The significance of multiplex PCR/heteroduplex analysis-based TCR-γ gene rearrangement combined with laser-capture microdissection in the diagnosis of early mycosis fungoides

Background: The diagnosis of early mycosis fungoides (MF) is a big challenge to dermatologists and dermatopathologists because it lacks specific clinicopathologic features. Methods: Fifty‐two paraffin‐embedded skin samples from 50 patients, including 31 with suspected MF, 10 with typical MF and 9 with benign inflammatory dermatosis (BID), were obtained from our archives. DNA was extracted both by traditional phenol‐chloroform method and by the laser‐capture microdissection (LCM)‐proteinase K approach. The T_VG/T_JG, V_2–5/V_8–12/JGT₁ and BIOMED‐2‐TCR‐γ primers were used to assess TCR‐γ monoclonal rearrangement as measured by polymerase chain reaction (PCR). Results: In the suspected MF group, clonal TCR‐γ gene rearrangements were detected in 11/31 cases (35.5%) by phenol‐chloroform DNA extraction and in 25/31 cases (80.7%) by LCM‐proteinase K extraction (p < 0.05). While T‐cell clonality was detected in 8/10 cases (80%) by the phenol‐chloroform method and 10/10 cases (100%) by LCM (p > 0.05) in the typical MF group, no TCR‐γ monoclonal rearrangement was detected in the BID group. Conclusions: The strategy of multiple PCR/heteroduplex analysis for TCR‐γ gene rearrangement combined with LCM increases the detection rate of clonal TCR‐γ gene rearrangement in early MF cases and could provide strong evidence to confirm the diagnosis of early MF. Yang H, Xu C, Tang Y, Wan C, Liu W, Wang L. The significance of multiplex PCR/heteroduplex analysis‐based TCR‐γ gene rearrangement combined with laser‐capture microdissection in the diagnosis of early mycosis fungoides.     

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.     

Detection of TCR-gamma gene rearrangements in early mycosis fungoides by non-radioactive PCR-SSCP

Early mycosis fungoides (MF) can mimic numerous benign inflammatory dermatoses on routine histological examination. In this study, a recently developed non-radioactive polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique was used to assess T-cell clonality in paraffin-embedded skin biopsies clinically and pathologically suspicious for early MF. Non-radioactive PCR-SSCP is a simple, sensitive, reproducible and rapid procedure requiring minimal instrumentation. DNA was extracted from 22 skin biopsies of 20 patients with suspected patch stage MF and 15 skin biopsies of inflammatory dermatoses. V gamma1-8, V gamma9, V gamma10, V gamma11 and J gamma1/J gamma2 consensus primers were used for T-cell receptor (TCR)-gamma gene rearrangement amplification. PCR products were analyzed by non-radioactive SSCP. Clonal TCR-gamma gene rearrangements were detected in 17 of 22 (77%) suspected MF specimens. Clonal SSCP banding patterns were different among individual patients. In addition, identical banded patterns were demonstrated in serial skin biopsies from the same patient. No dominant T-cell clones were found in the inflammatory dermatoses studied. Therefore, non-radioactive PCR-SSCP is a useful molecular diagnostic tool for assessment of T-cell clonality in paraffin-embedded specimens suspicious for early MF. The SSCP imprint of PCR products is specific for each TCR-gamma gene rearrangement, and may be used to evaluate concurrent/recurrent disease in individual patients.     

A new polymerase chain reaction-based method for the detection of T-cell clonality in patients with possible cutaneous T-cell lymphoma

OBJECTIVE: To evaluate a new, rapid, and sensitive method for the detection of T-cell clonality in patients with lesions suggestive of cutaneous T-cell lymphoma (CTCL). DESIGNS: Skin specimens were obtained from 48 patients with possible CTCL. Polymerase chain reaction amplification of the T-cell receptor gamma (TCRG) gene was performed using consensus primers to the V and J regions. Clonal populations having identical N-region sequences are detected by single-strand conformation polymorphism analysis using a semiautomated electrophoresis system with a silver-staining method for gel visualization. The results of clinicopathological assessment were compared with those of immunohistochemistry and polymerase chain reaction analysis. SETTING: Cutaneous lymphoma clinic at a university medical center. PATIENTS: Forty-eight patients with skin lesions suggestive of CTCL. RESULTS: Based on the clinicopathological assessment, 26 patients were diagnosed as having CTCL. Of them, clonality was detected in 19 patients (73%) and an abnormal phenotype in 17 (68%) of 25 patients. Combining both tests, abnormal results were noted in 24 (92%) of 26 patients with CTCL. Clonality was also identified in 2 (12%) of 17 patients with presumably benign lesions on clinicopathological assessment. CONCLUSIONS: Polymerase chain reaction and single-strand conformation polymorphism analysis of the TCRG gene is a rapid and sensitive method that can contribute to the diagnosis of CTCL. The new method is especially useful when used in conjunction with immunophenotyping.     

