Immunohistochemical detection of CDK4 and 9 16INK4 proteins in cutaneous malignant melanoma

Summary p16^INK4 gene, which encodes a specific inhibitor of cyclin-dependent kinase 4 (CDK4). has been recently reported as an important tumour suppressor gene. It is mapped to chromosome 9p21, which is frequently deleted or mutated in many tumour cell lines including malignant melanoma. Since the CDK4/cyciin I) complex propels a cell to go through the G₁ check point of the cell cycle, a critical phase of cell division, alteration of the p 16^INK4 gene could lead a cell to uncontrolled proliferation and malignant transformation. To clarify any role for p16d^INK4 and CDK4 proteins in the development of human malignant melanoma, we have examined, immunohistochemically, the expression of these two proteins in melanocytic neoplasms including 19 primary lesions of nonfamilial melanoma. Intense nuclear and/or cytoplasmic expression of the CDK4 protein was observed in 11 of 19 cases (58%) of melanoma. In contrast, virtually no nuclear or cytoplasmic staining for CDK4 protein was detected in 28 benign melanocytic naevi, including six Spitz naevi. Expression of p16^INK4 protein was observed in three of 19 melanomas (16%) and in 17 of 28 benign naevi (61%). Inverse expression of CDK4 and p16^INK4, at individual cell level, was detected in one case of melanoma. The present study suggests that CDK4 overexpression is characteristic for malignant melanoma, and probably reflects its autonomous accelerated cell proliferation. The expression rate of p16^INK4 protein in malignant melanoma was lower than that in benign naevi, although the significance of p16^INK4 deletion in melanoma development has not been definitely confirmed.     

The effect of the sun on expression of beta-catenin, p16 and cyclin d1 proteins in melanocytic lesions

BACKGROUND: The tumour suppressor gene product, p16, is often inactivated during melanoma malignant progression. Although the importance of p16 in melanomas is well documented, its relationship with cyclin D1, beta-catenin and ultraviolet radiation (UVR) remains unclear. AIM: To determine the role of these cell cycle-related proteins and high-risk sun exposure in the biological behaviour of melanocytic lesions. METHODS: We used immunohistochemistry to examine 28 melanocytic naevi (MN; 9 congenital and 19 acquired types) and 24 primary cutaneous malignant melanomas (CMM; 19 nodular melanomas, 3 lentigo maligna melanomas, 1 acral lentiginous melanoma and 1 superficial spreading melanoma) for the presence of p16, cyclin D1 and beta-catenin. The melanocytic lesions were classified into two groups to examine the effects of UVR on these three proteins: high risk of sun exposure (chronically sun damaged; CSD), or low risk of sun exposure (nonchronically sun damaged; non-CSD). We evaluated the relationship between the production of these proteins and the histopathological and clinical characteristics of the lesions. RESULTS: Production of p16 was repressed in most CMM, but not in MN (P < 0.0001). Cyclin D1 was overproduced in CMM but not in MN, and beta-catenin was frequently overproduced both in MN and CMM. Overproduction of beta-catenin was not common in CSD melanocytic lesions, but was more frequent in non-CSD melanocytic lesions (P = 0.027). CONCLUSION: An immunohistochemical panel including melanocytic markers enriched by p16 and cyclin D1 could be used to differentiate some borderline melanocytic lesions. In addition, the Wnt/beta-catenin pathway was more frequently activated in non-CSD than in CSD melanocytic lesions.     

Expression of matrilysin (matrix metalloproteinase-7) in primary cutaneous and metastatic melanoma

