The Markers Auxiliary in Differential Diagnosis of Early Melanomas and Benign Nevi Sharing Some Similar Features Potentially Leading to Misdiagnosis - A Review of Immunohistochemical Studies.

Although most melanocytic skin lesions are correctly diagnosed, numerous studies have shown interobserver disagreement. This review analyzes 20 molecules as immunohistochemical markers for distinguishing dysplastic and/or Spitz nevi from early melanomas (in situ, Clark level I or II and/or Breslow thickness at most 1 mm). The detected presence and/or level of tested molecules was significantly different in early melanomas than in dysplastic and Spitz nevi for six and seven potential markers, respectively. The most promising results were obtained for 5-hydroxymethylcytosine, cyclooxygenase-2 and PReferentially expressed Antigen in MElanoma whose levels were different in dysplastic and Spitz nevi compared to early melanomas.

The detectability of intraepidermal melanocytes-A narrative review of immunohistochemical studies.

Some intraepidermal lesions, even the nonmelanocytic ones, may mimic melanoma in situ. The cytoplasmic clarity in atypical basal cells, architectural disorder with cell aggregates or nest-like structures within the epidermis often cause challenge particularly in sun-damaged skin. The aim of this review is to evaluate the effectiveness of the most often used immunohistochemical melanocyte markers in the diagnosis of intraepidermal lesions. All the analyzed markers can be useful in detection of intraepidermal melanocytes that occur more densely in lentigo maligna than in solar lentigo or sun-damaged skin. However, the number of these cells is underestimated by HMB-45 and Mel-5. The cytoplasmic Melan-A staining may overestimate melanocyte number and highlight the dendritic process. Atypical melanized keratinocytes in actinic keratoses and nest-like structures in lichenoid dermatoses may mimic atypical melanocytes in melanomas in situ. S-100 remains a marker of high sensitivity but low specificity. The nuclear localization of MITF, SOX-10, and PRAME overcomes the problem of melanosome transfer to cells of other types. Neither MITF nor SOX-10 is detectable in keratinocytes, which makes them useful in distinguishing actinic keratoses from melanomas in situ. The use of markers considered as melanocyte specific suggests that lichenoid dermatoses constitute a heterogeneous group of lesions.

Near-infrared, wavelength, and illumination scanning holographic tomography.

We present a holographic tomography technique in which the projections are acquired using both wavelength and illumination scanning in the near-infrared region. We show how to process the acquired data to obtain correct values of three-dimensional refractive index distributions in both single-wavelength and multi-wavelength data acquisition schemes and how to properly account for the dispersion of the sample. We perform numerical and experimental comparisons of different illumination scenarios to determine the most efficient measurement protocol. We show that the multi-wavelength protocol is advantageous in terms of signal-to-noise ratio and contrast-to-noise ratio over single-wavelength protocols, even for the same number of projections used for reconstructions. Finally, we show that this approach is suitable for providing high-quality refractive index distributions of relatively thick colon cancer samples.

Expression of Cyclooxygenase-2 in Human Epithelial Skin Lesions: A Systematic Review of Immunohistochemical Studies.

Permanent, elevated expression of cyclooxygenase-2 (COX-2) in keratinocytes of epidermis can stimulate its hyperplasia and constitute a factor promoting cancer development, as demonstrated in animal models. Intratumoral level and localization of COX-2 in epithelial lesions of human skin was examined immunohistochemically in 26 studies. In squamous cell carcinomas (SCCs), strong staining was observed with great compatibility. High COX-2 detectability throughout the entire tumor mass could be helpful in the finding of SCC cells. However, in basal cell carcinomas, and precancerous lesions, frequency and detection level of this protein, as well as the type and/or localization of stained cells within the tumor, varied among different research groups. The discrepancies may be due to the heterogeneity of each of these 2 groups of lesions. However, differences in COX-2 staining in normal skin indicate also possible methodological reasons. In general, COX-2 levels were significantly decreased in basal cell carcinomas compared with SCCs, which could be used in the differential diagnosis of these cancers. Reduced, although heterogenous, COX-2 expression in precancerous lesions may suggest its association with SCC development. These observations are consistent with data on the efficacy of preventive and therapeutic effects of nonsteroidal anti-inflammatory drugs that are COX-2 inhibitors.

Immunohistochemical detectability of cyclooxygenase-2 expression in cells of human melanocytic skin lesions: A methodological review.

Increased cyclooxygenase-2 (COX-2) expression is thought to support tumorigenesis through various mechanisms and is analyzed as a potential cancer marker. In 18 studies, COX-2 expression in melanocytic lesions of human skin was examined immunohistochemically. However, results obtained by individual research groups differ in terms of detection frequency and level of this protein, as well as localization of stained cells within tumor. Possible reasons for the discrepancies are analyzed in this review: the application of different antibodies, the use of standard histopathological sections or tissue microarrays and the analyzes of staining results based on different algorithms. COX-2 level is significantly lower in nevi than in melanomas, increases gradually with progression of these malignant cancers and reaches the highest values in metastases. These gradual changes in COX-2 expression appear to be difficult to analyze based only on subjective assessment of staining intensity. The most convergent data were obtained using antibodies for N-terminal fragments of COX-2 protein and analyzing results based on calculation of percentage fraction of positive cells. The extent of stained area in specimen thus appears to be more important than the intensity of staining in terms of evaluation of COX-2 performance as a diagnostic and prognostic marker of cutaneous melanoma.

Enhanced intratumoral expression of RNF2 is a favorable prognostic factor for patients with cutaneous melanoma?

Recent studies involving melanoma cell lines suggest that enhanced expression of epigenetic regulator RNF2 supports proliferation and promotes metastasis. However, it is not clear to what extent those data apply to disease progression and prognosis for melanoma patients. Therefore the aim of the present study was to assess the prognostic power of RNF2 intratumoral expression by melanoma cells. RNF2 was detected immunohistochemically in standard formalin-fixed paraffin-embedded samples of 9 benign nevi, 60 melanomas and 24 nodal metastases. The lowest percentage of RNF2-positive melanocytes found in nevi was comparable to expression levels in normal skin. The RNF2 expression found in melanomas was significantly higher and it was even more enhanced in metastases. The increased occurrence of RNF2 expressing cells was positively correlated with longer patients' overall survival. Moreover, a negative correlation was found between intratumoral RNF2 expression and number of generated metastatic lesions. Our data indicate that development of melanoma is associated with significant changes in RNF2 intratumoral expression and imply that at least for some patients the enhancement of the expression levels of RNF2 in both primary and metastatic lesions may be considered a favorable prognostic factor in melanoma.

Prognostic significance of RBP2-H1 variant of JARID1B in melanoma.

BACKGROUND: Histone demethylase JARID1B plays several context dependent roles in epigenetic regulation of cellular differentiation in normal development and is highly expressed in multiple human cancers. The protein is a strong transcriptional repressor capable of downregulating numerous genes. There are three splicing isoforms of JARID1B, however the links between the protein structure and function are not clear. The expression pattern of JARID1B in human melanoma seems to be different from observed in other cancers. Moreover, up to now no data on the impact of JARID1B expression in cutaneous melanoma on the patients' prognosis have been reported. METHODS: We investigated immunohistochemically the association of intratumoral expression of total JARID1B protein and its RBP2-H1 isoform in primary and metastatic melanomas with prognosis for the patients. RESULTS: Expression of both total JARID1B protein and its RBP2-H1 variant was found in all the melanomas investigated. Our results indicate, however, that only high (above 90% of the cells) intratumoral expression of RBP2-H1 can be considered prognostic factor associated with worse overall survival of the patients. CONCLUSIONS: Such results if considered together with data demonstrating a switch to enhanced expression of RBP2-H1 at early stages of malignant transformation of melanocytes are in agreement with hypothetical crucial role of JARID1B in the course of melanoma development and progression and suggest that altered splicing of JARID1B may be important factor increasing melanoma aggressiveness.

Intratumoral expression of cyclooxygenase-2 (COX-2) is a negative prognostic marker for patients with cutaneous melanoma.

Because of the well-known heterogeneity of melanomas, prognosis of the disease is often difficult to assess even for lesions classified in similar stages. The aim of this study was to assess the usefulness of COX-2 as a melanoma prognostic marker and to establish an optimum algorithm for analysis of COX-2 expression levels in lesions of interest. Expression of COX-2 was detected immunohistochemically in standard sections of formalin-fixed paraffin-embedded tissue samples of 85 primary melanomas, 36 lymph node metastases, and five skin metastases including 39 cases of paired primary and metastatic lesions obtained from the same patient. Enhanced expression of COX-2 in primary melanomas is an indicator of poorer prognosis. A significant correlation was found between high expression of COX-2 in primary lesions and shorter survival. The enhancement of COX-2 expression is also positively correlated with other prognostic factors such as tumor thickness and infiltration level, ulceration, high mitotic index, more invasive histologic type, vertical growth phase, and lymph node metastasis. On the whole, the results suggest that intratumoral expression of COX-2 is a strong negative prognostic marker for patients with melanoma. Moreover, our work shows that a simple and objective immunohistochemical scoring algorithm involving the determination of only a percentage fraction of positively stained cells is sufficient to obtain the prognostic information.