A realistic approach to the sensitivity of PCR-DGGE and its application as a sensitive tool for the detection of clonality in cutaneous T-cell proliferations

Abstract The practical value of the detection of clonality within the T-cell receptor gamma locus by polymerase chain reaction for the diagnosis of cutaneous T-cell lymphomas is well known. However, studies dealing with this subject so far, with special emphasis on the sensitivity of the technique in comparison to, for example, Southern blotting have used mixtures of DNA in various concentrations instead of using mixtures of the cells involved, which would reflect the in vivo situation in a more realistic scope. The purpose of this study was therefore to determine the sensitivity and the limitations of the PCR assay by dilution experiments, using mixtures of cells. Furthermore we studied its applicability to cutaneous T-cell proliferative disorders. Two clonal T-cell lines (MyLa and Jurkat) served as positive control. Dilutions of MyLa cells, cultured normal human keratinocytes and peripheral blood mononuclear cells from lymphoma negative volunteers were used to assess the sensitivity of the PCR-DGGE assay. Skin samples from 4 patients with cutaneous T-cell lymphoma, 1 lesional lymph node, 2 blood samples from a patient with Sézary syndrome and 4 lymphoma-negative tissue samples were analysed. Two samples were uncertain for diagnosis of lymphoma. The PCR-DGGE assay consisted of a 2-round nested PCR with consensus primers within the TCR-gamma locus followed by electrophoretic separation of the product along a denaturing urea/formamide gradient gel. PCR-DGGE sensitivity was, to our knowledge, for the first time investigated for mixtures of lymphocytes (clonal and polyclonal) and keratinocytes. Clonal T-cells were detected in a concentration between 1–0.1% in keratinocytes, whereas the sensitivity was generally lower upon dilution in peripheral blood mononuclear cells or in a mixture of keratinocytes and peripheral blood mononuclear cells. Nevertheless, T-cell clonality was detected in 2 blood samples of a patient with Sézary syndrome, which were negative by Southern blot analysis. The crucial point of this work was the new approach to establish the sensitivity of the PCR-DGGE, in a way which more closely mimics the condition of clinical specimens. Instead of mixing and amplifying DNA extracted from clonal T-cell lines and polyclonal bone marrow cells, we amplified DNA from clonal and polyclonal cells which had been mixed in various ratios before DNA extraction. Polymerase chain reaction in conjunction with denaturing gradient gel electrophoresis is a sensitive and versatile molecular tool for the assessment of clonality of suspect cutaneous lesions. The determination of sensitivity using DNA extracted from premixed cells more closely corresponds to the actual test situation when testing skin samples.     

Cutaneous involvement in patients with angioimmunoblastic lymphadenopathy with dysproteinemia: a clinical, immunohistological, and molecular analysis

OBJECTIVE: To determine whether cutaneous involvement in patients with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is related to a clonal T-cell proliferation. DESIGN: Retrospective study. SETTING: University hospitals. PATIENTS: Ten patients with AILD and cutaneous involvement. MAIN OUTCOME MEASURE: The T-cell receptor-gamma (TCRG)gene rearrangement was studied with the use of polymerase chain reaction and denaturing gradient gel electrophoresis in blood, nodal, and skin samples. Skin and nodal samples were investigated also for the presence of Epstein-Barr virus (EBV) RNA by in situ hybridization. RESULTS: A transient morbilliform eruption of the trunk was seen most often. Other cutaneous features were infiltrated plaques and purpuric or urticarial lesions. A clonal TCRG gene rearrangement was detected in 7 skin samples, corresponding to a maculopapular eruption with a histological pattern of nonspecific mild lymphoid dermal infiltrate in 6 patients, and to erythematous plaques with histological findings of typical cutaneous lymphoma in 1 patient. In the 5 patients in whom a TCRG gene rearrangement was evidenced in skin and lymph node samples, identical clones were detected in both. Five patients died by the end of the study, with a mean survival of 33.2 months. Four of these 5 patients had a clonal infiltrate in skin and lymph nodes. The EBV RNA was detected in only 1 of 10 skin biopsy specimens and in 5 of 8 lymph nodes tested. CONCLUSIONS: Cutaneous involvement is often related to a clonal T-cell proliferation in AILD, even when clinical and histological features are nonspecific. Cutaneous infiltrate seems to be clonally related to the nodal T-cell proliferation. The role of EBV infection in skin lesions was not evidenced.     

Automated kappa and lambda light chain mRNA expression for the assessment of B-cell clonality in cutaneous B-cell infiltrates: its utility and diagnostic application

INTRODUCTION: Primary cutaneous B-cell lymphoma (1 degrees CBCL) accounts for 25% of all lymphomas. The difficulty in distinction of reactive from neoplastic B-cell infiltrates prompts the use of molecular diagnostic adjuncts. While T-cell clonality can be seen in various reactive states, clonal B-cell infiltrates are often neoplastic; standard assays employed include polymerase chain reaction (PCR) or Southern blot analysis to assess heavy chain rearrangement. We sought to assess the utility of kappa (kappa) and lambda (lambda) mRNA expression using the Ventana automated assay (Ventana Medical Systems, Tucson, AZ, USA) in the analysis of atypical cutaneous B-cell lymphoid infiltrates. MATERIALS AND METHODS: Multiple 4 micro m sections of paraffin-embedded, formalin-fixed skin biopsies from 31 patients with CBCL were placed on silane-coated slides, deparaffinized, then digested in pepsin (5 mg/ml) for 30 min at 37 degrees C. Fluorescein-tagged oligoprobes and tissue mRNA were denatured at 80 degrees C for 5 min, hybridized for 2 h at 37 degrees C, and incubated with antifluorescein alkaline phosphatase conjugates. Detection of the probe target complex employed nitroblue tetrazolium and bromochloroindolyl phosphate conjugates with a nuclear fast red counterstain. A kappa : lambda ratio > 3 : 1 was held to represent kappa light chain restriction and a kappa : lambda ratio 相似文献   

Improved sensitivity of T-cell clonality detection in mycosis fungoides by hand microdissection and heteroduplex analysis

BACKGROUND: The presence of a dominant T-cell clone is an important diagnostic criterion in cutaneous T-cell lymphomas (CTCLs) and in atypical T-cell cutaneous infiltrates. Procedures based on polymerase chain reaction (PCR) are the most sensitive to detect clonality, but heteroduplex analysis is less sensitive than other methods such as denaturing gradient gel electrophoresis. OBJECTIVE: To assess whether a gross hand microdissection of the superficial (papillary) portion of the dermal infiltrate may improve the sensitivity of T-cell clonality detection by PCR-heteroduplex analysis in CTCL. SETTING: Regional university hospital (secondary or tertiary referral center).Patients A total of 29 patients with a definite diagnosis of mycosis fungoides based on typical histologic and immunophenotypic features were selected with patch (16) or plaque (13) stages. MAIN OUTCOME MEASURES: Assessment of T-cell clonality by PCR amplification of the T-cell receptor gamma chain followed by heteroduplex analysis using DNA extracted from the entire biopsy specimen and after gross microdissection of the subepidermal bandlike dermal infiltrate. RESULTS: T-cell clonality was demonstrated in 24 of 29 cases when hand microdissection was used compared with 16 of 29 cases with DNA analysis from entire biopsy specimens; thus, hand microdissection resulted in a sensitivity improvement of approximatively 50%. CONCLUSIONS: Hand microdissection substantially improves the detection of a T-cell clone in CTCL when using a PCR-heteroduplex analysis and could be used routinely in the clinical evaluation of T-cell infiltrates. Importantly, the microdissection method was found to be more useful for the detection of T-cell clones in early patch stages of CTCL than in plaque-stage disease.     

