Markers of melanocytic tumour progression

Melanoma progression markers can be defined as molecules with a preferential expression in one or a few stages of melanocytic tumour development. These molecules include growth factors, growth factors receptors, adhesion molecules, proteases and related components. Immunohistochemical studies suggest that some of these molecules are useful as prognostic markers in melanoma patients. In cutaneous melanocytic lesions, the distribution of E-cadherin, a member of a family of cell adhesion molecules that mediate cell-cell interactions by means of Ca2+ dependent, homophilic interactions, appears to be complex. Although a decrease of E-cadherin would be expected with invasive tumour growth in advanced primary melanoma and eventually in metastasis, surprisingly an increase is found, whereas alpha- and beta-catenin, (cytoplasmic) molecules functionally associated with E-cadherin, are detected in all benign and malignant lesions. A possible interpretation includes a difference in the morphogenesis and function of melanocytic cells, compared with epithelial cells. Further research is needed to clarify the role of E-cadherin/catenin during melanoma progression.     

Immune-phenotypical markers for the differential diagnosis of melanocytic lesions

For specific subsets of melanocytic proliferations, there are morphologic limitations in the histological diagnosis, especially for borderline melanocytic tumors. In particular, Spitzoid proliferations can be difficult to diagnose. For these reasons, in the last years, clinic research has focusedattention on discovery of new diagnostic markers. Published gene expression and proteomic profiling data indicate new candidate molecules involved in melanoma pathogenesis, and useful in differential diagnosis of difficult melanocytic lesions. Recently, the diagnostic power of galectin-3 was demonstrated in series of melanocytic lesions, with a strong increasing of expression in malignant lesions compared with benign lesions. Similarly, the accumulation of Collagen XVII antibody was detected in vertical melanoma fronts and associated with invasive phenotype. Moreover, overexpression of cyclin D1 and p21 was detected in Spitz nevi compared with non-spitzoid melanomas; Ki-67 appears highly expressed in deep areas of non-spitzoid melanomas. In this review,werealizedan overviewofthe main molecular markersthat canbe a usefultoolfor the differential diagnosisofbenign, borderlineand malignant melanocytic lesions, related to their biological behavior, useful also for predicting the evolutionof the disease.     

Malignant melanoma with neuroendocrine differentiation: clinical, histological, immunohistochemical and ultrastructural features of three cases

AIM: To document the clinical, histological, immunohistochemical and ultrastructural features of three malignant melanomas showing neuroendocrine differentiation. METHODS AND RESULTS: Three patients, two with primary cutaneous melanoma and one with nasal mucosal melanoma, subsequently developing or simultaneously presenting with metastatic malignant melanoma, were studied by conventional histological technique, immunohistochemistry of formalin-fixed paraffin-wax embedded tissues, and electron microscopy of epoxy-resin-embedded tumour tissue. Tumours showed either small cell or conventional malignant melanoma cell morphology. One of the three primary melanocytic lesions (the nasal melanoma) exhibited neuroendocrine differentiation immunohistochemically. All three metastatic malignant melanomas showed, in varying combinations, immunohistochemical and ultrastructural evidence for neuroendocrine differentiation: they were positive for the melanocytic markers, S100 protein, HMB-45, Melan-A and tyrosinase, and the neuroendocrine markers chromogranin, synaptophysin and neurofilament protein. Ultrastructural study in two of the metastases revealed neuroendocrine granules but no lattice-bearing melanosomes. CONCLUSIONS: The cases described are the most comprehensively investigated malignant melanomas showing neuroendocrine differentiation to date, and the first to document neuroendocrine differentiation ultrastructurally in these tumours. Malignant melanoma with neuroendocrine differentiation therefore needs to be recognized among the other, better known variants of malignant melanoma.     

Primary de-differentiated,trans-differentiated and undifferentiated melanomas: overview of the clinicopathological,immunohistochemical and molecular spectrum

