NGS-Based Minimal Residual Disease (MRD) after Stem Cell Transplantation (SCT) Is More Specific for Relapse Prediction Than qPCR and Suggests the Possibility of False-Positive qPCR Results

• Published in: Blood 2016-12, Vol.128 (22), p.3494-3494
• Main Authors: Kotrova, Michaela, van der Velden, Vincent H.J., van Dongen, Jacques J.M., Formankova, Renata, Sedlacek, Petr, Brüggemann, Monika, Zuna, Jan, Stary, Jan, Trka, Jan, Fronkova, Eva
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.3494.3494

Background MRD detection using qPCR for IG/TR rearrangements has been the gold standard approach for relapse prediction and for guiding post-transplant therapeutic interventions for already two decades. Despite its thorough standardization within the EuroMRD laboratories and very strict rules for defining MRD positivity (van der Velden, Leukemia, 2007) this method carries the risk of false-positive MRD results, mainly in post-transplant time-points with massive B-cell regeneration (Fronkova, Bone Marrow Transplantation, 2008). This may lead to application of unneeded therapeutic interventions and to the misinterpretations in the post-SCT survival analyses. Our previous study employing amplicon NGS-based MRD detection in frontline paediatric ALL patients (Kotrova, Blood, 2015) showed, that the sensitivity of NGS and qPCR is comparable, whereas NGS was proven to have better specifity for relapse prediction. We hypothesized that amplicon NGS of IG/TR rearrangements, providing the sequences of entire CDR3 regions, is a perfect tool for distinction between false and real post-transplant positivities. Aims To detect MRD by amplicon NGS in: 1) qPCR low-positive (non-quantifiable) samples from paediatric ALL patients after allogeneic SCT, who remained in long-therm complete remission 2) qPCR low-positive post-transplant samples from a control cohort of paediatric ALL patients who subsequently relapsed Methods Sequencing libraries were prepared from 500ng of bone marrow/peripheral blood DNA employing two-round PCR. In the first PCR virtually all IGH or TRG rearrangements present in the investigated sample are amplified using universal V(D)- and J-regions primers. In the second round, sequencing adaptors for Ion Torrent sequencers and sample-specific barcodes are added. Libraries were sequenced on Ion Torrent PGM/Ion Proton sequencers using the Hi-Q chemistry, according to the manufacturer's instructions. For the detection of clonal leukemia-specific IG/TR rearrangements we developed own in-house bioinformatics algorithm. NGS-MRD positivity was defined as the presence of leukemia-specific sequence(s) as identified at diagnosis in the follow-up samples. Results We sequenced 27 qPCR-positive (not quantifiable) post-transplant samples from 17 patients (14 patients carrying IGH and 3 patients TRG targets) who remained in complete remission after SCT with a median follow-up of 10.4 years (range 2.2-15.8). Only 1 of 27 (3.7%) follow-up samples was found to be MRD-positive