A New Polymerase Chain Reaction–Based Method for the Detection of T-Cell Clonality in Patients With Possible Cutaneous T-Cell Lymphoma

Guitart, Joan; Kaul, Karen L.

doi:10.1001/archderm.135.2.158

Cited by 34 publications

(21 citation statements)

References 23 publications

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“…14,15 The diagnosis of SS relies on the circulating Sézary cell count, as determined by cytomorphology, and T-cell clonality, as determined by molecular approaches. 16,[22][23][24][25] However, the malignancy to T-cell clones has never been determined in patients with SS, and it is unknown whether molecular studies would detect nonmalignant expanded populations.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, identifying T-cell clones in patients with SS relies on molecular analysis of the TCR␤ or TCR␥ chains at the DNA and RNA levels. Various polymerase chain reaction (PCR)-based techniques-including PCR-␤, 16 PCR-␥, 22 PCR-denaturing gradient gel electrophoresis-␥ (PCR-DGGE-␥ ), 23 PCR-polyacrylamide gel electrophoresis-␥ (PCR-PAGE-␥), 24 and PCR-single-strand conformation polymorphism-␥ (PCR-SSCP-␥) 25 -have been validated. Immunoscopy based on TCR V␤ mRNA spectratype analysis and sequencing of the CDR3 region is a sensitive technique that has been applied for the detection and monitoring of T-cell clones in different conditions, including SS.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, identifying T-cell clones in patients with SS relies on molecular analysis of the TCR␤ or TCR␥ chains at the DNA and RNA levels. Various polymerase chain reaction (PCR)-based techniques-including PCR-␤, 16 PCR-␥, 22 PCR-denaturing gradient gel electrophoresis-␥ (PCR-DGGE-␥ ), 23 PCR-polyacrylamide gel electrophoresis-␥ (PCR-PAGE-␥), 24 and PCR-single-strand conformation polymorphism-␥ (PCR-SSCP-␥) 25 26 The diagnostic value of TCR clonality studies in cutaneous T-cell lymphoma (CTCL) is usually admitted; however, assignment of malignancy to T-cell clones remains subjective. It is thought that a proportion of circulating T-cell clones is nonmalignant, 27 but T-cell clones in SS have never been accurately characterized.…”

mentioning

confidence: 99%

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Significance of circulating T-cell clones in Sézary syndrome