Primary cutaneous and mucosal melanoma shows a wide histological spectrum. The correct diagnosis depends upon the demonstration of melanocytic differentiation by recognition of an associated in-situ component or immunohistochemical evidence of a melanocytic phenotype using conventional melanocytic markers, such as S-100, SOX10, Melan-A and HMB-45. Exceptionally, melanomas lose their melanocytic phenotype, at least focally, and show differentiation towards other lineages. Review of the literature shows that de- and trans-differentiation in melanoma is rare but probably under-recognised and under-reported. These often large and frequently ulcerated tumours affect adults and show a wide anatomical distribution, including mucosal sites, although there is a predilection for sun-damaged skin of the head and neck. Histologically, the tumours are biphasic and contain a pre-existing conventional melanoma. The de-differentiated component closely resembles atypical fibroxanthoma, both morphologically and immunohistochemically. Trans-differentiated melanoma may show rhabdomyosarcomatous or spindle cell carcinomatous features. Undifferentiated melanomas are similar tumours in which the conventional melanoma component is absent. Their diagnosis depends entirely upon the clinical context and identification of a classical melanoma driver gene mutation, i.e. BRAF V600E. The diagnosis of these rare and unusual tumours is challenging, and requires thorough tumour sampling and recognition of the background of a pre-existing but often focal conventional melanoma together with molecular analysis.     

Histopathologic diagnosis of pigmented lesions of the skin

The present review deals with the histopathologic diagnosis of the commonest pigmented lesions of the skin with emphasis on melanocytic tumors. The discussed entities are systematized in pigmented lesions with and without an increase in the number of melanocytes, i.e. melanocytic hyperplasia, and in those that actually are melanocytic neoplasias. Histopathological, ultrastructural, and immunohistochemical features of relevance for the differential diagnosis are discussed in depth. One section is dedicated to the immunohistochemical markers of melanocytic lesions, the other one to the electron microscopy of malignant melanoma. Neoplasms simulating malignant melanoma and the less frequent variants of this tumor are also discussed. The diagnosis of pigmented lesions of the skin represents one of the most frequent and difficult challenges in dermatopathology, and useful clues for the differential diagnosis, especially of melanocytic dysplasias and neoplasias, are presented in the present article.     

Genetic factors in metastatic progression of cutaneous melanoma: the future role of circulating melanoma cells in prognosis and management

The greatest potential for improvement of outcome for patients with Cutaneous Malignant Melanoma lies in the prevention of systemic metastasis. Despite extensive investigation, current prognostic indicators either alone or in combination, although related to melanoma progression, are not sufficient to accurately predict the pattern of progression and outcome for any individual patient. Metastasis related death has been recorded in patients initially diagnosed with early stage tumour as well as in patients many years after initial tumour removal. The trouble finding a predictable pattern in the puzzle of melanoma progression may be linked to the fact that most of the material studied for prognosis is either, cutaneous primaries or metastatic deposits, rather than the melanoma cells in the circulatory system which are responsible for disease progression. In this review article we discuss the potential use of circulating tumour cell (CTC) detection and quantification for identifying patients at risk of metastatic deposits. We also discuss current therapies for the treatment of metastatic melanoma and analyse how CTCs may be used to evaluate the effectiveness of current therapies and to pinpoint patients who require further treatment.     

Atypical dermal nodules in benign melanocytic naevi

Aims : The prognosis of deeply invasive melanoma can be poor and to a large extent it is unresponsive to treatment once metastases have occurred. It is therefore important that any dermal melanocytic lesions that have some features suggestive of melanoma but are nevertheless benign, should be identified. Methods and results : A series of 40 benign melanocytic naevi is described in which the clinical presenting feature was a central focus of increased pigmentation. This was found histologically to correspond to dermal nodules of large melanocytes showing some, usually mild, nuclear atypia but low indices of cellular proliferation. The nodules are found within otherwise typical melanocytic naevi. The clinical and histological differential diagnosis included invasive melanoma but in follow-up, which is admittedly short (mean 24 months), none have recurred or metastasized. Conclusions : It is suggested that the nodules represent terminal differentiation of melanocytes rather than proliferative changes. They should be distinguished from melanoma and regarded as a variant of benign melanocytic naevi.     

An experience of malignant melanoma

The diagnosis of malignant melanoma involves its differentiation from many benign and malignant non-melanocytic tumours as well as from benign melanocytic lesions. Frequently knowledge of the clinical history and the site of the tumour is very valuable. The diagnostic value of many microscopic features is discussed.     

