DHPLC based fraction collection of TCR-γ rearrangments in childhood ALL: Direct sequencing of products amplified by a single or a multiplex PCR approach

Clonal T-cell receptor γ (TCR-γ) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-γ rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vγ segments in two distinct reactions either with a Jγ1.3/2.3 or a Jγ1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VγI-Jγ1.3/2.3 including 51 biclonal rearrangements. For characterisaton of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jγ primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-γ rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.