Differentiation between actinic reticuloid and cutaneous T cell lymphoma by T cell receptor gamma gene rearrangement analysis and immunophenotyping.

AIMS: Differentiation between actinic reticuloid and cutaneous T cell lymphoma can be extremely difficult. Demonstration of clonal T cell receptor (TCR) gene rearrangements has been suggested as a potential diagnostic criterion, but the results obtained thus far have been conflicting. This study investigated whether TCR gamma gene rearrangement analysis, using polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) and immunohistochemistry, can serve as a diagnostic criterion. METHODS: PCR/DGGE was performed on skin, peripheral blood mononuclear cells, and/or lymph nodes of seven patients with actinic reticuloid, 11 patients with Sézary syndrome, and 15 patients with a benign form of erythroderma. The results of PCR/DGGE and Southern blot analysis of TCR beta gene rearrangements were compared. In addition, CD4:CD8 ratios in skin and peripheral blood samples were investigated. RESULTS: Clonal T cell populations were detected in 19 of 21 samples obtained from patients with Sézary syndrome but were not detected in any of the 12 samples from patients with actinic reticuloid. Clonal T cells were detected in the peripheral blood of only one of 15 patients with a benign form of erythroderma. PCR/DGGE and Southern blot analysis gave concordant results in 28 of 29 samples. Immunophenotypic analysis demonstrated increased proportions of CD8+ T cells in skin (seven of seven cases) and peripheral blood (four of seven cases) of patients with actinic reticuloid. CONCLUSION: The results of this study demonstrate that gene rearrangement analysis, in combination with immunohistochemistry, may be an important adjunct in differentiating between actinic reticuloid and cutaneous T cell lymphoma. In patients suspected of having actinic reticuloid, application of both techniques is recommended.

Immunophenotyping and gene rearrangement analysis provide additional criteria to differentiate between cutaneous T-cell lymphomas and pseudo-T-cell lymphomas.

Differentiation between cutaneous pseudo-T-cell lymphomas and cutaneous T-cell lymphomas (CTCLs) may be extremely difficult. In this study, it was investigated whether demonstration of an aberrant phenotype and detection of clonal T-cell receptor gamma (TCR gamma) gene rearrangements can be used as additional differential diagnostic criteria. Immunohistochemical studies and TCR gamma gene rearrangement analysis using a polymerase chain reaction with primers specific for V gamma 1-8 and V gamma 9 gene segments in combination with denaturing gradient gel electrophoresis (PCR/ DGGE) were performed on frozen material of 11 pseudo-T-cell lymphomas and 17 CTCLs, including 9 cases of mycosis fungoides (MF) and 8 pleomorphic CTCLs. Clonal TCR gamma gene rearrangements were found in 66% of patch/plaque-stage MF and 100% of tumor-stage MF and pleomorphic CTCL, but not in any of 10 pseudo-T-cell lymphomas studied. Aberrant expression of CD2, CD3, and/or CD5 antigens was noted in 3 of 6 (50%) cases of patch/plaque-stage MF, all three cases of tumor-stage MF, and 5 of 8 (62%) pleomorphic CTCLs, but not in any of the 11 pseudo-T-cell lymphomas. Moreover, in pseudo-T-cell lymphomas exhibiting a nodular or diffuse growth pattern, a considerable admixture with reactive CD8+ T cells (15 to 60%), B cells (up to 20%), and macrophages was a characteristic finding. In conclusion, the results of this study suggest that demonstration of clonal TCR gene rearrangements and an aberrant phenotype, as well as demonstration of many admixed CD8+ T cells and B cells can be considered as useful additional criteria in the differentiation between pseudo-T-cell lymphomas and CTCLs.

Diagnostic and prognostic significance of clonal T-cell receptor beta gene rearrangements in lymph nodes of patients with mycosis fungoides.

In this study, 25 involved and uninvolved lymph nodes from 22 patients with mycosis fungoides (MF) and seven dermatopathic lymph nodes from patients with benign skin disorders were studied for the presence of clonal T-cell receptor beta (TCR beta) gene rearrangements by Southern blot analysis. These results were correlated with the histological classification, follow-up data, and survival. The results of the histological classification and Southern blot analysis were concordant in 26 of 32 cases. Clonal TCR beta gene rearrangements were found in all six MF lymph nodes showing (partial) effacement of the normal lymph node architecture, but in none of the eight uninvolved dermatopathic MF lymph nodes and in none of the seven dermatopathic control lymph nodes. In addition, in 5 of 11 dermatopathic MF lymph nodes that were considered to have early involvement by MF at histological examination, clonal TCR beta gene rearrangements were detected. In the group of MF patients with dermatopathic lymphadenopathy, patients with detectable clonal T-cell populations had a significantly shorter survival than patients without such a population (P < 0.01). The results of this study indicate that within the group of dermatopathic MF lymph nodes, prognostically different groups can be distinguished and that TCR beta gene rearrangement analysis may be an important adjunct in the early diagnosis of lymph node involvement by MF.

Demonstration of clonal immunoglobulin gene rearrangements in cutaneous B-cell lymphomas and pseudo-B-cell lymphomas: differential diagnostic and pathogenetic aspects.