Reduced expression of p16 and p27 is correlated with tumour progression in cutaneous melanoma

AIMS: To determine if the cyclin dependent kinase inhibitors (CDKIs) p16 and p27 show reduced expression in the progression from benign to malignant melanocytic tumours, and to correlate these findings with patient prognosis. METHODS: Ninety-two melanocytic tumours were assessed for immunohistochemical expression of p16 and p27. These specimens included nine compound naevi, 10 dysplastic naevi, 17 thin (<1 mm) melanomas, 22 thick (>1 mm) melanomas, nine in-transit metastases, 13 lymph node metastases, and 12 soft tissue metastases. Clinicopathological information on the 39 patients with melanoma primaries was obtained from the Sydney Melanoma Unit database. The median follow up period was 43.3 months. RESULTS: A significant loss of expression of p16 and p27 was found with tumour progression. Positive expression of p27 was found in all compound and dysplastic naevi but only 43.6% of melanoma primaries. Expression of p27 was greater in lymph node and in-transit metastases (63.6%), but lower in soft tissue metastases (36.4%). Positive expression of nuclear p16 was evident in 73.7% of benign naevi, 28.2% of primary melanomas and 14.7% of metastatic melanomas. Neither p16 nor p27 expression was significantly correlated with overall survival, disease free survival or other clinicopathological markers. CONCLUSIONS: The CDKIs p16 and p27 are associated with tumour progression in melanoma, but do not reliably predict recurrence or survival.     

Quality assessment by expert opinion in melanoma pathology: experience of the Pathology Panel of the Dutch Melanoma Working Party

Some cutaneous melanocytic lesions are notoriously difficult to diagnose by histopathology. For that reason, the Pathology Panel of the Dutch Melanoma Working Party was instituted and is regularly approached to provide an expert opinion on problem cases. In order to identify the most common diagnostic problems, 1069 consecutive referral cases of submitted lesions (1992 to 1994 inclusive) were analysed. About 60 per cent of the requests came from small laboratories, with up to three consultant pathologists. Two-thirds of the lesions reviewed concerned women and nearly 50 per cent of the patients were 30 years of age or younger. In 8 per cent of the cases, the referring pathologists felt unable to make a confident diagnosis; in 14 per cent, melanoma was suspected; and in 12 per cent, a differential diagnosis only had been formulated. The panel felt able to provide an unequivocal diagnosis in 93 per cent of the requests. Of the 158 lesions classified as ‘invasive melanoma’ by the referring pathologists, 22 were considered to be benign by the panel. Conversely, 108 invasive melanomas (panel diagnosis) had originally been considered as benign lesions, dysplastic naevi or melanoma in situ. These high numbers of discordancies reflect the intrinsic difficulty of the differential diagnoses in this selected material submitted to the panel. Diagnostic difficulties were most often encountered with Spitz naevi and dysplastic naevi. Although the rate of overdiagnosis and underdiagnosis is quite high, in the majority of cases the diagnosis of the referring pathologist matched the diagnosis of the panel. This may reflect a proper awareness of difficult melanocytic lesions in pathology practice. The activities of the Pathology Panel of the Dutch Melanoma Working Party contribute to the improvement of the quality of diagnosis in cutaneous melanocytic lesions, as they increase the number of unequivocal diagnoses and reduce the number of incorrect diagnoses. On the basis of the systematic comparison of the diagnosis by the referring pathologist and the panel, postgraduate teaching and quality control can be more focused on specific diagnostic problems. © 1997 John Wiley & Sons, Ltd.     

On the limited value of fine-needle aspiration for the diagnosis of benign melanocytic proliferations of the skin

This a retrospective study of 39 patients with pigmented cutaneous lesions with a subsequent histologic diagnosis of melanocytic neoplasia. The most important cytologic features seen in the fine-needle aspirates were assessed in the 26 cases deemed satisfactory for evaluation. Though cytology, along with essential clinical data, could enable a general diagnosis of melanocytic lesion and could differentiate it from other nonmelanocytic pigmented lesions, cytology could not provide a precise diagnosis of the different histologic types of benign melanocytic nevi, nor could it enable their differentiation from dysplastic melanocytic nevi or incipient malignant melanoma. Diagn. Cytopathol. 1998;19:441–445. © 1998 Wiley-Liss, Inc.     

Cutaneous pH landscape as a facilitator of melanoma initiation and progression

Melanoma incidence is on the rise and currently causes the majority of skin cancer‐related deaths. Yet, therapies for metastatic melanoma are still insufficient so that new concepts are essential. Malignant transformation of melanocytes and melanoma progression are intimately linked to the cutaneous pH landscape and its dysregulation in tumour lesions. The pH landscape of normal skin is characterized by a large pH gradient of up to 3 pH units between surface and dermis. The Na⁺/H⁺ exchanger NHE1 is one of the major contributors of acidity in superficial skin layers. It is also activated by the most frequent mutation in melanoma, BRAF^V600E, thereby causing pH dysregulation during melanoma initiation. Melanoma progression is supported by an extracellular acidification and/or NHE1 activity which promote the escape of single melanoma cells from the primary tumour, migration and metastatic spreading. We propose that viewing melanoma against the background of the acid‐base physiology of the skin provides a better understanding of the pathophysiology of this disease and allows the development of novel therapeutic concepts.     

