IGH TCR-DELTA PCR OLIGONUCLEOTIDE LIQUID HYBRIDIZATION, A FAST AND SENSITIVE ASSAY FOR MONITORING MINIMAL RESIDUAL DISEASE IN CHILDHOOD B-PRECURSOR ALL/

The detection of minimal residual disease (MRD) in childhood B-precurs or acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PC R) may turn out to be a powerful tool in the evaluation and guidance o f therapy. Most previously described techniques are highly sensitive b ut also too laborious for application in a routine setting. Here we de scribe a technique based on the determination of IgH V(H)DJ(H) and TCR V delta 2D delta 3 junctional regions by PCR-cycle sequencing analysi s and hybridization of junctional region oligonucleotide probes in a s tandard liquid hybridization (LH) assay. We systematically analyzed th e applicability of this simplified approach for the monitoring of MRD in a large patient group. IgH V(H)DJ(H) and TCR V delta 2D delta 3 jun ctional regions were amplified from presentation bone marrow samples o btained from 53 childhood B-precursor ALL patients. The combined appro ach allowed the identification of at least one tumor marker for 49/53 (92.5%) of patients. A total of 75 oligonucleotide probes (54 DJ(H), 2 1 V delta 2D delta 3) was tested in the LH assay. Sensitivity range wa s 10(-2)10(-5) and 10(-4)-10(-5) for DJ(H) and V delta 2D delta 3 junc tional region probes, respectively. A sensitivity of at least one mali gnant cell in 10(4) normal cells was obtained for 84.8% of evaluable p atients, applying on average 1.1 IgH and 0.47 TCR delta probes per pat ient. Comparison to a method based on the use of initial PCR product a s clone-specific probe showed that oligonucleotide LH was one log more sensitive in six of nine patients tested. The presented technique all ows the monitoring of MRD with acceptable sensitivities in over 90% of childhood B-precursor ALL patients. Moreover, the technique is suitab le for prospective patient studies in a routine setting as it is fast, reproducible and makes use of a standard hybridization protocol for d ifferent oligonucleotide probes.