Altered Splicing of JARID1B in Development of Human Cutaneous Melanoma?

Upregulated expression of histone H3K4 demethylase JARID1B has been found in several types of human cancer, but the expression pattern of this protein in benign naevi and human cutaneous melanomas seems to differ from that described for other tumors. We demonstrate that the apparent contradiction may be because of the fact that the malignant transformation of melanocytes is associated not so much with a general enhancement of a total expression of JARID1B but rather with a change in relative expression levels of individual splicing variants of the protein. Our data indicate that parallel immunohistochemical assays of the expression levels of all the isoforms and of the RBP2-H1 variant of JARID1B may be an efficient technique of differentiating between benign naevi and melanomas.

Stromal, rather than epithelial cyclooxygenase-2 (COX-2) expression is associated with overall survival of breast cancer patients.

BACKGROUND: Prognostic value of enhanced COX-2 expression in breast cancer has been controversial for a long time. The opinions vary widely between studies. Moreover, significant majority of studies considered only COX-2 expression in cancer epithelial cells. METHODS: We examined the prognostic value of COX-2 expression in both epithelial and stromal cells using three different antibodies and three algorithms of immunohistochemical scoring and categorizing the tumours into COX-2 overexpressing groups. RESULTS: Our results demonstrate that COX-2 expression in stromal cells is independent prognostic factor indicating worse overall survival of patients. Such a result was obtained using each of the three antibodies and two of the algorithms used for evaluations of COX-2 expression levels. We also show that immunohistochemical assessment of the prognostic value of COX-2 expression in cancer epithelial cells depends to a large extent on a combination of primary antibodies and algorithms used for determination of the COX-2 over-expressing tumours. CONCLUSIONS: Our results indicate that stromal expression of COX-2 is independent prognostic parameter relatively insensitive to variations in sensitivity of antibodies used for its determination. Wide scatter of the published results concerning prognostic value of COX-2 expression in breast cancer tissues seems to be due to a large extent to multitude of antibodies and scoring algorithms used by different groups.

Different detectability of cyclooxygenase-2 (COX-2) protein in standard paraffin sections and tissue microarrays of human melanomas and naevi - comparative study.

Cyclooxygenase-2 (COX-2), overexpressed in many types of human cancer, may be a valuable marker for human melanoma. However, there are discrepancies between expression levels detected by different groups. The majority of the studies were carried out using standard paraffin sections. Tissue microarrays (TMAs) might enable analysis of COX-2 expression in numerous lesions. Our study assesses to what extent reprocessing of tissue samples used for preparing TMAs may influence reproducibility of data obtained for standard sections. The study included TMAs and standard histopathologic sections. COX-2 was detected by immunohistochemistry with two primary antibodies targeting different epitopes. COX-2 expression levels detected with both antibodies in standard sections were similar as in our previous study. Surprisingly, results obtained in TMAs were significantly different. While one of the antibodies yielded for TMAs results similar to standard sections, COX-2 expression levels found with the second antibody were very low and expression patterns strikingly different from those observed for standard sections and for both TMAs studied with the first antibody. Good performance of the antibodies found in standard sections of human skin and melanocytic lesions does not guarantee similar results in TMAs. The finding discloses a new aspect of immunohistochemical assays involving TMAs.

JARID1B expression in human melanoma and benign melanocytic skin lesions.

It has been suggested that dynamically regulated expression of the JARID1B protein is required for the continuous growth of tumors and at the same time downregulated in melanoma. The majority of the data on a role of JARID1B in maintaining tumor growth has come from in-vitro and xenografting experiments, with only one immunohistochemical study involving human tissues. We compared JARID1B expression levels in human melanomas and benign nevi and analyzed patterns of spatial distributions of positive cells among different skin layers of the lesions. The expression of JARID1B was evaluated by immunohistochemistry in formalin-fixed paraffin-embedded samples of 30 nevi, 27 primary melanomas, four lymph node metastases, and one local recurrence of melanoma. Staining for JARID1B protein was stronger in melanomas compared with nevi. We also found a significant difference in the spatial distribution of positive cells in individual skin layers of nevi and melanomas. Staining of melanocytes located in granular and spinous layers of nevi was observed very rarely, whereas for melanomas, the mean percentage fractions of positive cells present in these layers exceeded the maximum values found for nevi. The spatial patterns and expression levels of JARID1B did not change significantly with melanoma progression and were similar for primary, metastatic, and recurrent melanomas. Contrary to earlier reports, this study shows enhanced expression of JARID1B by melanoma cells and indicates that such an enhancement may be an early event in the disease progression, is not correlated with melanoma invasiveness, and therefore may not be a suitable candidate as a prognostic marker.

