Targeted therapy in patients with PIK3CA-related overgrowth syndrome.

CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal naevi, scoliosis/skeletal and spinal syndrome) is a genetic disorder that results from somatic, mosaic gain-of-function mutations of the PIK3CA gene, and belongs to the spectrum of PIK3CA-related overgrowth syndromes (PROS). This rare condition has no specific treatment and a poor survival rate. Here, we describe a postnatal mouse model of PROS/CLOVES that partially recapitulates the human disease, and demonstrate the efficacy of BYL719, an inhibitor of PIK3CA, in preventing and improving organ dysfunction. On the basis of these results, we used BYL719 to treat nineteen patients with PROS. The drug improved the disease symptoms in all patients. Previously intractable vascular tumours became smaller, congestive heart failure was improved, hemihypertrophy was reduced, and scoliosis was attenuated. The treatment was not associated with any substantial side effects. In conclusion, this study provides the first direct evidence supporting PIK3CA inhibition as a promising therapeutic strategy in patients with PROS.

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.

The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Immunohistochemical expression of cyclooxygenase-2 (COX-2) in oral nevi and melanoma.

BACKGROUND: Cyclooxygenase-2 (COX-2) catalyses the conversion of arachidonic acid to prostaglandin, and its overexpression has been demonstrated in different malignant tumors, including cutaneous melanoma. However, no data about the expression of this protein in oral melanocytic lesions are available to date. The aim of this study was to evaluate the immunohistochemical expression of COX-2 in oral nevi and melanomas, comparing the results with correspondent cutaneous lesions. METHODS: COX-2 was evaluated by immunohistochemistry in 49 oral melanocytic lesions, including 36 intramucosal nevi and 13 primary oral melanomas, and in four cutaneous nevi and eight melanomas. RESULTS: All cases of oral and cutaneous melanomas were positive for COX-2. On the other hand, all oral and cutaneous melanocytic nevi were negative. CONCLUSION: COX-2 is highly positive in oral melanomas and negative in oral nevi and might represent a useful marker to distinguish melanocytic lesions of the oral cavity.

In-situ hybridization-based quantification of hTR: a possible biomarker in malignant melanoma.

AIMS: Telomerase is reactivated in most cancers and there is accumulating evidence that this is a driver event in malignant melanoma (MM). Thus, our aim was to evaluate if in-situ hybridization (ISH)-based quantification of telomerase RNA (hTR) could be used to distinguish MM from naevi, and if there was a correlation with the Breslow thickness. RESULTS AND METHODS: We created a tissue microarray (TMA) from formalin-fixed and paraffin-embedded tissue samples from 17 MM and 23 naevi, performed ISH targeting hTR, and quantified the signals. We found a more than eightfold greater number of hTR signals per nucleus in the MM samples compared to the naevi, and a positive correlation (P = 0.0381) between the number of hTR signals per nucleus and the Breslow thickness. CONCLUSION: Quantification of hTR ISH signals clearly distinguish MM from naevi (P < 0.0001) and the number of signals per nucleus correlates with the Breslow thickness, suggesting that hTR might be a valuable biomarker in MM. Furthermore, as ISH-based detection requires the presence of both hTR and telomerase reverse transcriptase (hTERT), it might be an indicator of active telomerase and thus have future relevance as a predictive biomarker for anti-telomerase treatment.

Dysregulation of redox-state-regulating enzymes in melanocytic skin tumours and the surrounding microenvironment.

AIMS: To investigate redox-regulating enzymes that may have a special role in melanoma pathogenesis due to continuous exposure to microenvironment-produced and ultraviolet radiation-induced oxidative stress. METHODS AND RESULTS: We assessed immunohistochemically the expression of antioxidant enzymes peroxiredoxins (Prxs) I-IV, sulfiredoxin (Srx) and redox-regulated proto-oncogene DJ-1 in material consisting of 30 benign naevi, 14 lentigo malignas and 67 malignant melanomas. Evaluation of immunostaining was performed with special attention paid to protein expression in different tumour compartments. In particular, the expression patterns of nuclear Prx I and Prx II and cytoplasmic DJ-1 were decreased significantly in melanomas compared with dysplastic and benign naevi. In multivariate analysis, several prognostic factors were identified: Prx III expression in the cytoplasm of stromal fibroblasts was associated with shortened melanoma-specific survival [hazard ratio (HR) 6.730; 95% confidence interval (CI) 1.579-28.689], while cytoplasmic Prx IV expression in endothelial cells (HR 6.563; 95% CI 1.750-24.620) and Srx expression in the cytoplasm of keratinocytes (HR 6.988; 95% CI 1.559-31.324) were associated with better prognosis independently of ulceration, thickness of melanoma or its diagnostic type. CONCLUSIONS: Redox-regulating enzymes have the potential to serve as novel prognostic factors and targeting them may offer new therapeutic options in malignant melanoma.

