P-039: Adaptation of a myeloma minimal residual disease multi-parametric flow assay for real world practice

Abstract

<h3>Background</h3> Achieving minimal residual disease (MRD) negativity during treatment for multiple myeloma (MM) is prognostic for disease free and overall survival. There is limited testing of MRD outside clinical trials, and the need to process fresh samples for multicolour flow cytometry (MCF) assays presents a challenge to laboratories. We set up an 8-colour MCF assay, in collaboration with a reference centre (Leeds HMDS), to identify and quantify malignant plasma cells in bone marrow (to limit of detection 10-5), using a BD LSRFortessaTM Flow Cytometer. We developed a fixation protocol, removing the need for immediate flow cytometer access and carried out a pilot study of MRD status in patients treated with standard non-trial protocols. <h3>Methods</h3> Initially, 43 fresh samples were processed in parallel with duplicate samples sent to Leeds, with 100% concordance in MRD result. We optimised a novel fixation technique, processing and staining samples within 24 hours, before using 0.4% paraformaldehyde (PFA) to fix, and storing at 4°C, permitting data acquisition up to 6 days later. We tested this on 24 samples and confirmed concordance between fresh and fixed samples, with good correlation in MRD level (Pearson's correlation co-efficient; r=0.95 (95% CI 0.89-09.8); r2=0.91; p=<0.0001), minimal bias (Bland-Altman; 0.04), and no change in plasma cell phenotype by flow markers. There was no difference comparing fresh samples with those fixed for 1-3 days or 4-6 days. <h3>Results</h3> Between July 2020 and June 2021, MRD was performed on 76 samples, with 24% (18/76) fixed to facilitate processing. All samples were from transplant eligible MM patients, with 58 post-induction therapy, on aspirates performed for restaging prior to autologous stem cell transplantation (ASCT). This cohort represents a UK real-world snapshot, with complete response in 28% (16/58) post-induction with NICE-approved bortezomib-based triplet regimens. 17% (10/58) of samples were MRD negative, with no difference in post-induction MRD level between patients with or without R-ISS-defined high-risk cytogenetics (p=0.72). 18 post-ASCT samples were analysed, and 8 (44.4%) were MRD negative; in 8 of these with matched pre-ASCT samples (all MRD positive), 4 were MRD negative post-ASCT. The number of MRD negative samples was significantly higher post-ASCT (p=0.02). <h3>Conclusions</h3> In summary, we describe an adaptation of the Leeds HMDS 8-colour MRD assay to include a novel fixation step, permitting batching for flow cytometric analysis, without affecting sample validity. This real-world snapshot suggests that less than 20% of patients are MRD negative post standard UK induction therapy, but this increases post-ASCT. Further work on larger cohorts and longer follow up will confirm the prognostic value of MRD assessments outside the trial setting and establish its utility in real world practice.