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.

Different expression of cyclooxygenase-2 (COX-2) in selected nonmelanocytic human cutaneous lesions.

The aim of our study was to elucidate the possible involvement of COX-2 in the development and/or progression of nonmelanocytic skin lesions. To evaluate the usefulness of that enzyme as a potential molecular marker, we examined the intensity and spatial distribution of COX-2 expression in selected types of such tumors using the same immunohistochemical procedure as in our earlier studies of melanocytic cancers. We examined 20 benign epithelial lesions, 11 precancerous lesions, 21 basal cell carcinomas (BCC), 14 squamous cell carcinomas (SCC) and eight fibromas. The levels of COX-2 expression detected in benign lesions and in normal skin were comparable. Elevated expression of this protein may play a role in the development of SCC, as indicated by strong immunostaining both in SCCs and precancerous lesions. Significantly stronger staining in SCCs compared to BCCs may indicate a role of COX-2 in cancer malignancy and serve as an indicator useful for differential diagnostics of the two types of cancer. Strong staining in all skin layers of SCC may help in detecting cancer cells infiltrating surrounding skin layers.

Cyclin-dependent kinase 2 (CDK-2) expression in nonmelanocytic human cutaneous lesions.

Lesions originating from different types of skin cells differ significantly with respect to their pathologic importance. The aim of this work was to examine as to what extent the differences in the origin are reflected in expression levels of CDK-2 and to investigate whether CDK-2 expression might be considered as potential marker useful for diagnostics and assessment of invasiveness of human nonmelanocytic lesions. We conducted comparative immunohistochemical studies of expression of cyclin-dependent kinase 2 (CDK-2) in 16 benign epithelial skin lesions, 11 precancerous lesions, 19 cases of basal cell carcinoma (first such study), 14 squamous cell carcinomas (SCCs), and 7 fibromas. Development of benign epithelial skin lesions was not associated with considerable increase of the CDK-2 expression. Increase of the CDK-2 level was observed in precancerous lesions, and the expression was strongest in SCCs. The level of CDK-2 may be related to invasiveness of skin cancers, as squamous cell carcinomas expressed the enzyme significantly stronger than basal cell carcinomas. Higher percentage fraction of CDK-2 positive cells observed in SCC compared with precancerous lesions may be useful for histopathologic diagnostics of this cancer. Moreover, strong immunohistochemical CDK-2 staining of the cancer cells present deep in dermis may facilitate their detection in histopathologic examinations.

Cyclooxygenase-2 immunohistochemistry in human melanoma: differences between results obtained with different antibodies.

Several groups have reported that cyclooxygenase-2 (COX-2) expression is significantly enhanced in human melanomas, and that the expression of this protein may be useful as diagnostic and prognostic marker for the disease. At the same time, collective analysis of immunohistochemical data on the COX-2 expression in melanomas, presented by different researchers, shows a clear lack of consistency of reported results commonly assigned to differences in protocols used for the staining. This paper describes a study involving the parallel use of three different primary anti-COX-2 antibodies targeting different COX-2 epitopes. A surprising outcome is that although the three antibodies gave very consistent results for the COX-2 expression in keratinocytes, they showed significant differences in immunoreactivity for both melanocytic naevi and melanomas. This phenomenon has not been described before, and has implications for the selection of antibodies for studies on the diagnostic potential of COX-2 for melanoma.

Cyclooxygenase-2 (COX-2): first immunohistochemical marker distinguishing early cutaneous melanomas from benign melanocytic skin tumours.

