Resolving T-cell receptor clonality in two and genotype in four multiplex polymerase chain reactions

Department of Pathology, Lund University Hospital, Lund, Sweden. michael.dictor@pat.lu.se

BACKGROUND AND OBJECTIVES: The diagnosis of T-cell neoplasia requires the use of immunohistochemistry on tumor sections or molecular genetic analysis of T-cell receptor (TCR) clonality. Multiplex polymerase chain reactions (PCR) offer a sensitive and expeditious approach to determining clonality early in the diagnostic work-up. We determined the sensitivity and specificity of four multiple PCR for genotyping lymphoid neoplasms at the TCR loci gamma (TCRG), delta (TCRD) and beta (TCRB, including complete [Vbeta-Jbeta] and incomplete [Dbeta-Jbeta] rearrangements). DESIGN AND METHODS: Template DNA was derived from frozen or formalin-fixed tissue and from imprints of aspirates or cut tissue surface on a FTA MicroCard. Each multiplex PCR was performed for 36 cycles in a single tube with multiple previously reported fluorescently labeled primers (TCRG and TCRD) or novel homologous primers (TCRB) and analyzed on electropherograms (Genescan), applying stringent criteria for interpreting clonal peaks. Two hundred and eleven clinically and immunohistochemically well-characterized benign and malignant non-T-cell lymphoid proliferations, including 138 B-cell lymphomas, were analyzed to determine specificity. The results were compared with those of 28 peripheral and immature T-cell neoplasms and two NK/T-cell lymphomas to determine sensitivity and compute predictive values. RESULTS: In all T-cell tumors, one or more TCR loci showed clonal rearrangement, which was not evident in two NK/T-cell lymphomas. TCRG was the single most informative locus (clonal rearrangement in 89%), followed by TCRB (79%) and TCRD (39%). Multiplex PCR targeting of TCRG and TCRD together resolved clonality in all T-cell neoplasms, whereas the TCRB locus was clonal in two of three cases with polyclonal TCRG. Unexpectedly, in B-cell lymphomas single clonal incomplete TCRB (Dbeta-Jbeta) peaks were 20 times more likely to occur than clonal TCRG. INTERPRETATION AND CONCLUSIONS: Clonality can be accurately determined in nodal T-cell lymphoma with two single-tube multiplex PCR targeting TCRG and TCRD. TCRB analysis should be considered in equivocal cases in which a polyclonal background may obscure clonal TCRD, but clonal incomplete TCRB rearrangement alone is insufficient for presuming T-cell lineage. In the absence of objective evidence of B-cell neoplasia, multiplex PCR of T-cell receptor genes may be used early in the diagnostic work-up, including for fine needle aspirates.

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