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Clinical Chemistry 47: 2003-2011, 2001;
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(Clinical Chemistry. 2001;47:2003-2011.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Denaturing HPLC for Identification of Clonal T-Cell Receptor {gamma} Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia

Udo zur Stadt1,1a, Johannes Rischewski1,1, Reinhard Schneppenheim1 and Hartmut Kabisch1

1 Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany.

aAuthor for correspondence. Fax 49-40-42803-8931; e-mail zurstadt{at}uke.uni-hamburg.de.

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 °C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 °C.

Results: For V{gamma}I-J{gamma}1.3/2.3 rearrangements, an analysis temperature of 60 °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.






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