Novel analysis of minimal residual disease in leukemia with TCR beta rearrangement--detection of monoclonality by single strand conformation polymorphism and PCR using a clonotype primer of leukemic T cell receptor beta-chain RNA.
Several means of analyzing minimal residual disease (MRD) in leukemia involving the rearranged T cell receptor (TCR) gene have been described. We investigated MRD in leukemia with TCR beta rearrangement by examining TCR beta-chain RNA. A complementary DNA (cDNA) corresponding to the variable region of the TCR beta-chains originating from the peripheral blood or bone marrow from four patients was amplified. Single strand conformation polymorphism (SSCP) analysis of amplified cDNA showed that all four patients had monoclonal leukemia with TCR beta rearrangement; two patients had Vbeta2+ leukemia, another patient had Vbeta14+ leukemia and the other had Vbeta9+ leukemia. Flow cytometry supported this finding. Sequencing of the Vbeta2-complementarity determining region 3 (CDR3), Vbeta9-CDR3 and Vbeta14-CDR3 revealed monoclonality. To investigate MRD using TCR beta-chain RNA, cDNA from each patient was diluted with the cDNA of a healthy person and amplified using a specific CDR3 clonotype primer. A band in the ethidium bromide-stained agarose gel was detected from samples diluted 10,000-fold. SSCP analysis determined which V region gene was utilized in monoclonal leukemic cells. The leukemic cell specific TCR, determined in such a manner, may be a target for immunotherapy. Because the MRD of T cell malignancy can be easily examined once the CDR3 clonotype primer is made, this novel analysis is considered to be a useful method.
Hanawa
H
H
First Department of Internal Medicine, Niigata University School of Medicine, Japan.
Koike
T
T
Sakaue
M
M
Kishi
K
K
Shibata
A
A
Abo
T
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D001483:Base Sequence; D005260:Female; D005434:Flow Cytometry; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D006801:Humans; D015458:Leukemia, T-Cell / Q000235:genetics / Q000473:pathology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D018365:Neoplasm, Residual / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012313:RNA / Q000235:genetics*
C034477:RNA primers; D012313:RNA