PERSONALIZED TEST FOR HIGH SENSITIVE DETECTION OF RESIDUAL PRIMITIVE LEUKEMIC CELLS BY DIRECT NESTED-PCR OF BCR-ABL1 REARRANGEMENT AT DNA LEVEL

SANCHEZ MB; RUIZ MS; GUTIERREZ L; KOILE D; YANKILEVICH P; MOSQUEIRA C; SANCHEZ AVALOS JC; MORDOH J; LARRIPA I; BIANCHINI M

Congreso; Reunión conjunta de sociedades de biociencias; 2017

Chronic myeloid leukemia (CML) patients who achieve a stable undetectable minimal residual disease (UMRD) by RT-qPCR are candidate for tyrosine kinase inhibitors (TKIs) suspension. In TKI-discontinuation trials, around 60% of patients loose UMRD and immediate TKI reinitiation is necessary. These results suggest that the risk of molecular relapse might be related to the achievement of deeper molecular response, below the detection limit of RT-qPCR. Molecular relapse has been attributed to the persistence of leukemic stem cells (LSCs). Thus, strategies that increase PCR sensitivity for LSCs detection may be relevant to better select patients with the highest likelihoods of maintaining treatment-free remission (TFR). Breakpoints that lead to BCR-ABL1 chimeric gene are highly dispersed over a ~150kbp region. Therefore, high quality DNA from newly diagnosed CML patients was necessary and amplified by a multiplex long-range PCR approach. The amplicons were sequenced using the Ion Torrent PGM platform. After alignment, the fusion point sequences were localized using CREST, and used for personalized PCR primers design (Primer3Plus). For LSCs isolation, 6-weeks-long term culture initiating cell (LTC-IC) assays were performed using CD34+ cells from fresh samples. Individual colonies plucked from methylcellulose were directly used for nested-PCR (dnPCR) without DNA extraction. We characterized the specific fusion breakpoint at DNA level in 4 out of 8 CML patients. For one of them, attaining MR3.0 at 18 months of Imatinib treatment, CD34+ cells were isolated for a LTC-IC assay; the proportion of residual LSCs (5 out of 20; 25%) was determined by personalized dnPCR.This study addressed two problems to improve deep monitoring of CML patients: determining the BCR-ABL1 breakpoint sequence and the detection of residual LSCs. Due to its complexity, the initial clinical role of DNA-PCR will be confined to monitor patients once they achieve RT-qPCR negativity and desire to enter TFR.