BACKGROUND: Matrilysin (MMP-7), a member of the matrix metalloproteinase (MMP) family of proteins, is expressed in various types of malignant tumours. There have been no previous studies of the correlation between matrilysin expression and melanoma. OBJECTIVES: Protein expression of matrilysin was evaluated in human cutaneous melanomas, metastatic melanomas, acquired common melanocytic naevi and Spitz naevi, and the data were corrected with the clinicopathological factors. METHODS: We retrospectively investigated 18 primary melanomas, 15 metastatic melanomas, 10 common melanocytic naevi and five Spitz naevi samples at our clinic using immunohistochemistry (IHC). Both promatrilysin and active matrilysin were found in the melanoma tissue extracts by Western immunoblotting. In situ hybridization demonstrated that melanoma cells selectively express matrilysin mRNA. RESULTS: Of the melanoma samples, 29 of 33 (87 x 9%) were positive for matrilysin, including 14 of 18 (77 x 8%) primary cutaneous melanomas and 15 of 15 (100%) metastatic melanomas. In contrast, matrilysin was not expressed in common naevi or Spitz naevi. The matrilysin IHC staining score in primary melanomas was associated with the presence of metastases, tumour thickness and TNM staging (P=0 x 001, 0 x 025 and 0 x 021, respectively). The 5-year overall survival was 26.3% for matrilysin-positive cases and 100% for matrilysin-negative cases among melanoma specimen. CONCLUSIONS: We found matrilysin expression in primary melanomas and in metastatic melanomas. We further demonstrated that the matrilysin IHC staining score was associated with invasive depth of primary melanoma lesions and metastases. Our observations indicate that matrilysin may be associated with melanoma progression, and may enhance melanoma tumour cell invasion. Therefore, matrilysin may be potentially valuable as a prognostic indicator to predict the clinical behaviour of melanoma.     

Increase in telomerase activity during progression of melanocytic cells from melanocytic naevi to malignant melanomas

Abstract During successive cell divisions of mortal cells the length of the telomeres (TTAGGG repeats in vertebrates) at the end of chromosomes decreases. It has been suggested that this process is responsible for cellular senescence. Expression of the ribonucleoprotein telomerase appears to prevent shortening of telomeres in germ-line cells and cancer cells. The purpose of this study was to investigate telomerase activity in melanocytic lesions and its possible role in the multistep tumor progression model of malignant melanoma. To quantify the level of telomerase activity both in cultured cells and in fresh tissue samples the TRAP (telomeric repeat amplification protocol) ELISA was used. Eight cell lines of malignant melanoma, 3 primary cultures of fibroblasts, 36 melanocytic naevi, 5 atypical melanocytic naevi, 3 Spitz’s naevi, 31 primary malignant melanomas and 13 metastases of malignant melanomas were investigated. Also 34 samples of skin (22 samples of perilesional skin and 12 samples of normal skin) were analysed. In our experiments all melanoma cell lines were strongly positive, whereas in fibroblasts telomerase activity could not be detected. Of the primary melanomas and metastatic melanomas, 90.3% and 92.3%, respectively, were strongly positive, and of the atypical melanocytic naevi, 80% were positive. Of the 36 common melanocytic naevi only 10 (27.7%) expressed weak telomerase activity and of the 34 samples of human skin, 4 (11.7%) expressed very weak telomerase activity. Our results indicate that telomerase activity increases from benign melanocytic naevi to atypical naevi and further to malignant melanoma and metastatic melanoma cells, and therefore may play a role in tumour initiation and progression. Received: 26 June 1998 / Received after revision: 28 October 1998 / Accepted: 2 November 1998     

Distribution and colocalization of markers for proliferation, invasion, motility and neoangiogenesis in benign melanocytic naevi and malignant melanomas

BACKGROUND: Melanomas are heterogeneous tumours, and differentiation from other melanocytic lesions may cause problems. It may be possible that the distribution and/or colocalization pattern of different markers in the lesions can enable a more accurate diagnosis of melanocytic tumours. OBJECTIVES: To test this hypothesis, melanocytic naevi, primary melanomas and metastases were investigated. METHODS: The distribution and colocalization of markers for proliferation, invasion, angiogenesis and motility of the tumour cells were investigated using antibodies directed against actin, cathepsin B (CatB), transforming growth factor-beta, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen/Ki-67 and basic fibroblast growth factor (FGF-2). In addition, melanoma markers (HMB-45 and Melan-A) and proteins unrelated to melanoma progression [epidermal growth factor (EGF) and cathepsin H] were investigated. RESULTS: Malignant melanomas tended to express more markers of malignancy compared with melanocytic naevi, and the differences were statistically significant for EGF and actin immunoreactivity: melanocytic naevi displayed clear EGF labelling more often (60% vs. 5%) and melanomas showed more intense actin labelling (70% vs. 0%). HMB-45+ cells to a large extent also stained with antibodies to CatB but not to EGF or actin; EGF-, FGF-2- and VEGF-immunoreactive cells were predominantly HMB-45-. Similar combinations were observed in melanocytic naevi and in melanomas. CONCLUSIONS: Labelling with EGF may improve the differential diagnosis of melanocytic neoplasias. However, we did not detect a clear-cut increase of markers of malignancy in melanoma. Cells expressing multiple malignancy markers were also found in some melanocytic naevi; this may confirm the dormant potential of melanocytic naevi for melanoma development.     

