Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia.
BACKGROUND
Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.


METHODS
TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C.


RESULTS
For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.


CONCLUSIONS
Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
zur Stadt
U
U
Department of Pediatric Hematology and Oncology, University Children's Hospital, Martinistrasse 52, D-20246 Hamburg, Germany. zurstadt@uke.uni-hamburge.de
Rischewski
J
J
Schneppenheim
R
R
Kabisch
H
H
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D020180:Heteroduplex Analysis; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*
D016692:Receptors, Antigen, T-Cell, gamma-delta