Gene expression patterns in melanocytic cells: candidate markers for early stage and malignant transformation.

Different stages of differentiation of human melanocytic cells, such as normal melanocytes, naevus and melanoma cells, reflect distinct gene expression patterns. A PCR-based subtractive hybridization and display method was applied to identify genes that are differentially expressed in melanocytic cells in relation to early stage and malignant transformation. This resulted in the identification of a number of candidate cDNAs differentially expressed among melanocytes, naevus cells, and (non)-metastatic melanoma cells. Out of this collection of cDNAs, 16 clones were screened that comprised 12 novel genes, one previously identified expressed sequence tag related to vesicular trafficking (Ras-related protein Rab5b). The other three were also known genes that were either related to cell motility (beta-tubulin), pre-mRNA splicing (small nuclear protein U1A), or of unknown function (the human TI227-H gene). The differential expression patterns of Rab5b and two novel gene fragments (pCMa1, pCMn2) were further assessed in melanocytic cells. pCMa1 was expressed more in metastatic melanoma than in primary melanoma cells. In contrast, pCMn2 was expressed in both non-metastatic and metastatic melanoma cells, but was not detectable in either normal melanocytes or naevus cells. The Ras-related protein Rab5b showed lower levels of expression in highly metastatic than in other melanoma cells. These three cDNAs may therefore be involved in the early stage and malignant transformation of melanocytes.     

The extracellular matrix in pigmented skin lesions: an immunohistochemical study

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.     

Immunohistochemical localisation of stem cell factor (SCF) with comparison of its receptor c-Kit proto-oncogene product (c-KIT) in melanocytic tumours

In order to characterise the distribution and role of stem cell factor (SCF), a recently-reported growth factor for normal melanocytes, we carried out an immunohistochemical study on benign and malignant melanocytic tumours with a comparison with the presence of its receptor c-Kit proto-oncogene product (c-KIT). In normal skin, SCF was mainly observed in endothelial cells of blood vessels but not frequently in basal melanocytes, whereas c-KIT was predominantly localised in tissue mast cells. In benign neoplastic melanocytes (common melanocytic naevi), localisation of SCF and c-KIT was complementary: SCF was mostly found in dermal naevus cells while c-KIT was revealed in epidermal naevus cells, although the expression of the latter antigen was not frequent. Malignant melanoma cells showed less frequent expression of these antigens than those in benign lesions. Of five cultured melanoma cell lines, SCF was observed in only one, and c-KIT was not found in any melanoma cells. No quantitative or qualitative alterations assessed by Western blot analysis were induced in the presence of phenotypic modifiers (sodium butyrate and HMBA). Present data suggest that loss of SCF expression in neoplastic melanocytes is commonly associated with malignant transformation of pigment cells rather than loss of its receptor c-KIT.     

Benign intradermal melanocytic naevus with osteoclast-like giant melanocytes: a case report of an unusual morphological pattern

Multinucleated osteoclast-like melanocytes are a relatively common feature in malignant melanoma, however reported cases of benign melanocytic naevi with giant cells melanocytes are scarce. Herein, we present an unusual case of an intradermal melanocytic naevus with numerous benign giant cell melanocytes. A 42-year-old female presented with a small papule on the forehead which was excised. Histologically, the lesion was formed of an intradermal proliferation of benign melanocytes without junctional activity. The melanocytes were arranged in small nests and showed neither cytological atypia nor mitotic activity. There were numerous multinucleated osteoclast-like giant cells with abundant eosinophilic granular cytoplasm and several small open face nuclei. These multinucleated cells showed immune reactivity to melanocytic markers. This case represents a rarely described morphological pattern of a benign intradermal melanocytic lesion showing multiple benign giant cell melanocytes. Reporting such a case provides further evidence to support the presence of this morphological entity and would aid in further understanding its biology and clinical significance.     