Ortonne

Huet

Gaudez

et al. 2006

Blood

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Identification of malignant Sé zary cells by T-cell receptor (TCR) clonality studies is routinely used for the diagnosis of Sé zary syndrome, but T-cell clones expressed in a single patient have never been accurately characterized. We previously reported that CD158k expression delineates Sé zary syndrome malignant cells, and, more recently, we identified vimentin at the surface membranes of Sé zary cells and normal activated lymphocytes. In the present study, T-cell clones from 13 patients with Sé zary syndrome were identified by immunoscopy and further characterized in the blood according to their TCR V␤, CD158k, and vimentin cell-surface expression. We found in most patients a unique malignant T-cell clone that coexpressed CD158k and vimentin and that, when patients were tested, was also present in the skin. However, in some patients we detected the presence of a nonmalignant circulating clone expressing high amounts of vimentin and lacking IntroductionSézary syndrome (SS) is an erythrodermic, aggressive, cutaneous T-cell lymphoma characterized by a malignant T-cell clone that localizes in the blood and skin. In the absence of a specific marker for malignant Sézary cells, a diagnosis of SS relies on the circulating Sézary cell count and on the molecular detection of a circulating T-cell clone. Given that the cytomorphology of Sézary cells is not specific to SS, 1,2 identification of a specific marker of malignant Sézary cells has been a challenging issue. Besides a T-cell memory phenotype, CD4 ϩ CD45RO ϩ , Sézary cells were shown to lack CD26 3-5 and to have diminished CD3 expression, 6 whereas the loss of CD7 3,7 appears not to be a specific feature of the malignant T-cell clone. 5,8 More recently, it has been shown that Sézary cells specifically overexpress T-plastin, which, however, cannot be used as a cell-surface marker because it is located in the cytoskeleton, in association with actin. 9 New markers of Sézary cells were described, including KIR3DL2/CD158k, the first positive cell-surface marker of Sézary cells. In patients with SS, KIR3DL2/CD158k is dimly expressed by circulating and cutaneous CD4 ϩ lymphocytes. [10][11][12] In healthy persons, CD158k is absent in CD4 ϩ lymphocytes, and only minor subsets of T CD8 ϩ and natural killer (NK) lymphocytes express high amount of this antigen. 13 SC5 monoclonal antibody (mAb) was shown to react with an unknown cell-surface antigen expressed by normal activated lymphocytes and Sézary cells, whereas it failed to stain most circulating T lymphocytes in healthy individuals. 14 We found that SC5 mAb specifically recognizes vimentin (VIM) at the cell surface of viable activated T lymphocytes and is effectively expressed by Sézary cells. 15 The finding of an expanded T-cell clone in the blood of patients with SS is commonly considered to be a diagnostic marker. TCR V␤ phenotyping with specific mAbs may identify a "clonotypic" lymphocyte subset as an expanded population bearing a single TCR V␤ chain, which may be of diagnostic value. 3,[16][17][18] This approach, h...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Therefore, identifying T-cell clones in patients with SS relies on molecular analysis of the TCR␤ or TCR␥ chains at the DNA and RNA levels. Various polymerase chain reaction (PCR)-based techniques-including PCR-␤, 16 PCR-␥, 22 PCR-denaturing gradient gel electrophoresis-␥ (PCR-DGGE-␥ ), 23 PCR-polyacrylamide gel electrophoresis-␥ (PCR-PAGE-␥), 24 and PCR-single-strand conformation polymorphism-␥ (PCR-SSCP-␥) 25 26 The diagnostic value of TCR clonality studies in cutaneous T-cell lymphoma (CTCL) is usually admitted; however, assignment of malignancy to T-cell clones remains subjective. It is thought that a proportion of circulating T-cell clones is nonmalignant, 27 but T-cell clones in SS have never been accurately characterized.…”

mentioning

confidence: 99%

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Significance of circulating T-cell clones in Sézary syndrome

Ortonne

Huet

Gaudez

et al. 2006

Blood

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“…Several benign lymphocyte-mediated dermatoses exist, including lymphomatoid papulosis, Mucha-Habermann disease, lymphomatoid drug eruptions, actinic reticuloid, and lichen planus, all of which demonstrate a dominant T-cell clone in some cases. 11,[32][33][34][35][36] Thus, although PCR may have a greater sensitivity in detecting T-cell clonality, the development of more predictive primer sets or entirely different methods 11 may be needed to help distinguish between benign and malignant T-cell clones. DelfauLarue et al 10 suggested "gating" the PCR-DGGE method to diminish sensitivity so that approximately 5% clonality would be required for the test to be positive.…”

Section: Commentmentioning

confidence: 99%

Analysis of T-Cell Receptor Gene Rearrangement for Predicting Clinical Outcome in Patients With Cutaneous T-Cell Lymphoma

Juarez¹,

Isenhath²,

Polissar³

et al. 2005

Arch Dermatol

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“…Both TCR-gamma PCR/HD-TGGE and TCR-gamma PCR/DGGE are well accepted as the preferred methods for the detection of clonality in T-cell populations [12,17,25,26], however the published sensitivities differ remarkably. The present study was aimed at a systematic evaluation of the capacity of HD-TGGE to detect clonally expanded T-cells.…”

Section: Discussionmentioning

confidence: 99%

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma

et al. 2001

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Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.

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A New Polymerase Chain Reaction–Based Method for the Detection of T-Cell Clonality in Patients With Possible Cutaneous T-Cell Lymphoma

Cited by 34 publications

References 23 publications

Significance of circulating T-cell clones in Sézary syndrome

Significance of circulating T-cell clones in Sézary syndrome

Analysis of T-Cell Receptor Gene Rearrangement for Predicting Clinical Outcome in Patients With Cutaneous T-Cell Lymphoma

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma

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