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Characterization of clone-specific rearrangement T-cell receptor gamma-
chain genes in lymphomas and leukemias by the polymerase chain reaction and
DNA sequencing
M Kneba, I Bolz, B Linke, J Bertram, D Rothaupt and W Hiddemann
Department of Internal Medicine, Georg-August University, Goettingen,
Germany.
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.
Volume 84,
Issue 2,
pp. 574-581,
07/15/1994
Copyright © 1994 by The American Society of Hematology
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