Micro-anatomy related antigen expression in melanocytic lesions

The in situ expression of antigens associated with melanosomes (gp-100), pigmentation (PAA), tyrosinase (TRP-1), melanoma (MAA-1/MAA-2), and HLA-DR was investigated immunohistochemically in frozen archival specimens of common acquired melanocytic naevi, in dysplastic melanocytic naevi, and in lymph node metastases of melanoma. Expression of these antigens was also studied in established cultured normal human melanocytes, naevus-derived melanocytes and melanoma cell lines of varying metastatic potential, by immunohistochemistry and flow cytometry. Compared with normal melanocytes, melanocytic naevi exhibited increased expression of gp-100, PAA, and TRP-1 in the lesional cells at or very near the dermo-epidermal junction, but with diminishing expression towards the intra-dermal base of the lesions. In contrast, expression of MAA-1 and MAA-2 was observed in melanocytes throughout the dermal part of the naevi. Melanocytes located at the basal layer of the epidermis were positive only for gp-100, PAA, and TRP-1 antigens. Dysplastic melanocytic naevi showed staining of gp-100, PAA, TRP-1, HLA-DR, MAA-1, and MAA-2 of junctional lesional melanocytes, but less intense than that of common acquired naevi. These antigens were not detectable in the dermal part of the dysplastic naevi. Expression of these antigens in lymph node metastases of melanoma was either positive or negative. Similar results regarding antigen expression were observed in all cultured melanocytic cells, both by immunohistochemistry and by flow cytometry. The present data suggest that analysis of these antigens may contribute to the discrimination of common acquired melanocytic naevi from their dysplastic counterparts. Furthermore, variations in the levels of expression in naevi may be consistently related to the micro-anatomy of the lesions, indicating that the micro-environment may have an influence on the expression levels of these antigens in different lesional melanocytes.     

Nucleolar organizer regions in melanocytic dysplasia and melanoma

Using silver (Ag) staining to demonstrate nucleolar organizer region-associated proteins (AgNORs), pigmented naevi exhibiting features of melanocytic dysplasia have been examined and compared with benign intradermal and compound naevi and with malignant melanomas. A highly significant difference was found between the numbers of AgNORs demonstrated in benign naevus cells and atypical melanocytes and in malignant melanocytes, suggesting that this technique may have a role in differentiating between difficult melanocytic lesions.     

Histopathological characteristics of small diameter melanocytic naevi

BACKGROUND/AIMS: The clinical definition of an atypical naevus ("dysplastic naevus" or "naevus with architectural disorder and cytological atypia of melanocytes") stresses size larger than 5 mm in diameter as a major diagnostic criterion. Because malignant melanomas and their precursors may arise in smaller lesions, a histological study of melanocytic lesions smaller than 4 mm in diameter was conducted to evaluate their histological appearance. METHODS: Two hundred and sixty one naevi smaller than 4 mm in diameter were collected and characterised by histological examination into benign naevi without architectural disorder and naevi with architectural disorder and mild, moderate, and severe atypical melanocytes according to criteria used on larger lesions. RESULTS: Small melanocytic naevi covered the same complex histological spectrum from benign naevi to severely atypical naevi when compared with larger lesions. A high proportion of small naevi (72%) exhibited features diagnostic for naevi with architectural disorder and cytological atypia. CONCLUSION: There is a discrepancy between histological and clinically defined atypical naevi. The same generally accepted criteria for the histological diagnosis of atypical naevi should be used for small melanocytic naevi in addition to large ones. Thus, small naevi exhibiting atypical features on histological examination should be categorised as atypical naevi, regardless of their small diameter.     

Dermal dendritic melanocytic proliferations: an update

Dermal dendritic melanocytic proliferations are a broad group of congenital and acquired melanocytic lesions characterized by the presence of dermal spindled and dendritic cells resembling melanocytes migrating from the neural crest to the epidermis. Historically, they were subdivided into dermal melanocytoses (naevus of Ota, Ito, Mongolian spot and related conditions), blue naevi and malignant blue naevi. The purpose of this review is to provide an update on recent developments in the field with emphasis on new entities and their differential diagnosis.     

Deep penetrating naevus: clinicopathological study of 31 cases with further delineation of histological features allowing distinction from other pigmented benign melanocytic lesions and melanoma