Evaluation of tumour-infiltrating CD4+CD25+FOXP3+ regulatory T cells in human cutaneous benign and atypical naevi, melanomas and melanoma metastases

BACKGROUND: CD4+CD25+FOXP3+ regulatory T cells (Tregs) are thought to induce immunotolerance in melanoma. They have not yet been investigated in the entire spectrum of melanocytic cutaneous lesions within a tumour site. OBJECTIVES: To evaluate CD4+CD25+FOXP3+ Tregs among tumour-infiltrating lymphocytes in cutaneous melanocytic lesions. METHODS: We analysed 128 lesions (10 benign junctional common naevi, 10 benign compound common naevi, 10 compound Spitz naevi, 10 junctional atypical naevi, 20 compound atypical naevi, 20 radial growth phase melanomas, 30 vertical growth phase melanomas and 18 melanoma metastases). Tregs were identified by CD25-FOXP3 double immunostains. RESULTS: This study indicates that CD4+/CD25+FOXP3+ Tregs are present in all groups of lesions. Junctional atypical naevi, compound atypical naevi and radial growth phase melanomas showed the highest percentages of CD4+CD25+FOXP3+ Tregs (junctional atypical naevi vs. junctional common naevi, compound common naevi, compound Spitz naevi, melanoma metastases: P < 0.0001; junctional atypical naevi vs. vertical growth phase melanomas: P = 0.001; compound atypical naevi vs. junctional common naevi, compound common naevi: P < 0.0001; compound atypical naevi vs. compound Spitz naevi, melanoma metastases: P = 0.002; compound atypical naevi vs. vertical growth phase melanomas: P = 0.02; radial growth phase melanomas vs. junctional common naevi, compound common naevi, compound Spitz naevi, melanoma metastases: P < 0.0001; radial growth phase melanomas vs. vertical growth phase melanomas: P = 0.008). CONCLUSIONS: The strong prevalence of CD25+FOXP3+ Tregs both in junctional and compound atypical naevi and radial growth phase melanomas, suggests that they induce immunotolerance early during melanoma genesis, favouring melanoma growth. Their evaluation within a tumour site could be useful for prognostic and therapeutic purposes.     

Loss of CDKN2A and p14ARF expression occurs frequently in human nonmelanoma skin cancers.

BACKGROUND: The CDKN2A locus on human chromosome 9p21 encodes two proteins named p16INK4a and p14ARF, known to function as tumour suppressors via the retinoblastoma (Rb) or the p53 pathway. The p53 tumour suppressor gene is the most commonly mutated gene in human and mouse cancers. Disruption of the p53 and Rb pathways is a fundamental trend of most human cancer cells. Recent studies have shown that the CDKN2A gene plays an active role in the p53 and Rb tumour suppressor pathways. Genetic abnormalities in CDKN2A have been well documented in human melanoma, but their involvement in nonmelanoma skin cancer (NMSC) is less clear. OBJECTIVES: To determine whether genetic abnormalities in CDKN2A and p53 genes play a role in the development of NMSC. METHODS: We analysed 40 primary NMSCs in 40 patients (21 squamous cell carcinomas, 17 basal cell carcinomas and two actinic keratoses) for p16INK4a and p14ARF protein expression and for genetic alterations in exons 1alpha, 1beta and 2 of CDKN2A. RESULTS: Immunohistochemical analysis revealed loss of expression of p16INK4a and p14ARF proteins in 38 and 39 of 40 NMSCs, respectively. Amplification of genomic DNA by polymerase chain reaction revealed homozygous deletion of exon 1beta in 20% of tumours and of exon 2 in 82.5% of tumours. Of 22 NMSCs with p53 mutations, 13 (59%) had ultraviolet (UV) signature mutations in the p53 gene; all of them were strongly positive for p53 immunostaining. CONCLUSIONS: In addition to mutations in the p53 gene, loss of expression of CDKN2A via deletion also plays an important role in the pathogenesis of human NMSC. While p53 mutations are induced by UVB, deletions in CDKN2A could arise spontaneously, perhaps during tumour progression.     