Polymerase chain reaction (PCR) for human herpesvirus 8 and heteroduplex PCR for clonality assessment in angiolymphoid hyperplasia with eosinophilia and Kimura's disease

BACKGROUND: Recently, human herpesvirus 8 (HHV-8) has been isolated from almost all cases of Kaposi's sarcoma. It has not been found in most cutaneous hemangioproliferative disorders other than Kaposi's sarcoma. Benign vascular lesions including Kimura's disease were not found to contain the HHV-8 DNA sequence. However, there has been contradictory data concerning the presence of HHV-8 in angiolymphoid hyperplasia with eosinophilia (ALHE). Clonality studies in ALHE and Kimura's disease were rare. METHODS: We performed polymerase chain reaction (PCR)-based analysis to determine whether HHV-8 is present and heteroduplex analysis of rearranged T-cell receptor (TCR) gene for clonality assessment in paraffin-embedded skin biopsy samples of 7 ALHE and 2 Kimura's disease, taken from immunocompetent patients. RESULTS: HHV-8 could not be identified in all the cases of ALHE and Kimura's disease. Although 2 cases (2/7) of ALHE and 2 cases (2/2) of Kimura's disease showed positive result for PCR analysis of TCR, all the cases were negative for heteroduplex-PCR. CONCLUSIONS: We suggest that HHV-8 may not involve in a pathogenetic role in ALHE and Kimura's disease and the failure to demonstrate clonality may be consistent with the reactive nature of these diseases and lack of malignant transformation. In addition, heteroduplex-PCR can be applied to confirm doubtful cases of lymphoma in that heteroduplex-PCR is more specific than PCR as seen in our study.     

HTLV-1-associated cutaneous disease: a clinicopathoiogical and molecular study of patients from the U. K.

The clinieopathological features of eightpatients with cutaneous disease associated with HTLV-1 infection are reviewed. All were U. K. residents of West Indian extraction, and two are currently alive. Disease remained confined to the skin in two patients. Five patients with a cutaneous prodromal phase developed leukaemia after a medianduration of 124 months (3months-21years), and in one of these combination chemotherapy produced a sustained clinical remission for 20 months. Two patients developed cutaneous disease after remission of their leukaemia. Cutaneous lesions were heterogeneous and included localized papules, a generalized papulonodular eruption, diffuse and localized erythematous plaques, pompholy x-like lesions on the palms and soles, and tumours. The histology of the skin lesions was also variable, and consisted of aheavy dermal infiltrate with lymphocytes, histiocytes, plasmacells, eosinophils and cytologically atypical mono-nuclear cells. Epidermotropism was present in biopsies from five patients. Tumour cells with large, densely staining, pleomorphic nuclei, arranged in rows between collagen bundles, were present in the majority of cases. In one patient the infiltrate also consisted of epithelioid cells and muitinucleated giant cells. Six cases were classified histologically as pleomorphic T-cell lymphoma, and two as cerebriform or mycosis fungoides type. Molecular studies revealed a clonal T-cell population associated withmonoclonal integration of HTLV-1 provirus in tissue DNA from six patients. In two patients HTLV-1 integration was established retrospectively using enzymatic in vitro amplification of a specific HTLV-1 pol genesequence in DNA extracted from paraffin-embedded sections. This study indicates that the clinical and pathological features of HTLV-1-associated cutaneous disease are diverse. Patients may have disease confined to the skin for prolonged periods, either at presentation or following clinical relapse—cutaneous adult T-celll ymphoma. Molecular techniques allow distinction from other types of cutaneous T-cell lymphoma, and provide an opportunity for retrospective studies of archival material.     

T-Cell clonality in pityriasis lichenoides et varioliformis acuta: a heteroduplex analysis of 20 cases

BACKGROUND: Cutaneous lesions of pityriasis lichenoides et varioliformis acuta (PLEVA), a T cell-mediated cutaneous inflammatory condition, are clinically similar to lymphomatoid papulosis (LyP), leading some authors to hypothesize that they are part of the same spectrum of lymphoproliferative disorders, although reports of the development of cutaneous lymphoma in patients with PLEVA are not as frequent as they are for patients with LyP. Furthermore, unlike in cases of LyP, no systematic search for a dominant T-cell clone has been carried out in cases of PLEVA, whereas clones have been detected in a few cases of PLEVA using mainly Southern blot analysis. OBJECTIVE: To investigate T-cell clonality in a series of archival PLEVA lesions. TISSUES: Archival paraffin-embedded biopsy specimens from 20 clinically and pathologically typical cases of PLEVA were selected. MAIN OUTCOME MEASURE: Identification of a dominant T-cell clone by polymerase chain reaction and heteroduplex analysis targeted on the TCRgamma gene. Peripheral blood mononuclear cells (PBMCs) and Jurkat cells were used as negative and positive controls. Serial dilutions of Jurkat T-cell lymphoma DNA in PBMC DNA were used to assess the sensitivity of the method. RESULTS: Analysis of 13 (65%) of 20 PLEVA biopsy specimens revealed the presence of a dominant T-cell clone. Positive and negative controls confirmed the specificity of the procedure. The sensitivity was determined to be between 1% and 5% of the total T-cell infiltrate. CONCLUSIONS: This study provides further evidence for the presence of a dominant T-cell clone in skin lesions of some patients with PLEVA and supports the hypothesis that PLEVA is part of the spectrum of clonal-T-cell cutaneous lymphoproliferative disorders.     

Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias

Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.     

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR/DGGE)-based detection of clonal T-cell receptor gamma gene rearrangements in paraffin-embedded cutaneous biopsies in cutaneous T-cell lymphoproliferative diseases.

Polymerase chain reaction (PCR)-based amplification of T-cell receptor (TCR)-gamma genes is a novel technique that can detect a clone of T cells comprising less than 1% of the total T cells in a lymphoid infiltrate. Besides greater sensitivity than Southern blotting, this technique can be performed with smaller quantities of lower molecular weight genomic DNA as template. We retrospectively analyzed 12 paraffin-embedded biopsies of cutaneous T-cell lymphoma (CTCL), 1 case suspicious for CTCL, 1 case of granulomatous slack skin, and 8 cases of inflammatory skin diseases to determine if PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis can detect TCR-gamma gene rearrangements on paraffin-embedded specimens. We were able to amplify Vgamma1-8 TCR sequences in each case and detected a dominant clone in 9 of 12 cases of CTCL and in granulomatous slack skin. We analyzed Vgamma9 sequences in 9 cases of CTCL and detected a dominant clone in 4 cases. This study demonstrates that PCR-DGGE can easily be applied retrospectively to cutaneous biopsies of lymphoproliferative diseases when fresh tissue is not available.     

Early mycosis fungoides: molecular analysis for its diagnosis and the absence of p53 gene mutations in cases with progression

The histological diagnosis of initial mycosis fungoides (MF) and the molecular mechanisms that are responsible for its progression and transformation to the more highly malignant variants of MF remain largely unknown. Because of the rare occurrence of these tumours, the need for snap frozen skin biopsy specimens and the difficulty to obtain suitable material for karyotypic and genotypic analysis, specific cytogenetic and molecular lesions have not yet been identified. In particular the role of known oncogenes and tumour suppressor genes, including the p53 gene, in the pathogenesis and clinical progression of MF has not been extensively investigated. The present study was carried out using the polymerase chain reaction (PCR) technique combined with temperature gradient gel electrophoresis (TGGE) to detect mutations of the p53 gene in 58 patients with MF. TGGE analysis was also used in combination with clonality analysis by means of T-cell receptor gamma (TCRG) gene rearrangement studies to distinguish parapsoriasis en plaque and initial MF from patch/plaque stage MF. More than 83% of the diagnoses of initial MF could be confirmed using PCR-TGGE analysis. However, although the sensitive TGGE analysis was used for all exons, p53 gene polymorphisms were found in 4 and p53 gene mutation in only 1 of 58 biopsy specimens. It appears unlikely that p53 gene mutations play a role in either the pathogenesis of parapsoriasis and initial MF or their progression to advanced stages of MF. However, TCRG gene rearrangement studies by means of TCR-TGGE analysis may be useful for distinguishing histologically discordant cases of initial MF.     

Detection of Epstein-Barr virus genome in primary cutaneous T and B cell lymphomas and pseudolymphomas

The Epstein-Barr virus (EBV) genome has recently been identified in Hodgkin's disease (HD) and nodal non-Hodgkin's lymphomas (NHL). In order to elucidate the possible aetiopathogenetic role of EBV in benign and malignant lymphoproliferative disorders we investigated skin specimens from 24 patients with a primary cutaneous lymphoproliferative disorders (10 T-cell lymphomas 6 B-cell lymphomas and 8 pseudolymphomas) and from 22 normal individuals for the presence of EBV DNA using the polymerase chain reaction (PCR) technique and in situ hybridization (ISH) on formalin-fixed paraffin-embedded tissue sections. EBV DNA was identified by PCR in one of two cases of mycosis fungoides, in one of seven cases of pleomorphic T-cell lymphomas, in one case of centro-blastic (CB) lymphoma of six B-cell lymphomas, and in three of eight pseudolymphomas. The EBV genome was also found in 2 of 22 specimens of normal skin. The small EBV-encoded nuclear RNAs, EBERs, were not detected in any PCR-positive sample by ISH. Based on our PCR and ISH findings, EBV does not seem to play a significant role in the development of cutaneous lymphomas.     

