Detection of clonally rearranged T-cell-receptor gamma chain genes from T-cell malignancies and acute inflammatory rheumatic disease using PCR amplification, PAGE, and automated analysis

Abstract

Clonal expansions of T cells carrying identical T-cell-receptor (TCR) genes are the hallmark of T-cell malignancies, but they can also result from a strong immune reaction to a dominant epitope. The basis for the molecular detection of clonal T cells is amplification of the V-(D)-N-J region of the TCR gene. We evaluated PCR amplification of the rearranged gamma TCR from genomic DNA extracted from peripheral blood and subsequent polyacrylamide gel electrophoresis (PAGE) in an automated DNA sequencer. We determined the sensitivity for the detection of clonal T cells and propose a standardized evaluation procedure for the electrophoretic profiles generated by the DNA sequencer. The sensitivity of our method was 0.6-1.25% of clonal T cells within a polyclonal background. Sixteen patients with T-cell malignancies, ten with acute inflammatory rheumatic diseases, and twelve healthy controls were examined. Among the systemic T-cell malignancies, all but one patient with T-PLL (8/ 9) revealed a clonal PCR signal. No clonal signal was detectable in any patient in clinical complete remission (5/5) or in either of the two patients with lymphomas limited to cutaneous sites. However, clonal T cells were detected in one patient with polymyalgia rheumatica and in one with reactive arthritis. A polyclonal signal was found in the remaining eight patients with acute inflammatory rheumatic diseases and in 12 healthy controls. Taking our results together, the PCR/PAGE assay is able to reliably distinguish clonal from polyclonal T-cell populations. However, although the sensitivity is limited to approximately 1%, clonal T cells can be found in the peripheral blood of some patients with autoimmune diseases and not only in T-cell malignancies.

Significance of circulating T-cell clones in Sézary syndrome

“…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.…”

“…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.…”

“…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.…”

An Automated Method for the Analysis of T-Cell Receptor Repertoires: Rapid RT-PCR Fragment Length Analysis of the T-Cell Receptor β Chain Complementarity-Determining Region 3

“…The analysis of expressed TCR (3 gene repertoires provides far more information than the comparable analysis of genomic DNA by established Southern blot or PCR techniques. [1][2][3] Monoclonal, oligoclonal, and polyclonal patterns of TCR expression can be discriminated easily by repertoire analysis, and the specific V(3 gene families involved in each pattern of expression are identified. This information, which extends traditional TCR gene family rearrangement studies, is necessary for the analysis of complex T-cell populations, such as those associated with autoimmune disease and benign T-cell lymphoproliferative disorders.…”

“…The analysis of genomic TCR rearrangement patterns is useful for the diagnosis of monoclonal lymphoproliferative disorders, such as T-cell lymphoma. [1][2][3] The examination of TCR transcripts is more informative in autoimmune diseases and nonmalignant lymphoproliferative disorders in which complex populations of T cells may be contributing to the disease.…”