Genome profiling of melanocytic tumors using multiplex ligation-dependent probe amplification (MLPA): Its usefulness as an adjunctive diagnostic tool for melanocytic tumors

BACKGROUND: The histopathology of melanocytic tumors sometimes presents diagnostic problems. Applicable parameters other than routine pathology are needed. OBJECTIVE: We assessed the feasibility of multiplex ligation-dependent probe amplification (MLPA), a novel PCR-based genome profiling method, in the classification of melanocytic tumors. METHOD: We extracted DNA from paraffin-embedded tissue sections of 24 primary melanomas, 14 Spitz nevi and 17 common melanocytic nevi. We analyzed the copy number gains or losses of a total of 76 genes spanning almost all chromosome arms using commercially available MLPA kits. RESULTS: Although four melanocytic nevi and three Spitz nevi did not yield sufficient DNA for reliable analysis due to small tumor size, the MLPA analysis was feasible and applicable to the remaining 88% of samples. We found multiple genetic aberrations in primary melanomas. The total number of aberrations in each tumor ranged from 1 to 32 (average, 12.04). All but two melanomas showed aberrations at more than three genetic loci. Seventeen (70.8%) of the 24 melanomas showed a copy number loss of either the CDKN2A or CDKN2B gene on chromosome 9p21. All the Spitz nevi and 7 (50%) of 14 common melanocytic nevi had copy number changes at one or two gene loci (average, 1.04). The receiver operator characteristic curve analysis showed that the threshold value of copy number aberrations corresponding to 98% specificity for melanoma was 2.42 and the sensitivity using this threshold value was 92.5%. CONCLUSIONS: MLPA could be used as an adjunctive diagnostic tool for melanocytic tumors.     

Loss of beta-catenin expression associated with disease progression in malignant melanoma

BACKGROUND: beta-catenin plays a crucial role in the function of cell adhesion molecules and also participates in growth regulatory signalling pathways that may be involved in malignant transformation. OBJECTIVES: To examine beta-catenin expression in lesions of melanocytic origin for associations with clinicopathological markers of disease progression and for its significance as a predictor of disease recurrence and prognosis. METHODS: beta-catenin expression was examined by immunoperoxidase staining in 50 melanocytic naevi and 91 primary and 50 metastatic melanomas. RESULTS: beta-catenin was expressed in 96% of melanocytic naevi, in 94% and 65%, respectively, of radial and vertical growth phase primary melanomas, and in 38% of metastatic melanomas. Benign and malignant melanocytic lesions had distinct patterns of beta-catenin localization. Most lesions expressing beta-catenin exhibited cytoplasmic staining; however, over 40% of benign lesions also displayed nuclear staining, which was present only in 10% of primary and 15% of metastatic melanomas. Absent or weak expression of beta-catenin in primary melanomas was associated with several markers of disease progression, including tumour thickness and presence of lymph node metastases. A similar but not statistically significant trend was observed for the association of beta-catenin expression with disease recurrence and prognosis. CONCLUSIONS: These results suggest that loss or downregulation of beta-catenin expression in melanoma cells plays a significant role in progression of the disease.     