Lymphomatoid papulosis associated with mycosis fungoides: clinicopathological and molecular studies of 12 cases

The association of mycosis fungoides and a primary cutaneous CD30+ lymphoproliferative disorder has been reported and probably represents different clinical aspects of a unique T-cell monoclonal expansion. In this study, 12 patients (6 men and 6 women) presented with lymphomatoid papulosis and mycosis fungoides. A TCRgamma gene rearrangement study was performed by an automated high-resolution PCR fragment analysis method on skin biopsy specimens taken from the different clinical lesions in each patient. An indolent clinical course was observed in the majority of patients. T-cell clonality was identified in 7 of 12 lymphomatoid papulosis lesions (58%) and in 6 skin biopsies of plaque stage mycosis fungoides (50%). In each individual case, where T-cell clonality was detected, both mycosis fungoides and lymphomatoid papulosis specimens exhibited an identical peak pattern by automated high-resolution PCR fragment analysis, confirming a common clonal origin. Only one case showed a clonal TCRgamma rearrangement from the lymphomatoid papulosis lesion, which could not be demonstrated in the mycosis fungoides specimen. The demonstration of an identical clone seems to confirm that both disorders are different clinical manifestations of a unique T-cell monoclonal proliferation. Our results also seem to confirm that the association of mycosis fungoides with a primary cutaneous CD30+ lymphoproliferative disorder usually carries a favourable prognosis.     

Detection of clonal rearrangement of T-cell receptor genes in the diagnosis of primary cutaneous CD30 lymphoproliferative disorders

BACKGROUND: Detection of clonality has been reported to be a helpful tool in the diagnosis of cutaneous lymphomas. Monoclonal rearrangement of T-cell receptor genes (TCR) was reported in fresh frozen tissue of lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large-cell lymphoma (ALCL), but the diagnostic value of T-cell clonality in formalin-fixed, paraffin-embedded biopsies has so far not been assessed. METHODS: Detection of clonal rearrangement of TCRgamma genes by highly sensitive polymerase chain reaction-based automated high-resolution fragment analysis (AHRFA) in archival LyP (n = 18) and ALCL (n = 17) tissue. RESULTS: Detection of clonality differed significantly among the histologic forms of LyP as well as between LyP and ALCL with clonality found in none of the 10 biopsies of LyP type A and B, in 4/8 (50%) of the LyP type C specimens, and in 11/17 (65%) of ALCL cases. CONCLUSIONS: T-cell clonality can only be found in a minority (four of 18; 22%) of archival LyP specimens, even when employing a highly sensitive detection method and is thus of limited diagnostic value. Final diagnosis of LyP has to be based mainly on clinical, histologic, and immunohistochemical findings rather than on results of clonality studies.     

PCR-heteroduplex analysis of T-cell receptor gamma gene rearrangement in paraffin-embedded skin biopsies

We developed a rapid, simple, and sensitive method for the detection of T-cell receptor-gamma (TCRgamma) gene rearrangements in paraffin-embedded skin biopsies. Available techniques often require either fresh tissue, several primer pairs, nested amplifications, or specialized electrophoresis steps such as denaturing gradient gel electrophoresis. Our method is based on heteroduplex analysis of polymerase chain reaction (PCR) products of the TCRgamma in a nondenaturing modified polyacrylamide gel using a single pair of primers and is adapted for paraffin-embedded tissue. When tested against Southern blot analysis, the PCR results correlated in 8 of 9 cases. Six mature cutaneous B-cell lymphomas and 29 inflammatory skin disorders all resulted in a polyclonal amplification pattern. When analyzing 3-mm or 4-mm punch biopsies of 51 cases of cutaneous T-cell lymphoma, 37 (72.5%) showed a clonal rearrangement with this technique. For 7 cases of patch stage mycosis fungoides, frozen tissue and formalin-fixed and paraffin-embedded tissue was available, and in 5 of 7 cases (71%), the results in frozen and paraffin-embedded tissue were concordant. One case showed a clonal pattern in frozen tissue but not in paraffin-embedded tissue, and one case was polyclonal in frozen tissue but monoclonal in paraffin-embedded tissue. Using serial dilutions of DNA from a T-cell ALL in a polyclonal background (tonsil), we established a sensitivity of 0.5%. Heteroduplex PCR of the TCRgamma is a rapid, sensitive, and inexpensive screening procedure as well as a useful adjunct to histologic analysis and immunophenotyping of cutaneous T-cell proliferations.     

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.