Prevalence of Merkel cell polyomavirus DNA in cutaneous lymphomas, pseudolymphomas, and inflammatory skin diseases.

Several groups confirmed Merkel cell polyomavirus (MCPyV) as the likely causative agent of Merkel cell carcinoma. Hematolymphoid disorders are known to be a substantial risk factor for Merkel cell carcinoma, and vice versa. The association between MCPyV and hematologic neoplasms is poorly analyzed, as well as the speculation that lymphocytes may serve as reservoir for MCPyV. Therefore, we investigated the prevalence of MCPyV DNA in primary cutaneous T- and B-cell lymphomas, pseudolymphomas (PLs), and inflammatory skin diseases with dominant lymphocytic infiltrate. We performed a molecular pathology study in 22 tissue samples and 1 blood sample of different cutaneous lymphomas from 19 patients (17 mature T-cell neoplasms, 5 mature B-cell neoplasms, and 1 immature hematopoietic malignancy), 13 PLs from 12 patients, and 25 various inflammatory skin diseases from 23 patients. All tumors were analyzed for the presence of MCPyV DNA by polymerase chain reaction, confirmed by Southern blot hybridization of polymerase chain reaction products. We detected MCPyV DNA in 4 of 23 (17.4%) cutaneous lymphoma tissue samples (3 of 17 mature T-cell neoplasms and 1 of 5 mature B-cell neoplasms), in 2 of 13 (15.4%) PL tissue samples, and 2 of 25 (8%) inflammatory skin conditions (1 drug reaction and 1 erythema multiforme). We conclude that MCPyV DNA is infrequently, but consistently present in lesional tissue from patients with primary cutaneous lymphomas, PLs, and inflammatory skin diseases; prevalence is in the range of 8%-17%. Our results suggest that MCPyV does not play a significant role in the pathogenesis of cutaneous lymphoproliferative disorders.

Prevalence of MCPyV in Merkel cell carcinoma and non-MCC tumors.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the likely causative agent of Merkel cell carcinoma (MCC). However, the prevalence of MCPyV in non-MCC population and its possible role in the pathogenesis of other skin cancers are not known yet. METHODS: A molecular pathology study was performed in 33 MCC samples and 33 age- and sex-matched samples of sun exposed non-MCC tumors [12 seborrheic keratoses (SK), 11 basal cell carcinomas (BCC) and 10 lentigo maligna melanomas (LMM)]. All tumors were analyzed for presence of MCPyV-DNA by polymerase chain reaction (PCR) and Southern-Blot hybridization of PCR products. RESULTS: MCPyV sequences were detected in 21 MCC samples (64%) and in 2 non-MCC tumors of sun exposed skin (6%; both SK-patients). Neither the tissue samples from BCC nor LMM proved positive for MCPyV sequences. CONCLUSION: We were able to confirm prior data on prevalence of MCPyV-DNA in MCC. Furthermore, a female predominance of MCPyV-positive MCC-patients was detected. There was no relevant association of MCPyV with SK, BCC and LMM. Speculative, prevalence of MCPyV in an age- and sex-matched non-MCC population could average up to 6%.

Heterogeneity of T-cell clones infiltrating primary malignant melanomas.

It is established that primary malignant melanomas (pMM) can be infiltrated by T-cell populations with predominantly one T-cell clone. As pMM generally express multiple tumor-associated antigens (TAA), here we used laser-capture microdissection (LCM) to isolate different tumor-infiltrating lymphocyte (TIL) clusters in order to determine whether pMM are infiltrated only by one single clone or whether the TAA may attract various T-cell populations. As T-cell receptor (TCR) clonality is a useful tool for the demonstration of specific T-cell clones, we analyzed 56 pMM, three cutaneous melanoma metastases, and 15 pairs of pMM with a sentinel lymph node (SLN) for clonal rearrangements of the (TCR) gamma chain gene. We detected the clonality of TCR gamma chain gene in 25 of 56 pMM, and in 10 of 17 SLN studied. In four of the 15 pairs of primary tumor and SLN, we found clonal TCR gamma in both the melanoma and the SLN, with two pairs harboring the identical clone. As we detected different clones in pMM and the corresponding SLN, we subsequently performed LCM in 21 malignant melanomas with multiple lymphocytic clusters for the presence of focal clonal T cells in different regions of the melanoma. In seven melanomas, both clusters of TILs showed the same rearranged TCR gamma chain gene and in five of the seven biopsies the clonal rearrangement occurred in different variable (V) regions of the TCR gamma chain gene. These tumors showed infiltration by more than one clone. In 10 biopsies TCR clonality was restricted to one cluster, while the second microdissected sample of the infiltrate was polyclonal. In conclusion, within one primary malignant melanoma several T-cell clones with different rearrangements may occur. The balance between these clones may decide on the progress of melanoma.

