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Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) gamma and TCRbeta PCR as well as an in-house TCRgamma PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRgamma PCR revealed 81% clonality, the in-house TCRgamma method revealed 86% clonality, and the Biomed-2 TCRbeta revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRbeta rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRgamma PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRgamma assay verifies our previously published findings on clonally expanded T-cells in CTCL.

Polymerase chain reaction (PCR)-based detection of clonal T- and B-cells is widely used in the diagnosis of various lymphomas, including those of the skin. A large number of corresponding methods have been published. Recently, for the first time, standardized PCR protocols were developed in common by 14 European centers of lymphoma diagnosis and research (Biomed-2 protocols). Here, we have applied Biomed-2 immunoglobulin heavy chain (IgH)-PCR for clonality detection in primary cutaneous B-cell lymphoma (CBCL) and compared it with previously established methods. The DNA of 43 paraffin-embedded lesional skin biopsies of confirmed CBCL cases [27 follicle center cell lymphoma (FCCL), 11 marginal zone B-cell lymphoma/immunocytoma (MZL/IC) and five large CBCL of the lower leg (CBCL-LL)] were amplified by the Biomed-2 IgH-PCR protocols as well as using four other assays, priming also the three IgH framework regions (FR) 1-3. All PCR products were analysed by fluorescence fragment analysis. Twenty-nine of 43 (67%) CBCL samples (5/5, 100% of CBCL-LL; six of 11, 54.5% of IC/MZL; 18 of 27, 66.7% of FCCL) showed monoclonal B-cell presence complementary in all of the IgH-PCR. The three Biomed-2 PCR indicated together clonality in 24 of 43 samples (56%). Considering each method separately, the Biomed-2 FR3-PCR showed the highest rate of clonality detection (20 of 43, 47%). In conclusion, the Biomed-2 FR3-PCR is recommended for detecting B-cell clonality in archival skin samples of CBCL but should be completed by FR1- and/or FR2-PCR.

During T-cell maturation, T-cell receptor (TCR) gene segments rearrange, resulting in a new, unique DNA configuration. The recombined TCR gene loci display a high degree of nucleotide sequence variability. Molecular biological clonality assays focus on this cell-specific DNA pattern. The finding of an identical TCR rearrangement in a large number of T lymphocytes signals a malignant proliferation, although clonality is not always equivalent to malignancy. Thus, detection of clonal TCR gamma rearrangements by polymerase chain reaction (PCR), followed by high-resolution electrophoresis is a valuable tool in the diagnosis of cutaneous and other T-cell lymphomas. For the clonality assay described here, all rearrangements of T cells present in a given sample are amplified by a set of only three TCR gamma-PCRs. The products are investigated by either heteroduplex temperature gradient gel electrophoresis (HD-TGGE) or fluorescent fragment analysis (FFA) on a capillary DNA sequencer (or by both methods), for clonality. Both electrophoresis techniques show highly reproducible results and are comparatively easy to conduct, however, specific instruments are required. Concerning lower detection thresholds, the methods need a minimum of about 1% of clonal T-cells in mixtures with polyclonal T-cells for revealing clonality.

Detection of a dominant T-cell clone by T-cell receptor ( TCR) gene rearrangement analysis is often essential for the diagnosis of cutaneous T-cell lymphomas ( CTCL). The occurrence of T-cell clones in addition to the diagnostic T-cell clone during the course of CTCL has been reported, but the data of these studies have been contradictory. We retrospectively evaluated the data of 114 lesional skin biopsies from 26 patients with Mycosis fungoides and two patients with primary cutaneous anaplastic large cell lymphoma, which were analysed with the standardized Biomed-2 PCR for the TCR γ and TCR β locus. A dominant T-cell clone was repetitively detected in 93% (26/28) of patients. Additional T-cell clones appeared temporarily in 39% (11/28) of patients. Correlation with the clinical data did not show an association of the presence of additional T-cell clones with age, number of treatments, progression of disease or survival. Our findings demonstrate that a persistent T-cell clone, most likely the disease causing tumor clone, is detectable in almost all CTCL patients. In addition, transiently appearing T-cell clones frequently occur during the course of disease. The biological relevance of these additional clones has still to be determined. However, it is important to take the possibility of additional T-cell clones into account for diagnostic analyses. [ABSTRACT FROM AUTHOR]