A study of tumor progression: The precursor lesions of superficial spreading and nodular melanoma

Six evident lesional steps of tumor progression form the neoplastic system that affects the human epidermal melanocyte: 1) the common acquired melanocytic nevus; 2) a melanocytic nevus with lentiginous melanocytic hyperplasia, i.e., aberrant differentiation; 3) a melanocytic nevus with aberrant differentiation and melanocytic nuclear atypia, i.e., melanocytic dysplasia; 4) the radial growth phase of primary melanoma; 5) the vertical growth phase of primary melanoma; and 6) metastatic melanoma. The common acquired melanocytic nevus is viewed as a focal proliferation of melanocytes, destined in most instances to follow a programmed pathway of differentiation that leads to disappearance of the nevus. If the pathway of differentiation is not followed, characteristic lesions result, and such lesions are regarded as the formal histogenetic precursors of melanoma. Such a developmental flaw is termed aberrant differentiation, and the resultant precursor lesion is designated melanocytic dysplasia. The vast majority of melanocytic nevi showing melanocytic dysplasia are terminal lesions that do not progress to melanoma. If melanoma is to develop via a precursor lesion, however, the nevus with melanocytic dysplasia is that precursor. When melanomas do develop, they develop focally within the precursor. The resultant primary melanoma itself does not follow a pathway of inexorable expansion of a population of melanoma cells in space and time. Rather, primary melanomas, with the exception of nodular melanoma, also evolve in a stepwise fashion. The first step, termed the radial growth phase, is characterized by the net enlargement of the tumor at its periphery, along the radii of an imperfect circle. Tumors in this stage of development show a characteristic pattern of growth within the epidermis and a distinctive form of invasion of the papillary dermis. Such melanomas are not associated with metastasis, and it is hypothesized that such tumors do not have competence for metastasis. For a melanoma to acquire competence for metastasis it must progress to the next step of tumor progression--the vertical growth phase. This lesional step is characterized by the appearance of a new population of cells within the melanoma, not an expansion of the cells forming the pre-existing radial growth phase. The net growth of the cells of the vertical growth phase is perpendicular to the directional growth of the radial growth phase. As a rule, the cells of the vertical growth phase grow in an expansile fashion, expansile as a balloon expands: a growth form characteristic of metastases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expert review remains important in the histopathological diagnosis of cutaneous melanocytic lesions

Aims:  To assess the type of problems encountered in diagnosing melanocytic lesions and to evaluate the contribution of expert review.
Methods and results:  Data from 1887 lesions submitted for consultation to one of the expert pathologists of the Dutch Melanoma Working Group Pathology Panel between 1991 and 2004 were analysed. Referring pathologists can voluntarily submit lesions which are difficult to classify to the panel. Most cutaneous melanocytic lesions ( n  = 1217) were submitted with a presumed diagnosis by the referring pathologists. Relevant underdiagnoses of melanoma ( in situ ) and overdiagnoses of naevi were prevented in 12% (144/1217) and 15% (178/1217) of cases, respectively. Problematic melanocytic lesions were (i) spitzoid and dysplastic lesions, (ii) lesions with histological features that hampered the diagnosis such as regression, lymphocytic infiltrate, or a combination with other melanocytic lesions, and (iii) lesions with unusual clinical features, e.g. childhood melanoma. Remarkably, the features of the lesions that were submitted and the types of over- and under-diagnosis remained consistent from 1991 to 2004.
Conclusions:  A second opinion from an expert pathologist on problem-prone melanocytic lesions improves patient care, in our series in 27% of cases.     

Serological reagents for the immunohistochemical analysis of melanoma metastases in sentinel lymph nodes

For the immunohistochemical analysis of melanoma, various serological reagents are available. Melanocyte differentiation markers are reactive with cells and tumors of melanocytic lineage. HMB45 to gp100 has been the most commonly used melanocyte differentiation marker. Recently it was complemented by reagents such as antibodies to Melan-A/MART-1 and tyrosinase. Other reagents, whose reactivity is not strictly confined to melanocyte differentiation antigens, are also commonly used. Among them, the most prominent is S100. Other reagents are D5 to MITF or PNL-2. The properties of these reagents are presented, and their usefulness as markers in the setting of metastatic melanoma in sentinel lymph nodes is discussed.     