Laser-capture microdissection: applications in routine molecular dermatopathology.

Advances in molecular pathology with the introduction of the Southern blot technique and the polymerase chain reaction (PCR) have emerged as important tools, which are frequently used in routine dermato-histopathology. Applications for PCR-based diagnostics are particularly helpful for the determination of clonality in cutaneous lymphocytic infiltrates and for detection of infectious agents, such as herpes simplex virus (HSV), varicella zoster virus (VZV), Borrelia burgdorferi, Mycobacteria, Leishmania, and Treponema pallidum. As biopsies are always composed of different cells, the cells of interest are often only a minor population. As a consequence, their specific DNA is diluted by the majority of contaminating cells. Another problem is the time- and labor-intensive DNA extraction, because usually only formalin-fixed, paraffin-embedded tissue is available, which makes molecular diagnostics a time and labor consuming, and consequently a cost-intensive procedure. To overcome these shortcomings and to eventually shorten the time to generate a result, we introduce a laser-capture microdissection (LCM)-based method for the detection of infectious agents and clonality. Only the cells of interest for the particular indication are microdissected (e.g. epidermal cells for HSV and VZV and lymphocytes for clonality analysis) and subjected to PCR amplification. Due to an accelerated DNA-extraction procedure which generates DNA in 5 h (compared to 3-4 days using conventional DNA extraction), we are able to generate a result within one working day.

Improved detection of clonality in cutaneous T-cell lymphomas using laser capture microdissection.

BACKGROUND: The diagnosis of cutaneous T-cell lymphoma is a challenge for both the pathologist and the clinician. This is particularly true for distinguishing early-stage mycosis fungoides from dermatitis. In this clinical setting, the presence of a clonal T-cell population supports lymphoma. METHODS: Usually, routinely processed paraffin-embedded material is available for gene rearrangement analysis, and polymerase chain reaction (PCR)-based methods to assess clonality can be performed. One drawback of this approach is that sensitivity is suboptimal in biopsy specimens in which the lymphocytic infiltrate represents only a small percentage of all cells present. Another drawback is that DNA extraction from routinely processed, paraffin-embedded tissue is a time-consuming and labor-intensive procedure which can take up to 5 days in our laboratory. To bypass these problems, we used laser capture microdissection (LCM) to obtain lymphocytic infiltrates from tissue sections of formalin-fixed, paraffin-embedded skin biopsy specimens. This approach allows for more specific PCR assessment of the lymphocytic infiltrate and for rapid DNA extraction and PCR analysis. RESULTS: Using the LCM approach, we could demonstrate clonal T-cell receptor gamma gene rearrangements in biopsy specimens that did not show clonality using DNA extracted by conventional methods from full tissue sections. In addition, DNA extraction and PCR analysis can be performed in 11 h. CONCLUSION: In conclusion, applying LCM to clonality analysis of cutaneous lymphocytic infiltrates is rapid and more sensitive than conventional methods, and we recommend introducing this approach into the routine diagnostic setting.

Mutations of the BRAF gene in benign and malignant melanocytic lesions.

A single-point mutation in exon 15 of the BRAF gene has recently been reported in a high percentage in cultured melanoma cells and in 6 of 9 primary melanomas examined. To evaluate the impact of the T1796A BRAF mutation, we screened primary melanomas, various types of nevi and lesions where a melanoma developed in an underlying nevus. We could detect the mutation in 28 of 97 (29%) melanomas and in 39 of 187 (21%) nevi, including blue nevi (0/20) and Spitz nevi (0/69), which did not carry the mutation. In melanomas with an underlying nevus, either the mutation was present in both the laser-microdissected nevus cells and the laser-microdissected melanoma cells (3/14) or both lesions were negative for the BRAF mutation except one case. In conclusion, mutations in exon 15 of the BRAF gene are nonspecific for progression of a nevus to a melanoma. Other so far unknown cofactors seem to be of importance.