SummaryLichen sclerosus et atrophicus is a chronic dermatosis of unknown etiology and pathogenesis. Lichen sclerosus et atrophicus associated skin lesions show T cell enriched infiltrates, sometimes resembling the histologic picture of early mycosis fungoides. It is supposed that the infiltrating T cells participate in the pathogenesis of atrophy and sclerosis. We investigated skin biopsies from 39 lichen sclerosus et atrophicus patients by histology, immunohistochemistry and, in order to establish the status of T cell clonality, by polymerase chain reaction amplifying the T cell receptor-γ rearrangements. A stage-dependent shift of the CD3-positive T cells was observed from a predominantly CD4-positive to a predominantly CD8-positive phenotype. The increase of CD8-positive cells was associated with more pronounced epidermotropism and basal degeneration. Nearly all CD8-positive cells expressed cytotoxic granules (TIA1), possibly causing the basal destruction. In the late fibrotic stage of the disease, only a weak or no infiltrate was found. Regarding the T cell receptor-γ polymerase chain reaction, the presence of clonally expanded T cells was demonstrated in 19 of 39 patients (49%) by at least one of two different high resolution electrophoresis techniques applied to separate the amplification products. Thus, for the first time clonally expanded infiltrating T cells were detected in lichen sclerosus et atrophicus. Furthermore, this is one of the first reports on the detection of clonally expanded infiltrating T cells in an inflammatory skin disease. The clonal T cells could not be assigned to the CD4 or CD8 subtype. Most likely, their presence is not the result of a malignant transformation but a response to an as yet unknown lichen sclerosus et atrophicus associated antigen. [ABSTRACT FROM AUTHOR]

Recently, a Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered. We evaluated by PCR 14 paraffin-embedded specimens with the histological diagnosis of endemic, classic and HIV-associated Kaposi's sarcoma (KS) for the presence of the KSHV DNA sequence. In addition, biopsies of adjacent, histologically unaffected skin, peripheral-blood mononuclear cells (PBMCs) of HIV-infected KS patients, PBMCs of one classic KS patient, and specimens of patients with hemangioproliferative disorders other than KS as well as samples of cutaneous T- and B-cell lymphoma were analyzed for KSHV. In all cases of KS, independent of the KS subtype, KSHV was detected in lesional skin. No KSHV was found in biopsies of the adjacent unaffected skin or PBMCs of HIV-infected KS patients. We found KSHV in the PBMCs of a patient with classical KS. All specimens of cutaneous T- and B-cell lymphomas or lymphomatoid papulosis were negative for KSHV. In addition, the samples with hemangioproliferative disorders other than KS were negative for KSHV. There was one borderline case of KS or acroangiodermatitis that was positive for KSHV. Additional histological sections and clinical evaluation confirmed the diagnosis of classic KS. In summary, the data indicate that PCR for KSHV should be a useful diagnostic tool in cases of hemangioproliferative disorders. [ABSTRACT FROM AUTHOR]

Analyzes the mycosis fungoides skin biopsy samples by polymerase chain reaction. Detection of clonal T cells in the peripheral blood; Provision of prognostic information on multivariate analysis; Performance of survival analysis from the date of diagnosis.

Analyzes CD30[sup+] cells in four patients with primary cutaneous CD30[sup+] large T cell lymphoma by single cell polymerase chain reaction of T cell receptor-gamma genes followed by sequencing. Observation related to the presence of T cell receptor-gamma gene in large CD30[sup+] atypical cells; Findings of comparative genomic hybridization among patients; Observation related to CD30[sup+] population in T cell lymphoma.

Abstract: For diagnosis and monitoring of Sézary syndrome flow cytometric quantification of CD7− and CD26− T-cells is widely used. Because antigen loss is a characteristic but not disease-specific finding we investigated the significance of this approach. Therefore we analyzed the prevalence of tumor cells in FACS-sorted CD7+/− as well as CD26+/− circulating T-cells applying a clone-specific qualitative and quantitative T-cell receptor PCR. Tumor cells varied considerably in the CD7+ and CD7− cell subset but were largely confined to the CD26− population. We conclude that quantification of CD26− T-cells reflects the tumor cell amount more accurate and should be preferred in the clinical setting. [Copyright &y& Elsevier]