AIMS: To examine a series of deep penetrating naevus (DPN) and discuss the differential diagnosis of pigmented, deep penetrating melanocytic lesions and their biological potential. DPN has been described as a variant of common acquired intradermal melanocytic naevus. DPN remains poorly recognized by pathologists, partly attributable to its relatively rare occurrence. METHODS AND RESULTS: Thirty-one cases of deeply pigmented lesions were studied. The patients included 17 females and 14 males with an age range between 3 and 56 years (mean 25.8, median 23). The common clinical sites were face (n = 10) back (n = 6) and lower extremity (n = 7). The clinical diagnoses included various benign melanocytic naevi, and malignant melanoma, as well as non-melanocytic lesions. Histologically, all cases presented as wedge-shaped lesions composed of fusiform cells but also epithelioid melanocytes, with pale cytoplasm and oval nuclei. Pigment was identified in melanophages but also within lesional melanocytic cells. Nine cases contained mitotic figures. Nine cases showed the coexistence of 'ordinary' common acquired naevocytes, and seven lesions showed overlapping features with either ordinary blue naevus or Spitz naevus. In 13 lesions there was at least one feature that may cause concern as to the biological nature of the tumour. These include asymmetry, cytological atypia, inflammation, or an 'expansile' advancing margin. Each tumour was treated by simple excision; one lesion recurred after 1 year. No tumour metastasized. CONCLUSIONS: DPN is a distinct variant of melanocytic naevus. In some cases the histological features overlap with other benign melanocytic lesions. Criteria for recognizing malignant examples remain unclear, but cytological atypia and low mitotic activity do not necessarily portend a sinister outcome.     

Inducible nitric oxide synthase expression in benign and malignant cutaneous melanocytic lesions

Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) and plays an important role in tumour growth. In this study, inducible NOS (iNOS) expression was evaluated by immunohistochemistry in 34 melanocytic naevi (13 common melanocytic naevi, six Spitz naevi, and 15 so-called 'dysplastic naevi'), ten cutaneous melanomas in situ, 50 stage I invasive melanomas, and eight subcutaneous metastases of melanoma. In addition, four samples of melanocytic naevi and four samples of invasive melanomas were collected in order to perform western blot and northern blot analysis. By immunohistochemistry, melanocytic naevi never expressed iNOS. Among cases of melanoma in situ, two were negative, seven displayed staining in less than 20% of melanoma cells, and positivity was observed in 21-50% of melanoma cells in only one case. iNOS expression was detected in 46 out of 50 invasive melanomas (92%). Among these cases, 18 showed positivity in less than 20% of melanoma cells, 18 showed positivity in 21-50% of melanoma cells, and ten showed iNOS expression in more than 50% of cells. Statistical analysis revealed a significant difference in iNOS expression between melanocytic naevi and cutaneous melanomas (p<0.001). In addition, iNOS expression was significantly higher in invasive melanomas than in melanomas in situ (p=0.01). Among primary cutaneous melanomas, no significant correlation was found between iNOS expression and histopathological parameters (histotype, level, thickness and presence of regression/inflammatory infiltrate) and disease-specific survival. In subcutaneous melanoma metastases, iNOS expression was diffuse in more than 50% of cells. Statistical analysis revealed that subcutaneous melanoma metastases showed greater iNOS immunoreactivity than invasive melanomas (p=0.02). Molecular analyses confirmed that iNOS mRNA and protein were highly expressed in melanoma samples. In conclusion, iNOS was constantly absent in melanocytic naevi, whereas it was frequently expressed in melanomas, with up-regulation of the enzyme paralleling tumour progression. These data suggest that iNOS may play a role in the malignant transformation of melanocytes and in tumour growth. In addition, iNOS may be useful as an immunohistochemical marker for malignant melanocytic lesions.     

Histopathological characteristics of malignant melanoma affecting mucous membranes: a unifying concept of histogenesis

AIMS: To investigate histopathological characteristics of melanocytic lesions affecting mucous membranes in various anatomical sites. Particular attention was paid to elucidation of morphological characteristics of early phases of mucosal melanoma in order to contribute to effective detection of this highly malignant neoplasm in the curable stages. METHODS: A total of 87 melanocytic lesions of mucous membranes were investigated histopathologically. There were 55 malignant melanomas including eight lesions of melanoma in situ, three in the radial growth phase (RGP) and 44 in the vertical growth phase (VGP), and 28 benign melanocytic lesions including four melanotic macules, 19 melanocytic naevi and five blue naevi. In addition, this series also included four equivocal lesions for which diagnoses were not definitely determined. With regard to malignant melanoma, histopathological patterns of in situ phase and RGP were intensely evaluated. RESULTS: Histopathological features of benign melanocytic lesions were essentially the same as those of the corresponding lesions of the skin. In the vast majority of mucosal melanomas, irrespective of anatomical sites, the main histopathological pattern seen in melanoma in situ and in RGP was the lentiginous pattern, which shows proliferation of atypical melanocytes in the lower layer of more or less acanthotic epithelium, though subtle variations of the pattern were detected. No association of melanocytic naevus was detected in any cases of melanoma. Based on these findings, we have proposed a unifying concept of de novo histogenesis of mucosal malignant melanoma. CONCLUSIONS: Our concept of histogenesis of mucosal melanoma assists in the identification of this highly malignant neoplasm in the early curable stages.     