Twenty-five patients with a benign or malignant cutaneous B-cell lymphoproliferative disease, including seven cutaneous pseudo-B-cell lymphomas, eight primary cutaneous B-cell lymphomas (CBCL), and 10 secondary cutaneous B-cell lymphomas, were investigated for the presence of clonal immunoglobulin (Ig) gene rearrangements using Southern blot hybridization analysis. The selection of pseudo-B-cell lymphomas was based on the presence of polyclonal light-chain expression with immunohistochemical analysis. All cases of CBCL demonstrated monotypic light-chain expression or absence of detectable Ig on CD20+ B cells. Clonal rearrangements of one or more Ig genes were demonstrated in four of seven cases of cutaneous pseudo-B-cell lymphomas, six of eight cases of primary CBCL, and in all cases of secondary CBCL. The observation that cutaneous pseudo-B-cell lymphomas as defined by immunohistochemical criteria often contain occult monoclonal B-cell populations implies that differentiating between pseudo-B-cell lymphomas and CBCL is not always possible by means of gene-rearrangement analysis. These findings may support the concept that cutaneous pseudo-B-cell lymphomas and primary CBCL are part of a continuous and progressive spectrum of B-cell lymphoproliferative skin disorders.

Frequency and prognostic significance of clonal T-cell receptor beta-gene rearrangements in the peripheral blood of patients with mycosis fungoides.

BACKGROUND: Previous studies have shown that peripheral blood involvement is a poor prognostic sign in patients with cutaneous T-cell lymphoma. However, evaluation of the results of these studies is difficult. In this study peripheral blood mononuclear cells from 45 patients with various stages of mycosis fungoides (MF) were investigated for the presence of clonal T-cell populations by T-cell receptor beta (TCR beta)-gene rearrangement analysis. RESULTS: Clonal TCR beta-gene rearrangements were found in five (11%) of 45 patients with MF, including one (3%) of 31 patients without MF and four (27%) of 15 patients with histologically confirmed lymph node involvement. With respect to skin stage, clonal T-cell populations were detected in one (4%) of 23 patients with plaque stage disease, two (10%) of 19 patients with tumor stage disease, and two (50%) of four patients with erythrodermic MF. In the group of patients with lymph node involvement the median survival of patients with detectable clonal T-cell rearrangements in the peripheral blood was much shorter (3 months) than that of patients without clonal rearrangements (16 months). CONCLUSIONS: The results of this study indicate that clonal TCR beta-gene rearrangements, as detected by Southern blot analysis, are uncommon in the peripheral blood of patients with MF, in particular in patients without histologically documented lymph node involvement. The presence of clonal T-cell populations in the peripheral blood of MF patients with lymph node involvement is usually associated with rapidly fatal disease.

Oral etoposide in the treatment of cutaneous large-cell lymphomas. A preliminary report of four cases.

The favourable results of oral etoposide as single-agent therapy in four patients with a cutaneous lymphoma other than mycosis fungoides are reported. In all cases other chemotherapeutic options were limited because of prior chemotherapy or the age of the patients. Therapy with etoposide resulted in an initial complete remission in all patients, and was associated with minimal side-effects.

Diagnostic value of T-cell receptor beta gene rearrangement analysis on peripheral blood lymphocytes of patients with erythroderma.

Differentiation between Sézary's syndrome (SS) and benign forms of erythroderma may be extremely difficult. In this study T-cell receptor beta (TCR beta) gene rearrangement analysis was performed on peripheral blood lymphocytes (PBL) from 32 patients with erythroderma, including 10 patients with SS, three patients with another type of cutaneous T-cell lymphoma, and 19 patients with a benign form of erythroderma. The aim of this study was to define the sensitivity and specificity of this technique in the diagnosis of SS. Clonal TCR beta gene rearrangements were found in eight of 10 patients with SS, one T-CLL patient, one of two patients with erythrodermic mycosis fungoides, and only one of 19 patients from the benign group. In the two "false-negative" cases of SS clonal TCR beta gene rearrangements were detected in PBL obtained during follow-up. The results indicate that TCR beta gene rearrangement analysis on PBL is a sensitive and highly specific technique, that may contribute significantly to the differential diagnosis of patients with erythroderma. However, because both "false-positive" and "false-negative" results may occur, the results of gene-rearrangement analysis should always be considered in conjunction with clinical, histologic, and immunophenotypical data.

Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction.

A two-step polymerase chain reaction (PCR) procedure was used as a new screening strategy for the detection of human papillomavirus (HPV) genotypes in cervical scrapes omitting prior DNA extraction. Sample preparation consisted of a freeze-thaw step followed by boiling the cells before the PCR mixture was added. This pretreatment was as efficient and reproducible for HPV DNA amplification as DNA purification. By using crude cell suspensions, a prescreening of the samples with the general primer-mediated PCR method (GP-PCR) was performed to detect a broad spectrum of sequenced and still unsequenced HPV types at the subpicogram level. HPV-containing scrapes by GP-PCR were subjected to HPV 6, 11, 16, 18, 31, and 33 type-specific PCR (TS-PCR) to identify the sequenced HPV types. This direct GP/TS-PCR method was tested on a large group of cervical scrapes (n = 459) from women visiting a gynecologic outpatient clinic. The results were compared with HPV data obtained by a method using modified filter in situ hybridization and TS-PCR in which the PCR was mainly used to confirm HPV positivity. A substantially higher HPV prevalence rate was found by direct GP/TS-PCR strategy. The results indicate that GP/TS-PCR is a rapid, sensitive, and reliable detection method for HPV in cervical scrapes. The easy performance on crude cell suspensions makes this strategy applicable for large HPV-screening programs.