PS1313 FEASIBILITY AND PRELIMINARY RESULTS OF A PROSPECTIVE EVALUATION OF RESIDUAL DISEASE IN DIFFUSE LARGE B-CELL LYMPHOMAS USING DEEP SEQUENCING OF CELL FREE DNA AND DNA FROM FORMALIN FIXED BIOPSIES
Authors: C. Carniti, G. Biancon, S. Banfi, C. Vella, M. Magni, M. Pennisi, D. Anna, A. Guidetti, P. Corradini
Journal: HemaSphere (2019)

Background: Minimal residual disease (MRD) detection in mantle cell lymphoma (MCL) provides valuable prognostic information, leading to the design of MRD-based therapeutic strategies in prospective clinical trials. Quantitative polymerase chain reaction (qPCR) of IGH or BCL1-IgH rearrangements is the gold standard for MRD monitoring in MCL. Its major limitation is its relative quantification and the high proportion of follow-up (FU) samples with a low tumor burden that fall below the quantitative range (BQR) of the standard curve. Multiparameter flow cytometry (MFC) is a rapid absolute quantification alternative to qPCR which showed promise on retrospective testing. (Cheminant M. et al.) Aims: We here compare qPCR, prospective MFC and ddPCR in MCL samples from three laboratories of the Euro-MRD consortium. The main aim was to compare prospective MFC and ddPCR, particularly in FU samples with a low tumor burden, defined as BQR by qPCR Methods: Samples were collected (with informed consent) in three prospective EU-MCL network clinical trials. qPCR and ddPCR were performed in Paris-Necker, Creteil and Torino according to Euro-MRD guidelines, as described (van der Velden VH. et al, Drandi D. et al). Based on Poisson assumption, we stratified results with alternatively positive or negative replicates into three subgroups- for qPCR: 3/3 BQR vs. 2/3 vs. 1/3 positive replicates; for ddPCR: 2/3 or 1/3 replicates with > = 3 positive events vs. 2/3 replicates with <3 events vs. 1/3 replicates with <3 events. ddPCR results with <3 replicates and > = 3 positive events were considered to be below the limit of quantification (BQL); those with <3 events were considered negative. MFC was performed, as described (Cheminant M. et al), with a robust quantifiable sensitivity (<30 cells in the “Mantle box”) of 10E-4. Samples positive between 5.10E-5 and 10E-4 were considered positive BQF (Fluorescent). Results: 198/242 FU samples from 116 MCL with both qPCR and ddPCR with 3 replicates were analysed. Of these, 115/198 (58%) were BQR by qPCR. Overall, ddPCR scored positive (in all 3 replicates) in 28/115 (24%). ddPCR positive results belonged mostly to the BQR 3/3 triplicate positive samples (20/47, 42%) whereas only 7/43 (16%) of 2/3 and 1/25 (4%) of 1/3 triplicate positive results by qPCR were ddPCR positive, raising the possibility of non-specific false positivity by qPCR. 5/42 (12%) qPCR negative samples were positive by ddPCR (Table 1). Prospective MFC and ddPCR were performed on 68 FU samples in one center. All 12 samples positive >10E-4 by ddPCR were also MFC positive, whereas only 2/16 samples positive by ddPCR below 10E-4, including 3 BQL, were positive by MFC. Conversely 5/40 ddPCR negative samples were MFC positive, but 4/5 were selected on the basis of MFC+/qPCR negativity, with ddPCR confirming the qPCR result.Summary/Conclusion: ddPCR, recently introduced into the Euro-MRD network, is comparable to qPCR and MFC quantification for high level MRD positivity (>10E-4) but is more sensitive than MFC for lower level positivity. Compared to qPCR, it reduces the incidence of BQR positivity by over 80% and, when using the criteria defined, here splits qPCR BQR results into 24% positive, 13% BQL and 63% negative results. The prognostic relevance of ddPCR needs to be compared to qPCR, but it appears better adapted to pre-emptive surveillance, since more sensitive than MCF, less subject to grey-zone positivity than qPCR and capable of absolute quantification of low-level positivity. Its place is currently being assessed in the context of prospective trials within the European MCL Network.