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.     

Benign atypical naevi: diagnostic difficulties and continued controversy

Benign melanocytic naevi exhibit a wide spectrum of histological appearances. Some share significant clinical and histological features and are recognized as entities. Included among these are pagetoid/junctional Spitz naevus, pigmented spindle cell naevus, halo naevus, recurrent and traumatized naevus, ultraviolet (UV) irradiated naevus, naevus in infants, acral naevus, genital naevus and naevi from other specific anatomic locations. However, there still remains a diagnostic grey area of acquired predominantly junctional naevi with architectural and cytological atypia. Only a small percentage of these will fulfil the criteria for dysplastic naevus if criteria are strictly applied. Therefore, there exists a group of otherwise ordinary acquired naevi with atypical junctional activity, mostly mild, whose biological significance is unclear. In older individuals, although junctional activity in otherwise benign naevi does occur, extra care should be exercised in order to prevent the diagnosis of melanoma in situ being overlooked.     

Melanocytic lesions in lymph nodes associated with congenital naevus

Two cases of melanocytic lesions in lymph node associated with congenital naevus are presented. The first was a 30-year-old man with a nodular melanoma arising in a small congenital naevus. The second was a 2-year-old male infant with a giant congenital naevus. In both cases, naevus cell aggregates were observed in the capsule, trabeculae, perisinusoidal areas and lymphatic vessels surrounding the nodes. In the first case, clusters of large atypical melanocytes were present amongst naevus cell aggregates in the perisinusoidal areas as well as in the lymphoid parenchyma. Between the naevus cells and large atypical melanocytes, transitional forms were observed which supports the idea that the presence of large atypical melanocytes is indicative of benign naevus cells. In the second case, marginal sinuses were packed with clusters of large melanin-rich cells. Immunohistochemically, these cells were S-100 protein negative, but ultrastructural studies proved them to be melanocytes. They were considered indicative of spread of benign naevus cells via lymphatic vessels. Arrested migration of naevus cells during embryogenesis and benign spread of naevus cells are possible explanations for the histogenesis of naevus cell aggregates in lymph nodes associated with congenital naevus.     

Complementary expression of melanosomal antigens and constant expression of pigment-independent antigen during the evolution of melanocytic tumours

Summary We have generated monoclonal antibodies (MoAbs) against melanosomal proteins (MoAb 1C11 and MoAb HMSA-1) and a cytoplasmic protein strongly synthesized in neoplastic melanocytes but not associated with melanogenesis (MoAb 7H11). An immunohistochemical study of paraffin sections showed that nearly 90% of epidermal neoplastic melanocytes, including melanomas, expressed 1C11 antigen, whereas this antigen was poorly preserved in dermal melanocytic cells except melanomas. HMSA-1 antigen was expressed in a complementary manner to 1C11 antigen, being found in dermal naevus cells but not generally in the epidermal regions, except for dysplastic naevi and melanomas. In contrast, 7H11 antigen was distributed in nearly 90% of melanocytic tumours except solar lentigo and lentigo maligna lesions. The failure of MoAb 1C11 to react with dermal melanocytes may reflect a subtle alteration in melanogenesis during tumour evolution. Overall, the combined use of MoAbs serves as an accurate diagnosis of melanocytic tumours, the pigment-independent MoAb 7H11 being particularly useful for amelanotic and metastatic lesions.     

