Rapid, multifluorescent TCRG Vgamma and Jgamma typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations.
Detection of clonal T cell receptor gamma (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10(-3) at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent Vgamma and Jgamma PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vgamma and Jgamma segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10(-2)-10(-3) level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TAL1-negative and -positive cases. A combination of fluorescent multiplex and Vgamma-Jgamma-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.
Delabesse
E
E
Biological Hematology, Hôpital Necker-Enfants Malades and Université Paris V, France.
Burtin
M L
ML
Millien
C
C
Madonik
A
A
Arnulf
B
B
Beldjord
K
K
Valensi
F
F
Macintyre
E A
EA
D000293:Adolescent; D001483:Base Sequence; D002648:Child; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000276:immunology*; D016133:Polymerase Chain Reaction; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D016692:Receptors, Antigen, T-Cell, gamma-delta