p53 immunoreactivity in human malignant melanoma and dysplastic naevi

Expression of the tumour suppressor protein, p53, was determined in 77 cutaneous melanocytic lesions, and in five lymph node metastases from malignant melanoma, in an immunohistochemical study employing CM-1, an antiserum raised against recombinant human p53 protein. Because wildtype p53 protein is rapidly degraded in normal cells, p53 immunoreactivity suggests the presence of an abnormally stable p53 protein. This may occur through either post-translational mechanisms or gene mutation. A highly significant correlation was found between p53 immunoreactivity and malignancy in melanocytic lesions (P<0.0001). Overall, p53 immunoreactivity was observed in 63% of tumour specimens examined, but not in benign melanocytic naevi, although occasional foci of weak nuclear p53 immunoreactivity were observed in a minority of dysplastic naevi and a solitary Spitz naevus. A significant correlation was also found between strong p53 immunoreactivity and malignant melanomas associated with a poor prognosis (P=0.008). These data suggest an important role for p53 tumour suppressor protein in the biology of human cutaneous malignant melanoma.     

Nevus associated malignant melanomas--diagnostic validation and prognosis

The diagnosis and prognosis of naevus-associated malignant melanomas are examined in the present study. For this purpose, 581 cases of primary malignant melanoma seen in the University Department of Dermatology, Berlin Steglitz, were histologically investigated for naevus association. A naevocytic association was proven in 135 (23%) of the malignant melanoma biopsies. Naevocytic malignant melanomas were found at a significantly higher rate (P less than 0.01) in patients under 50 years of age. The 5-year survival rate for naevocytic melanomas was not significantly different from that for other malignant melanoma types: around 80% in both groups. An immunohistological evaluation of the diagnosis of naevus-associated melanoma was also performed on the basis of specimens from 89 melanocytic lesions. The use of HMB-45 for diagnosis of melanocytic tumours made it possible to differentiate resting dermal naevus cells from malignant melanoma infiltrates in paraffin sections in the present study, thus simplifying the diagnosis of naevus-associated malignant melanomas. However, dysplastic naevi, junctional naevi and juvenile melanomas are also stained by HMB-45, which means that malignant melanomas associated with junctional melanocytic naevi still cannot be reliably identified even today.     

Expression of endoglin in human melanocytic lesions

Angiogenesis plays an important role in progression of various tumours including malignant melanoma. It is possible that the immunostaining of angiogenic markers could differentiate benign and malignant melanocytic lesions or that the immunostaining pattern with angiogenic markers could vary with tumour thickness and thus be a prognostic marker. We were interested to see whether there was any correlation between endoglin (CD 105; EDG) expression with tumour thickness in primary cutaneous malignant melanomas (MM), any correlation between EDG expression and clinical outcome in patients with primary cutaneous MMs and any difference in staining pattern between cutaneous MMs and Spitz naevi which could be of diagnostic value. Tissue sections from 14 primary cutaneous MM lesions with a Breslow thickness of > 2 mm, 10 primary cutaneous MM lesions with a Breslow thickness of 1-2 mm, and six Spitz and 10 compound naevi were stained for EDG. EDG expression was compared with survival in patients with primary cutaneous MMs. Overall, EDG staining was positive in 96% of MMs and 94% of benign melanocytic naevi. Very strong (++++) and strong (+++) EDG staining was found in 58% of MMs and 56% of benign melanocytic lesions. EDG expression did not vary significantly with the thickness of the lesion in primary cutaneous melanoma. Survival of melanoma patients did not correlate with the degree of EDG expression. Therefore, expression of this marker alone is not sufficient to differentiate benign and malignant melanocytic lesions.     

Genetic and epigenetic alterations in the differential diagnosis of malignant melanoma and spitzoid lesion

