Molecular Diagnosis of Cutaneous T-Cell Lymphoma: Polymerase Chain Reaction Amplification of T-Cell Antigen Receptor β-Chain Gene Rearrangements

Abstract

The goal of our study was to molecularly diagnose CTCL, by cloning the T-cell antigen receptor beta chain (TCR-beta) gene rearrangement from the malignant T cells of a patient with Sézary syndrome, in order to generate a specific oligonucleotide probe capable of detecting CTCL cells through polymerase chain reaction (PCR) amplification. Total RNA isolated from peripheral blood lymphocytes was reverse transcribed and resultant first strand cDNA was PCR amplified utilizing a concensus primer to the TCR-beta variable region (V beta) and a 3' primer to the TCR-beta constant region (C beta). PCR reaction products were subcloned into a plasmid vector and sequenced. Sequence analysis revealed that the patient's in-frame TCR-beta gene rearrangement utilized V beta 6.4, D beta 1.1, J beta 2.2, and C beta 2.1 gene segments. Oligo-primers to V beta 6.4 and J beta 2.2 were utilized to PCR amplify genomic DNA taken from the patient's blood and involved skin. Screening the amplified DNA with an oligo-probe specific for the patient's V-D-J junctional sequences resulted in the detection of the patient-specific sequences. No sequences were detected from DNA from other malignant or benign infiltrates. Thus, we have defined a "molecular fingerprint" specific for a patient's malignant T-cells and can molecularly diagnose CTCL through PCR amplification.