We have reported recently that changes in expression level of COX-2 are correlated with development and progression of human melanoma. In this study, we investigated whether the COX-2 expression level might be a useful immunohistochemical marker for distinguishing cutaneous melanomas from benign melanocytic lesions. Up to now, immunohistochemical markers have not ensured satisfactory sensitivity and specificity of differential pathologic diagnosis of melanoma. The expression of COX-2 was determined immunohistochemically in formalin-fixed, paraffin-embedded specimens of 33 early Clark I/II melanomas and 58 naevi. Mean COX-2 expression in melanomas was significantly stronger than in naevi (P approximately 10(-13)). A simple diagnostic algorithm using threshold values of the COX-2 expression level allows for differentiation between early melanomas and naevi with high sensitivity (Se) and specificity (Sp) (for Se between 91 and 100%, Sp values change between 96.5 and 51.7%). Areas under the receiver operating characteristic curves were, respectively, 0.97+/-0.02 and 0.86+/-0.04 for the COX-2 expression in central and border regions of the lesions. For all the melanomas (not only the early ones),the respective areas under the ROC curve values were 0.98+/-0.01 and 0.97+/-0.02. In conclusion, COX-2 is the first immunohistochemical marker that allows the distinguishing of early melanomas from benign melanocytic lesions with both high sensitivity and specificity.

Cyclin-dependent kinase 2 expression in human melanomas and benign melanocytic skin lesions.

Cyclin-dependent kinase 2 (CDK-2) is strongly involved in regulating the progression of the cell cycle through G1/S checkpoint and S phase. Numerous studies demonstrated increased levels of CDK-2 (and also of its regulatory cyclins E and/or A) in different types of human tumours. Correlations found between the expression of those cell cycle regulators and progression and/or invasiveness of some tumours indicated the importance of CDK-2 as a potential prognostic marker. At the same time, in vitro studies of melanoma cell lines revealed melanocyte-specific regulation of CDK-2. The present study was aimed at examining levels of CDK-2 in human melanomas and benign pigmented lesions to evaluate whether it might be considered a potential molecular marker of melanoma progression. Expression of CDK-2 was determined immunohistochemically in formalin-fixed paraffin-embedded specimens comprising 76 lesions including 41 primary cutaneous melanomas, 15 lymph node melanoma metastases (in eight cases correlated with primary tumours), three melanoma recurrences (two cases correlated with both primary and metastatic melanomas) and 17 nevi. Our results demonstrate that development and progression of melanoma are associated with changes in CDK-2 expression level. Statistical significance of the observed correlations indicates that CDK-2 may be a suitable prognostic marker for melanoma and perhaps also a target for chemotherapeutic drugs.

Different expression of lysosome-associated membrane protein-1 in human melanomas and benign melanocytic lesions.

Lysosome-associated membrane protein-1 is a protein with a significant content of beta1,6-branched N-glycans. It is thought that enhanced expression of lysosome-associated membrane protein-1 in tumour cells may promote invasion by influencing both adhesion to extracellular matrix and perhaps also binding to endothelial cells. The present study was aimed at examining levels of lysosome-associated membrane protein-1 in human melanomas and benign pigmented lesions to evaluate whether this protein might be considered a potential molecular marker of melanoma progression. The expression of lysosome-associated membrane protein-1 was for the first time determined immunohistochemically in formalin-fixed paraffin-embedded specimens comprising 42 primary cutaneous melanomas, 15 lymph node melanoma metastases (11 correlated with primary tumours), three melanoma recurrences (correlated with both primary and metastatic melanomas), 27 nevi and four epithelial tumours (two seborrhoeic keratoses and two basal cell carcinomas). Our results demonstrate that development and progression of melanoma are associated with changes of the lysosome-associated membrane protein-1 level. The expression was strongest in melanoma recurrences and lymph node metastases, weaker in primary cutaneous melanomas and not detectable in melanocytes of pigmented nevi. Nodular melanomas expressed lysosome-associated membrane protein-1 at higher level than superficially spreading melanomas.

Expression of cyclooxygenase-2 in benign naevi and during human cutaneous melanoma progression.

Cyclooxygenase-2 (COX-2) is an enzyme that plays an important role in the production of prostaglandins. Numerous studies have demonstrated increased levels of COX-2 in human cancers of different types. It is thought that COX-2 may be involved in the development and progression of malignant tumours. However, data on the changes in COX-2 expression during the development and progression of human melanoma are relatively limited. Moreover, the results reported by different groups disagree to a large extent. The aim of this work was to evaluate whether COX-2 protein might be considered a potential molecular marker of melanoma progression. The expression of COX-2 was determined immunohistochemically in formalin-fixed, paraffin-embedded specimens of 64 human melanocytic skin tumours (17 naevi, 36 primary cutaneous melanomas and 11 lymph node melanoma metastases, with six pairs of primary and metastatic lesions obtained from the same patients). It was found that the expression level of COX-2 was dependent on both the stage and histopathological type of the melanoma. Collectively, our data indicate that changes in the expression level of COX-2 are correlated with the development and progression of human melanoma, and imply that the COX-2 protein may be considered a potential prognostic and predictive marker in malignant melanoma.