TGF-ß signaling, activated stromal fibroblasts, and cysteine cathepsins B and L drive the invasive growth of human melanoma cells.

Accumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional-Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane-permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-ß production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-ß signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas.

Expression of fatty acid synthase (FASN) in oral nevi and melanoma.

OBJECTIVE: The aim of this study was to determine the expression of fatty acid synthase (FASN) in oral nevi and melanomas, comparing the results with correspondent cutaneous lesions. MATERIALS AND METHODS: Expression of FASN was evaluated by immunohistochemistry in 51 oral melanocytic lesions, including 38 intramucosal nevi and 13 primary oral melanomas, in 10 cutaneous nevi and in 14 melanomas. RESULTS: Fatty acid synthase was strongly expressed only in melanomas, either of the oral mucosa or cutaneous. On the other hand, most oral and cutaneous nevi were negative, with a few oral cases showing focal and weak expression. CONCLUSION: Fatty acid synthase is expressed in malignant melanocytes, and it can be a helpful marker to distinguish oral melanomas from oral melanocytic nevi.

Different patterns of expression of the erbB family of receptor tyrosine kinases in common nevi, dysplastic nevi, and primary malignant melanomas: an immunohistochemical study.

erbB receptors contribute to tumor formation and progression. Variable expression of erbB1, erbB2, and erbB3 has been reported in nevi and melanomas; erbB4 has hardly been investigated. We examined the expression of all 4 erbB receptors in common and dysplastic nevi and melanomas. Formalin-fixed, paraffin-embedded tissues of 100 melanomas, 27 common nevi, and 23 dysplastic nevi were immunostained with antibodies against the 4 erbB receptors. erbB3 and erbB4 showed stronger positivity in nevi than in melanomas, and in common than in dysplastic nevi. Staining pattern was more orderly in nevi than in melanomas. Common nevi showed more prominent membranous staining for erbB3 than dysplastic nevi followed by melanomas. In melanomas, greater thickness was associated with more widespread erbB2 and erbB3 staining in the vertical than in the radial growth phase, and in the dermal than in the epidermal component. Higher mitotic counts were associated with more widespread and intense erbB2 expression in the vertical growth phase than in the radial growth phase and in the dermal than in the epidermal component. Melanomas with more widespread erbB2 staining had heavier lymphocytic infiltrates. erbB1 expression was negligible in all groups. erbB2, erbB3, and erbB4 are expressed in all subtypes of melanocytic lesions, but with quantitative and qualitative differences. Receptor expression seems to decrease and to become less mature and orderly with tumor progression. The complex patterns of erbB receptor expression in melanocytic lesions warrant further investigation.

Detection of mRNA of Telomerase Protein in Benign Naevi and Melanomas Using RNAscope.

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.

Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion.

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma.

Expression of cyclo-oxygenase-2 in macrophages associated with cutaneous melanoma at different stages of progression.

The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2 S): cystathionine γ lyase (CSE) and cystathionine ß synthase (CBS). A third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), synthesizes H2 S in the presence of the substrate 3-mercaptopyruvate (3-MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non-lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2 S donors, the most active of which was diallyl trisulfide (DATS). The main pro-apoptotic mechanisms involved were suppression of nuclear factor-κB activity and inhibition of AKT and extracellular signal-regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l-cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l-cysteine/CSE/H2 S pathway is involved in melanoma progression.

Decreased collagenase production by regional fibroblasts cultured from skin of a patient with connective tissue nevi of the collagen type.

