EX-VIVO CLONOTYPE PRIMER-DIRECTED GENE AMPLIFICATION TO IDENTIFY MALIGNANT T-CELL REPERTOIRES

A novel strategy that utilizes input genomic DNA and overcomes limitations encountered with traditional RNA reverse transcription-polymerase chain reaction (PCR) amplification methodology is described to screen for T cell receptor (TCR) repertoires. The methodology has been developed to identify individual T cell clonotypes with regard to their unique receptor beta chain variable/diversity/joining (VDJ) region gene rearrangement. The technique avoids preselection for a given antigen specificity and is therefore independent of artificial bias introduced by in vitro cell population expansion. This technique was used to detect and identify genetically of malignant clones from heterogeneous mononuclear cell populations from an array of hemato-oncological disorders, including mycosis fungoides/Sezary Syndrome, adult T cell leukemia, and large granular lymphoproliferative disease. An initial primary PCR, directed by a TCR-Jbeta generic primer and a complement of family-specific TCR-Vbeta primers, defines predominant T cell receptor variable gene usage. Use of a TCR-Jbeta generic primer supplants the use of a constant region primer anchor and thus eliminates the need to target mRNA. The process of variable gene screening also expedites gene sequencing. By sequencing through the VDJ juxtaposed region, i.e., the third complementarity determinant region, clonotype-specific primers are developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, unique T cell clonal repertoires. Analysis of the products of the CPD-PCR permits the detection of a single malignant cell among one million polyclonal cells and supercedes the constraints of prior studies that provide a limited evaluation of family variable gene repertoire usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones.