Characterization of clone-specific rearrangement T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing.
The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T-NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T-cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.
Department of Internal Medicine, Georg-August University, Goettingen, Germany.
M
M
Kneba
I
I
Bolz
B
B
Linke
J
J
Bertram
D
D
Rothaupt
W
W
Hiddemann
D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000737:chemistry*; D004586:Electrophoresis; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D008223:Lymphoma / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics
D016692:Receptors, Antigen, T-Cell, gamma-delta; D004247:DNA