A 10-yr-old female presented with cerebriform tumors covering the plantar surfaces of both feet. Histologically, the lesions consisted of thick collagen fibers and the content of collagen per surface area of skin was increased about 8-fold. Examination of the collagen by SDS-polyacrylamide gel electrophoresis, after limited pepsin proteolysis, showed that the lesions consisted almost exclusively of type I collagen, the predominant collagen type in human skin. Thus, a diagnosis of connective tissue nevi of the collagen type was made. Fibroblast cultures were established from the affected and normal-appearing areas of the skin, and examined for the rate of collagen synthesis, production of collagenase and growth kinetics of the cells. Cell cultures derived from the lesion and from control skin synthesized procollagen at the same rate and in a normal type I/type III procollagen ratio. However, the production of enzymatically active and immunologically detectable collagenase was reduced by 70-82% in the cultures derived from the lesion as compared to controls (p less than 0.005). Fibroblasts derived from the lesions also displayed a mean population doubling time of 1.17 +/- 0.08 days compared to 1.83 +/- 0.24 and 1.92 +/- 0.09 days for control cell strains and cells derived from normal skin of the patient, respectively (p less than 0.025). These results suggest that the excessive deposition of collagen in this case may have resulted from decreased local degradation of collagen. Enhanced proliferative capacity of the regional fibroblasts may have contributed to the accumulation of collagen in these lesions.

Telomerase activity and expression of apoptosis and anti-apoptosis regulators in the progression pathway of human melanoma.

The stages defining the progression pathway of human melanoma are atypical nevi, the precursor lesions and risk markers of melanoma, melanoma in situ and melanoma in the radical growth phase (RGP), which represent the early stages of melanoma development, and primary melanoma in the vertical growth phase (VGP) and melanoma in the metastatic growth phase (MGP), which are the advanced stages of the disease. Unlike cells obtained from VGP and MGP melanomas, which can be established as cell lines, cells derived from atypical nevi, melanoma in situ, and RGP melanoma cannot be propagated in vitro. Thus, information regarding molecular markers that may be differentially expressed in the early versus the advanced stages of this disease can only be obtained from the analysis of specimens. Since activation of telomerase and deregulation of apoptosis contribute to the pathogenesis of a significant number of human malignancies, we conducted a study, using nevus and melanoma specimens, to determine at what stage in the progression pathway of melanoma, telomerase activity can first be detected, and whether concordant with telomerase activation, one might observe a stage-specific switch from expression of promoters to inhibitors of apoptosis. The findings described here, demonstrate telomerase activity in some but not all MGP melanomas and not in any of the preceding pathological stages, and no apparent imbalance between pro- and anti-apoptotic markers in telomerase-positive MGP melanomas compared to telomerase-negative nevi and telomerase-negative VGP and MGP melanomas.

Immunohistochemical studies of beta-hexosaminidase in neoplastic and normal tissues with monoclonal antibodies.

A total of 87 human specimens with 10 histological types of primary neoplasm were studied immunohistochemically with monoclonal antibodies specific for beta-hexosaminidase (Hex). High levels of Hex were found in malignant neoplasms of the skin, cervix, colorectum and in benign as well as neoplastic plasma cells, while no activity was detected in normal epidermis, normal colorectal epithelium or benign naevi. The strongest immunohistochemical reaction was revealed in tumor cells of malignant melanoma. Adenomas and adenocarcinomas of the colorectum showed high levels of Hex with a basal pattern of immunoreactivity more frequent in the tumor cells of adenocarcinomas than adenomas. Fibroblasts and macrophages in the tumors often disclosed immunoreactivity. In most of the sections (including those from plasma cell neoplasms), 7E4 antibody showed low immunoreactivity compared to 2E3, except for non-neoplastic plasma cells, which were as a rule positive with 7E4 and largely negative with 2E3 antibody. This result probably indicated different isoenzymes in benign and neoplastic plasma cells.

Expression of activated TrkA protein in melanocytic tumors: relationship to cell proliferation and clinical outcome.