What naevus is dysplastic,a syndrome and the commonest precursor of malignant melanoma? A riddle and an answer

This assay examines critically the concept of 'dysplasia' as it applies to melanocytic neoplasia and concludes that the concept is flawed. Doubt is cast also upon the validity of the concepts of a dysplastic naevus and a dysplastic naevus syndrome. The author is of the view that most malignant melanomas arise de novo and not in pre-existing dysplastic naevi. Because so-called dysplastic naevi are considered by the author to be the commonest melanocytic naevi in man and devoid of nuclear atypia, he proposes that they be re-named 'common naevi' in the interest of simplicity, accuracy, and care of patients. Lastly, it is suggested that resolution of problems in the sphere of melanocytic neoplasia might be accomplished more readily were terms like dysplasia, dysplastic cells, dysplastic naevi, and the dysplastic naevus syndrome eschewed.     

Detection of the c-met proto-oncogene product in normal skin and tumours of melanocytic origin

The proto-oncogene c-met product (c-MET) is a receptor tyrosine kinase and functions as a receptor for hepatocyte growth factor (HGF). Although the function of c-MET has yet to be fully clarified, HGF stimulates the phosphorylation of tyrosyl residues on c-MET and triggers the signal transduction pathways, resulting in a contribution to the malignant progression of melanonocytes with synergic factors such as basic fibroblast growth factor and mast cell growth factor. Using immunohistochemical methods, we have studied the localization of c-MET in normal skin and various melanocytic tumours, c-MET was detected in keratinocytes, melanocytes, sebaceous cells, and other cells of the skin. In particular, basal melanocytes almost always showed nuclear labelling. Melanocytic naevi generally revealed predominantly nuclear staining of cells in the epidermis, whereas only a few cases showed a distinct cytoplasmic localization of c-MET in dermal naevus cells. The distribution pattern of c-MET in melanoma cells was basically similar to that of benign lesions, although the numbers tested were small. Cultured human melanoma cells also showed predominantly nuclear labelling, but were unresponsive to exogenous c-MET ligand HGF. Treatment with the glucosidase inhibitor castanospermine caused accumulation of protein at 220 kD, without diminishing the amount of normally-processed 190-kD c-MET. Although there was no significant difference in c-MET distribution between benign and malignant melanocytic lesions, it is suggested that malignant transformation of melanocytes may be associated with loss of response to HGF or other growth-regulating factors.     

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.     

Combined naevus: a benign lesion frequently misdiagnosed both clinically and pathologically as melanoma

BACKGROUND: Combined naevi are characterised pathologically by the presence of two or more different types of melanocytic naevi in a single lesion. They are prone to clinical and pathological misdiagnosis as melanoma. Misdiagnosis may result in inappropriate treatment, patient anxiety and medicolegal consequences. AIMS: With the aim of reducing the incidence of misdiagnosis, this study documents the clinical and pathological features of a large series of combined naevi and describes how to distinguish them from melanoma. PATIENTS AND METHODS: The slides of skin lesions from 220 patients that were coded as combined naevus between 1990 and 2001 were retrieved from the archival files of the Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia. The clinical notes, letters from referring pathologists (for consultation cases) and slides were reviewed and for each lesion clinical and pathological features were assessed. Thirty-eight cases were excluded either because they included only one naevus component or because there were atypical pathological features in the slides available for review. The remaining 182 cases formed the study population. RESULTS: The patients included 92 females and 87 males (1.1:1). In three cases the gender was not known. Mean and median patient ages were 29.6 and 28 years, respectively. The anatomical site of involvement was the trunk in 64 cases (35.2%), head and neck region in 43 cases (23.6%), upper extremity in 40 cases (22.0%), lower extremity in 18 cases (9.9%) and perineum and buttock region in eight cases (4.4%). In nine cases the site of involvement was not known. A pre-operative clinical diagnosis was recorded in 126 cases; of these, melanoma was suspected clinically in 33 cases (26.2%) while combined naevus was diagnosed clinically in only three cases (2.4%). Histologically, 180 cases included two different naevus components, and in two cases three different naevus components were present. The most common combination was a common acquired naevus of compound type associated with a blue naevus of deep penetrating naevus type; this occurred in 57 cases (31.3%). The referring pathologist recorded a preferred diagnosis in 88 of 122 consultation cases; of these, melanoma was suspected in 23 cases (26.1%) and in 23 cases combined naevus was favoured (26.1%). CONCLUSIONS: Combined naevus is an uncommon type of melanocytic naevus that is frequently misdiagnosed both clinically and pathologically. Knowledge and recognition of the pathological features of combined naevi and the important features that distinguish them from melanomas should reduce the frequency of misdiagnosis.