Comparison of BIOMED-2 versus laboratory-developed polymerase chain reaction assays for detecting T-cell receptor-gamma gene rearrangements

Abstract

Detecting clonal T-cell receptor (TCR)-gamma gene rearrangements (GRs) is an important adjunct test for diagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol), which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V), with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-gamma GR. The combination of TCR-beta, mono-V TCR-gamma and multi-V TCR-gamma detected more clonal cases (68/144, 47%) than any individual PCR assay. We detected clonal TCR-beta GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-gamma primers, the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-gamma primers improved the sensitivity for detecting clonality, 60/68 (88%). Combining either mono-V or multi-V TCR-gamma primers with TCR-beta primers also improved the sensitivity, 64/68 (94%). Significantly, TCR-gamma V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.

Figure 1

A: Schematic representation of the TCR-γ genomic DNA locus. Approximate positions of the primers for both mono-V and multi-V PCR strategies are marked by arrows. The bars represent consensus primer-binding sequences. * ← JP primer is only used in the mono-V assay. B:TCR-γ variable gene segments V1-8, V9, V10, V11 (forward primers) and J segments (reverse primers): Reference DNA sequences from GenBank (top), mono-V TCR-γ primers (middle), multi-V TCR-γ primers (bottom). All DNA sequences are in 5′ to 3′ orientation. C: Detection of TCR-γ PCR products by capillary gel electrophoresis. Mono-V (left column) and multi-V (right column) PCR assays amplified representative DNA samples from the same patient.

Figure 2

Comparison of TCR-γ V11 gene segment amplification by mono-V versus multi-V PCR assays in control DNA. All PCR assays were set in 25 μl reaction volume. The commercial polyclonal (A and B) and clonal (E and F) control DNAs obtained from In vivoScribe were used at 500 ng per reaction. The reactive tonsil DNA (C and D), Jurkat T-cell line DNA (G–I), and 10% Jurkat T-cell line DNA diluted in reactive tonsil DNA (J and K) were used at 250 ng per reaction. T_A, annealing temperature of the PCR reaction. I shows the same PCR products as H but with a 17.5-fold increase in scale sensitivity.

Figure 3

Effect of combining individual multi-V TCR-γ V9 and V11 BIOMED-2 primers. We synthesized multi-V TCR-γ V9 and V11 primers based on their published DNA sequences. We added the primers individually (A, D, G and B, E, H) and in combination with each other (C, F, I) to amplify TCR-γ GRs from control DNA. Reactive tonsil DNA (A–C), 100% Jurkat T-cell line DNA (D–F), and 10% Jurkat T-cell line DNA diluted in reactive tonsil DNA (G–I) were used at 1 μg in 50 μl reaction volume. All PCR reactions included a mixture of J1/2 and JP1/P2 primers.

Figure 4

Effect of annealing temperature (T_A) on TCR-γ V11 gene segment amplification using the V9 + V11 multi-V primer set. All PCR assays were set in 25 μl reaction volume. Reactive tonsil DNA (A–C), Jurkat T-cell line DNA (D–F), 10% Jurkat T-cell line DNA diluted in reactive tonsil DNA (G–I), and a representative clinical DNA sample (J–L; patient 3, Table 3) were used at 250 ng per PCR reaction. PCR amplification was performed at T_A of 60°C (A, D, G, J), 55°C (B, E, H, K), or 50°C (C, F, I, L).