over time. The effect of anatomic site on the mutation spectrum further suggests regional differences among cutaneous melanocytes.     

Vesicular Stomatitis Virus Variants Selectively Infect and Kill Human Melanomas but Not Normal Melanocytes

Metastatic malignant melanoma remains one of the most therapeutically challenging forms of cancer. Here we test replication-competent vesicular stomatitis viruses (VSV) on 19 primary human melanoma samples and compare these infections with those of normal human melanocyte control cells. Even at a low viral concentration, we found a strong susceptibility to viral oncolysis in over 70% of melanomas. In contrast, melanocytes displayed strong resistance to virus infection and showed complete protection by interferon. Several recombinant VSVs were compared, and all infected and killed most melanomas with differences in the time course with increasing rates of melanoma infection, as follows: VSV-CT9-M51 < VSV-M51 < VSV-G/GFP < VSV-rp30. VSV-rp30 sequencing revealed 2 nonsynonymous mutations at codon positions P126 and L223, both of which appear to be required for the enhanced phenotype. VSV-rp30 showed effective targeting and infection of multiple subcutaneous and intracranial melanoma xenografts in SCID mice after tail vein virus application. Sequence analysis of mutations in the melanomas used revealed that BRAF but not NRAS gene mutation status was predictive for enhanced susceptibility to infection. In mouse melanoma models with specific induced gene mutations including mutations of the Braf, Pten, and Cdkn2a genes, viral infection correlated with the extent of malignant transformation. Similar to human melanocytes, mouse melanocytes resisted VSV-rp30 infection. This study confirms the general susceptibility of the majority of human melanoma types for VSV-mediated oncolysis.