MATRIX METALLOPROTEINASE-2 (72 kD TYPE IV COLLAGENASE) EXPRESSION OCCURS IN THE EARLY STAGE OF HUMAN MELANOCYTIC TUMOUR PROGRESSION AND MAY HAVE PROGNOSTIC VALUE

Matrix metalloproteinase-2 (MMP-2), a member of the matrix metalloproteinase family, participates in degradation of the pericellular and extracellular matrix during neoplastic growth and metastasis. Experimental data have substantiated its role in melanoma invasion, but there is no information at present concerning its expression in histological specimens from human melanocytic tumours. This study describes the occurrence and immunolocalization of MMP-2 in human melanocytic lesions, defining distinct steps in melanoma progression. Paraffin-embedded sections from 118 melanocytic lesions were immunostained using a specific antibody to 72 kD type IV collagenase. The material included 34 common naevocellular naevi, 14 dysplastic naevi, 21 in situ melanomas, 20 primary malignant melanomas, and 29 melanoma metastases. Intracytoplasmic MMP-2 immunoreactive protein was found in the ‘naevocytic nests’ of common naevi, in junctional naevus cells, and in melanoma cells. The surrounding normal skin stained negatively, except for occasional macrophages, sweat glands, and hair follicles. The number of MMP-2-positive cells increased with decreasing architectural organization and increasing atypia in the melanocytic lesions. The MMP-2 positivity in the primary and subcutaneous melanoma lesions correlated with later haematogenous metastasis. The data suggest that MMP-2 expression is an early event in melanocytic tumour progression, but is nevertheless prognostic for haematogenous metastasis in melanoma.     

Expression of microtubule-associated protein 2 in benign and malignant melanocytes: implications for differentiation and progression of cutaneous melanoma

Cutaneous melanocytic neoplasms are known to acquire variable characteristics of neural crest differentiation. Melanocytic nevus cells in the dermis and desmoplastic melanomas often display characteristics of nerve sheath differentiation. The extent and nature of neuronal differentiation characteristics displayed by primary and metastatic melanoma cells are not well understood. Here, we describe induction of a juvenile isoform of microtubule-associated protein 2 (MAP-2c) in cultured metastatic melanoma cells by the differentiation inducer hexamethylene bisacetamide. Up-regulation of this MAP-2 isoform, a marker for immature neurons, is accompanied by extended dendritic morphology and down-regulation of tyrosinase-related protein 1 (TYRP1/gp75), a melanocyte differentiation marker. In a panel of cell lines that represent melanoma tumor progression, MAP-2c mRNA and the corresponding approximately 70-kd protein could be detected predominantly in primary melanomas. Immunohistochemical analysis of 61 benign and malignant melanocytic lesions showed abundant expression of MAP-2 protein in melanocytic nevi and in the in situ and invasive components of primary melanoma, but only focal heterogeneous expression in a few metastatic melanomas. In contrast, MAP-2-positive dermal nevus cells and the invasive cells of primary melanomas were TYRP1-negative. This reciprocal staining pattern in vivo is similar to the in vitro observation that induction of the neuronal marker MAP-2 in metastatic melanoma cells is accompanied by selective extinction of the melanocytic marker TYRP1. Our data show that neoplastic melanocytes, particularly at early stages, retain the plasticity to express the neuron-specific marker MAP-2. These observations are consistent with the premise that both benign and malignant melanocytes in the dermis can express markers of neuronal differentiation.     

Accuracy of frozen section measurements for the determination of Breslow tumour thickness in primary malignant melanoma

AIMS: Microstaging of primary malignant melanoma (MM) and the width of surgical margins depend mainly on Breslow tumour thickness (BTT). The use of frozen section (FS) measurements of BTT has been doubted, and previous reports have shown conflicting results regarding the comparability to paraffin sections (PS). To look for significant differences of BTT due to freezing or paraffin embedding, we evaluated a larger series of melanocytic lesions as far as possible excluding other technical influences. METHODS AND RESULTS: Paired 'mirror sections' of 112 melanocytic lesions (33 MM and 79 melanocytic naevi) were measured according to Breslow on single corresponding PS and FS of the same tumour specimen. Comparing measurements on FS and PS, we found very small differences of BTT on average and an almost equal distribution of BTT in the two sets of values with no statistically significant difference by applying the Wilcoxon signed rank test. Concerning the clinically most important 1 mm-threshold of BTT, 110 (98.2%) of the lesions gave equal measurements in FS and PS. CONCLUSIONS: Frozen sections can be used for accurate measurements of Breslow tumour thickness. Consequently, intraoperative frozen section diagnosis of thick melanoma immediately followed by excision with wide surgical margins is possible in experienced centres.