BACKGROUND: The histopathological differentiation of malignant melanoma and Spitz naevus often presents diagnostic problems. OBJECTIVES: We aimed to find out applicable diagnostic parameters other than routine pathology. METHODS: The cases included conventional melanomas and Spitz naevi as well as atypical spitzoid lesions that had posed diagnostic difficulties. We examined hotspots of mutation in the BRAF, NRAS and HRAS genes by polymerase chain reaction-based direct sequencing. We also analysed DNA copy number aberrations and the methylation of CpG sequences in several cancer-related genes by utilizing a novel methylation-specific multiplex ligation-dependent probe amplification method. RESULTS: Twenty three of 24 conventional melanomas showed at least one of the genetic and epigenetic alterations examined, although one acral melanoma did not show any alteration. By sharp contrast, 12 Spitz naevi with an unambiguous histopathology showed no or few chromosomal aberrations, no oncogene mutations and no methylation of CpG sequences. Of the 16 ambiguous spitzoid lesions, most of which were designated atypical Spitz tumour by one of the authors, all but one showed no mutations, no methylations and few copy number aberrations. However, three tumours showed copy number loss of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A), an alteration observed frequently in melanomas but not found in conventional Spitz naevi. These results show that, although most atypical Spitz tumours do not differ from conventional Spitz naevi showing virtually no genetic and epigenetic aberrations, some cases may have chromosomal aberrations that include copy number loss of the CDKN2A gene. CONCLUSIONS: Genetic and epigenetic analyses may be useful as an additional diagnostic tool to distinguish between melanoma and Spitz naevus, and may help to define subgroups in atypical Spitz tumours.     

Application of fluorescence in situ hybridization as a diagnostic tool in melanocytic lesions,using paraffin wax‐embedded tissues and imprint‐cytology specimens

Background. Accurate histopathological diagnosis of certain melanocytic skin lesions as benign or malignant can be notoriously difficult. Recently, four‐colour fluorescence in situ hybridization (FISH) has emerged as an important tool for classifying these lesions. Aim. To evaluate the sensitivity and specificity of a melanoma FISH probe kit for accurate diagnosis of melanocytic tumours, and to validate its use with imprint‐cytology specimens from the cut surface of tumours. Methods. In total, 50 melanocytic skin lesions (31 malignant melanomas, 10 benign melanocytic naevi, and 9 histologically challenging benign melanocytic skin lesions) were evaluated. The samples comprise 47 tissue specimens embedded in paraffin wax, and three imprint‐cytology specimens from the cut surface of melanomas. FISH was performed using four locus‐specific identifier probes [Ras responsive element binding protein (RREB)1, myeloblastosis viral oncogene homologue (MYB), cyclin (CCN)D1 and centromere of chromosome (CEP)6], and results were compared with the clinical long‐term follow‐up and histopathological diagnosis data. Results. The melanoma FISH probe distinguished between naevi and melanomas with a sensitivity of 100% and a specificity of 94.1%. The most sensitive criterion was a gain in 6p25 (RREB1), seen in 100% of cases, followed by CEP6‐related MYB loss (48.1%), CCND1 gain (37%) and MYB gain (22.2%). More than three‐quarters (77.8%) of melanomas were positive for two or more criteria. Positive FISH results were also obtained for the imprint‐cytology specimens. Conclusions. FISH is a valuable diagnostic tool for differentiating between benign and malignant melanocytic lesions, providing a high degree of sensitivity and specificity. The probes displayed exceptional discriminative capacity in difficult or ambiguous lesions. To our knowledge, his is the first reported use of imprint‐cytology specimens for FISH diagnosis.     

Microsatellite instability in malignant melanomas.

Microsatellite instability (MSI) is caused by deficient DNA mismatch repair, and results in a "mutator" phenotype. Recent studies have produced contradictory results about the frequency and significance of MSI in malignant melanomas. In this study, we therefore determined the time of onset and relative frequency of MSI during the progression of melanocytic tumours, including benign melanocytic naevi. We examined 7 different microsatellite loci in 9 melanocytic naevi, 25 primary malignant melanomas and 8 melanoma metastases. None of the melanocytic naevi showed MSI. In contrast, moderate frequency of MSI in 1/12 (8%) was detected in thin melanomas of <0.75 mm vertical thickness and in 1/8 (12%) of those with a thickness >0.75 mm and < 1.5 mm. The rate of MSI was increased in tumours thicker than 1.5 mm (2/5) and in melanoma metastases, with over 25% (2/8) of the lesions investigated. We conclude that MSI occurs in a considerable subset of malignant melanomas and that there is a pattern consistent with increasing frequency of MSI with progression of melanocytic tumours.     

Moesin and CD44 expression in cutaneous melanocytic tumours

The ERM (ezrin, radixin and moesin) family members, located just beneath the plasma membranes, are thought to be involved in the association of actin filaments with the plasma membrane. One of the family members, moesin, is reported to bind to CD44. Splice variants of CD44 are thought to be associated with tumour progression or differentiation. Our aim was to investigate immunohistochemically the expression of moesin together with CD44 on paraffin tissue sections of a series of melanocytic tumours. The material included 12 ordinary melanocytic naevi, six Spitz naevi, eight dysplastic naevi, six blue naevi, seven malignant melanomas in situ , 15 primary malignant melanomas, five metastatic melanomas to the skin and five lymph node metastases. In the normal skin and the melanocytic tumours the expression of moesin was largely similar to that of CD44 standard. Strong moesin staining was observed in benign melanocytic lesions and melanomas in situ . However, the expression was decreased in advanced malignant melanomas. The moesin labelling in melanoma cells was downregulated with the depth of dermal invasion. The immunoreactivity was also diminished in the skin metastases and the lymph node metastases of melanoma. These results suggest that in melanocytic tumours, the alternation in the expression of moesin may be involved in the progression of malignancy.     

Specific dermoscopy patterns and amplifications of the cyclin D1 gene to define histopathologically unrecognizable early lesions of acral melanoma in situ

OBJECTIVE: To define early lesions of acral melanoma in situ that cannot be recognized histopathologically. DESIGN: A retrospective review of the clinical, dermoscopic, and histopathological findings. SETTING: University department of dermatology. PATIENTS: Thirty-three patients with melanocytic lesions on acral volar skin that were clinically suspected of being early melanomas. MAIN OUTCOME MEASURES: Fluorescent in situ hybridization studies to detect the cyclin D1 gene amplification in proliferating melanocytes, which is a characteristic genetic aberration recently found in acral melanoma. RESULTS: Seventeen of 33 lesions were histopathologically diagnosed as either melanoma in situ (8 lesions) or benign melanocytic nevi (9 lesions). Amplification of the cyclin D1 gene was observed in 2 (25%) of the 8 melanomas in situ. None of the 9 nevi showed the amplification. The remaining 16 lesions were, however, difficult to classify histopathologically because most of them showed only a slight increase of nonatypical melanocytes in the basal cell layer of the epidermis. On dermoscopic examination, 9 of these 16 lesions exhibited the parallel ridge pattern that has been reported to be highly specific to melanoma in situ, and 4 (44%) of them had amplifications of the cyclin D1 gene. Amplifications were not found in any of the remaining 7 lesions that showed dermoscopic patterns suggestive of melanocytic nevi. CONCLUSIONS: Cyclin D1 gene amplification detected by fluorescent in situ hybridization identified a very early progression phase of acral melanoma that precedes histopathologically defined melanoma in situ. The present study also indicates the specificity of the parallel ridge pattern on dermoscopy to detect melanomas on acral volar skin at such a very early developmental phase.     

Quantitative surface topography as a tool in the differential diagnosis between melanoma and naevus

Background/aims: The mainstays of the clinical diagnosis of melanoma are asymmetry, border irregularity, color variegation, and a diameter >6 mm, and any major progress in diagnostic accuracy will probably be related to the development of additional criteria. Such independent criteria might arise from the study of the geometry of the tumour surface, because this quality has been substantially disregarded until now. Our work is aimed at utilizing the surface topography for the differential diagnosis between malignant melanoma and naevocytic naevus. Methods: Dynamically focusing optical profilometry was used to measure the surfaces of negative replicas of melanocytic skin tumours and of the surrounding normal skin. 21 silicone imprints of superficial spreading melanomas and 25 imprints of naevocytic naevi were examined. Results: Melanomas and naevi differed with respect to a variety of statistical surface parameters, and a linear discriminant analysis correctly allocated 19 out of 21 melanomas (90%) and 21 out of 25 naevi (84%). To get an unbiased estimate of the errors to be expected with this classification rule, we calculated bootstrap-corrections to the apparent errors. Estimated probabilities of correct allocation were 84.1% for melanomas and 77.1% for naevi. Conclusion: Our findings suggest that simple statistical parameters of surface topography can differentiate effectively between malignant melanomas and naevocytic naevi.