Melanocytic soluble adenylyl cyclase protein expression around lentigo maligna and in contralateral control skin.

Recurrence rates of both lentigo maligna (LM) and lentigo maligna melanoma (LMM) following conventional surgery are usually relatively high. We aimed to assess the frequencies of melanocytes in tumour-free margins around LM/LMM using soluble adenylyl cyclase (sAC) immunohistochemistry, and to compare these with those of matched healthy contralateral skin. Using the primary mouse-anti-human sAC antibody R21, we evaluated pan-nuclear melanocytic R21 immunostaining, and found that it was significantly (P < 0.001) higher in peritumoural melanocytes (median 20%; range 0-100%) than in contralateral healthy skin (mean 0%; range 0-20%). Accordingly, there was no correlation between peritumoural and contralateral R21 immunoreactivity (r = 0.12; P = 0.18). In conclusion, melanocytic R21 immunoreactivity in melanocytes is higher in tumour-free margins around LM/LMM than in site-matched contralateral skin. This observation may indicate that the biology of 'healthy'-appearing melanocytes around LM/LMM might be different from that of truly benign melanocytes.

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.

Soluble adenylyl cyclase antibody (R21) as a diagnostic adjunct in the evaluation of lentigo maligna margins during slow Mohs surgery.

Margin-controlled staged excision (slow Mohs) has emerged as a preferred method for the treatment of lentigo maligna (LM). The interpretation of margins for LM is one of the most challenging tasks faced by a dermatopathologist. R21 is a mouse monoclonal antibody against soluble adenylyl cyclase (sAC), overexpressed in the nuclei of LM but not in native melanocytes. The objective of this study was to validate the use of sAC immunohistochemistry in histological assessment of slow Mohs surgery margins for LM. Seventeen randomly selected cases of patients who underwent slow Mohs surgery for LM at Lahey Clinic, Burlington, MA, were studied. Ninety-nine margins were stained with R21 and microphthalmia transcription factor antibodies and reevaluated blindly by 2 observers. Sixteen of 17 lesions expressed sAC. In all cases, observers agreed on interpretation of R21 stains. In 85 (86%) margins, there was concordance between routine sections and R21 stains. In 14 margins (14%), the results were discrepant. In 2 margins, R21 identified foci of LM missed on routine sections. In 8 margins, atypical melanocytes, interpreted as positive in routine sections, were negative for R21 questioning the accuracy of the original interpretation. Microphthalmia transcription factor stained nuclei of melanocytes in all margins. We found significant correlation between assessment of margins by sAC immunohistochemistry and routine histology. Evaluation of sAC expression using R21 antibody is a useful diagnostic adjunct in the evaluation of margins of LM during slow Mohs surgery.

Tyrosinase immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution.

Monoclonal antibody T311 specifically detects tyrosinase protein expression. Tyrosinase-derived peptides are recognized by CD8+ T-cells and applied in immunotherapy. We examined formalin-fixed paraffin-embedded tissue of 50 melanoma (primary n=31, metastatic n=19) and 41 control cases (junctional, dermal, compound, Spitz, Reed, balloon-cell nevi) by immunochemistry using the alkaline phosphatase-anti-alkaline phosphatase method after antigen retrieval. Staining with mAb T311 showed a sensitivity of 94% for melanoma with a very high specificity for melanocytic cells. Immunopositivity (94% of melanomas overall) correlated inversely with clinical stage: clinical stage I and stage II showed 100%, stage III and stage IV 86% immunoreactivity each. Staining changed from an exclusively homogeneous pattern in early stages to a more heterogeneous pattern in later stages. Melanocytic control tissue like nevi of different subtypes all showed weak to moderate, homogeneous immunoreactivity with polarity towards the epidermis. RT-PCR ELISA analysis of short-term melanoma cell cultures displayed mRNA expression in only half of the originally immunopositive tumors only, suggesting rapid mRNA expression loss in culture. mAb T311 allows detection of melanoma-associated tyrosinase protein expression and thus profiling of melanomas using routine archival tissue suited for immunotherapy approaches involving tyrosinase derived epitopes.