Analysis of T-cell receptor-gamma gene rearrangements using oligonucleotide microchip: a novel approach for the determination of T-cell clonality

Abstract

T-cell clonality estimation is important for the differential diagnosis between malignant and nonmalignant T-cell proliferation. Routinely used methods include polymerase chain reaction (PCR) analysis of T-cell receptor-gamma (TCR-gamma) gene rearrangements followed by Genescan analysis, polyacrylamide gel electrophoresis, or heteroduplex analysis to visualize amplification products. Here, we present a new method for the analysis after PCR of TCR-gamma rearrangements using hybridization on oligonucleotide microchip. A microchip was designed to contain specific probes for all functional variable (V) and joining (J) gene segments involved in rearrangements of the TCR-gamma locus. Fluorescently labeled fragments of rearranged gamma-chain from patients and donors were obtained in a multiplex nested PCR and hybridized with a microchip. The results were detected using a portable microchip analyzer. Samples from 49 patients with T-cell lymphomas or leukemias and 47 donors were analyzed for T-cell clonality by microchip and single-strand conformation polymorphism analysis, which served as a standard reference method. Comparison of two techniques showed full concordance of the results. The microchip-based approach also allowed the identification of V and J gene segments involved in the particular TCR-gamma rearrangement. The sensitivity of the method is sufficient to determine 10% of clonal cells in the sample.

Figure 1

Scheme of the microchip for detecting T-cell clonality by rearrangements of the TCR-γ locus. Gel pads with oligonucleotides complementary to sequences of the Vγ genes of family 1 (Vγ 1-8) are shown dark gray, gel pads with oligonucleotides complementary to sequences of the Vγ genes of families 2, 3, and 4 (Vγ9, Vγ10, Vγ11) are shown light gray, and gel pads with oligonucleotides complementary to sequences of the Jγ genes are shown white. Each oligonucleotide is used in duplicate on the microchip.

Figure 2

a: Hybridization pattern obtained on the microchip with a polyclonal sample. b: Analysis of the results. The TS is shown with a horizontal line. None of the signals exceeds the TS.

Figure 3

a: Hybridization pattern obtained on the microchip with a clonal sample. b: Analysis of the results suggesting a clonal rearrangement of the TCR-γ locus. The TS is shown with a horizontal line. Signals from gel pads Vγ2, Vγ10, and Jγ1/2 exceed the TS and suggest a biallelic clonal rearrangement of the TCR-γ locus with the involvement of the Vγ2, Vγ10, and Jγ1/2 genes.

Figure 4

a: Hybridization pattern obtained on the microchip with oligoclonal sample. b: Analysis using the threshold signal (TS) equal to <J> + 3 × σ (TS is shown with a horizontal line). c: Analysis using the TS equal to <J> + 4 × σ.

Figure 5

Sensitivity assay. Hybridization pattern (a) and analysis of the results (b) for a sample containing 10% of Jurkat cells. Signals from gel pads Vγ8, Vγ11, and Jγ1/2 exceed the TS and suggest a biallelic clonal rearrangement of the TCR-γ locus with the involvement of the Vγ8, Vγ11, and Jγ1/2 genes. Hybridization pattern (c) and analysis of the results (d) for a sample containing 5% of Jurkat cells. Only signal from gel pad Vγ11 hardly exceeds the TS; there is no sufficient data in favor of clonal rearrangement.