We evaluated expression of activated nerve growth factor receptor tyrosine kinase (p-TrkA) by immunohistochemical analysis in 152 primary and 64 metastatic human melanoma biopsy specimens and 8 nevi. Membranous, cytoplasmic, and/or nuclear expression of p-TrkA was seen in 54.6% of primary melanomas and 30% of metastases. Membranous p-TrkA was detected in 21.7% of primary and 14% of metastatic melanomas and cytoplasmic immunoreactivity in 28.9% of primary tumors and in 22% of metastases. Significantly fewer metastases than primary tumors expressed nuclear p-TrkA (16% vs 39.5%; P = .006). A significantly higher percentage of nodular than superficial spreading melanomas expressed membranous (40% vs 11%; P < .0001) p-TrkA. Nevi expressed no membranous or cytoplasmic p-TrkA; 63% showed nuclear reactivity. p-TrkA expression varied significantly with thickness of primary tumors (lower expression in thinner lesions: membranous, P = .004; cytoplasmic, P = .001; nuclear, P = .031). An association between ulceration and membranous (P = .054), cytoplasmic (P < .0001), and nuclear (P = .022) p-TrkA expression was found. Membranous p-TrkA significantly predicted decreased overall survival (P = .002). A significant association between membranous p-TrkA and cyclin A (P = .004) and Ki-67 (P < .0001) and between cytoplasmic p-TrkA and cyclin A (P < .0001), Ki-67 (P = .004), and cyclin D3 (P = .027) was found. p-TrkA had no effect on MAPK(ERK1/2) activation. A significant inverse association between cytoplasmic beta-catenin and cytoplasmic p-TrkA levels (P = .006) and between nuclear p-TrkA and cytoplasmic E-cadherin (P = .022) was seen. We present the first evidence of a role for TrkA activation in a subset of melanomas as a predictor of an aggressive phenotype and poor outcome.

Expression of 12-lipoxygenase as a biomarker for melanoma carcinogenesis.

12-Lipoxygenase (12-LOX), through its metabolite 12( )-hydroxyeicosatetraenoic acid [12( )-HETE], has been demonstrated to play a pivotal role in experimental melanoma invasion and metastasis, and 12-LOX expression may be important in early human melanoma carcinogenesis. We have studied the differences in 12-LOX protein expression during the progression of melanoma from human melanocytic cells to benign and dysplastic naevi to malignant metastatic disease. 12-LOX expression was determined in normal human skin melanocytes and in melanocytes found in compound naevi, dysplastic naevi and melanomas using a platelet-type 12-LOX antibody with a diaminobenzidine immunoperoxidase system detection system and was quantified using the analysis software NIH Image 1.62. Mean cellular pixel densities for 12-LOX staining ( = 50 cells/histological type) were unchanged in compound naevi ( = 0.14) and were increased in dysplastic naevi and melanomas compared with normal skin melanocytes ( = 0.03 and = 0.01, respectively). Similarly, melanomas had higher levels of expression compared with dysplastic naevi ( = 0.03). 12-LOX expression was significantly different between compound naevus and dysplastic naevus melanocytes ( = 0.01). These data suggest that 12-LOX may be an important novel marker for cancer progression within the melanoma system, and therefore could be a useful biomarker and therapeutic target for melanoma chemoprevention.

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

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 multi-step 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.

The value of cyclooxygenase-2 expression in differentiating between early melanomas and histopathologically difficult types of benign human skin lesions.

Early cutaneous melanomas may present a substantial diagnostic challenge. We have already reported that expression of cyclooxygenase-2 (COX-2) may be useful for differentiating between cutaneous melanomas and naevi. The purpose of this study was to examine the value of COX-2 in a challenging task of differential diagnosis of early melanomas and melanocytic naevi considered by histopathologists as morphologically difficult to unequivocally diagnose as benign lesions. The material for the study comprised formalin-fixed paraffin-embedded samples of 46 naevi (including 27 cases of dysplastic, Spitz and Reed naevi) and 30 early human cutaneous melanomas. The expression of COX-2 was detected immunohistochemically. Melanomas expressed COX-2 significantly more strongly compared with naevi. The test, on the basis of determination of the percentage fractions of COX-2-positive cells, allows for differentiation of early skin melanomas and naevi with high sensitivity and specificity. Receiver operating characteristic analysis of the test results yielded areas under receiver operating characteristics curves (AUC)=0.946±0.030 for central regions and AUC=0.941±0.031 for the peripheries of the lesions. The performance of the test in differentiating between melanomas and the naevi group comprising dysplastic, Spitz and Reed naevi was also good, with AUC=0.933±0.034 and 0.923±0.037 for the central and the border regions of the lesions, respectively. Using a more complex diagnostic algorithm also accounting for the staining intensity did not result in an improvement in the resolving power of the assay. A diagnostic algorithm using differences in the percentage fractions of cells expressing COX-2 may serve as a useful tool in aiding the differential diagnosis of 'histopathologically difficult' benign melanocytic skin lesions and early melanomas.