P853: automated separation of cell clusters provides an easy and accurate strategy for the analysis of minimal residual disease in multiple mieloma in single 11-colors flow cytometry combination.

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Minimal residual disease (MRD) by flow cytometry (FCM) is an important prognostic factor in patients with Multiple Myeloma (MM). Thanks to the incorporation of new fluorochromes and the development of new generation cytometers, it is feasible to perform the MRD studies in a single tube, increasing the number of fluorescences and applying reproducible multidimensional analysis strategies. Aims: -To design a simple and reproducible analysis strategy, applicable to a single tube of 11 colors, for the study of EMR in MM in patients who have received anti-CD38 therapies. -To determine the most significant parameters for the discrimination between populations of polyclonal and aberrant plasma cells. -To identify possible phenotypic changes in persistent populations compared with the phenotype at the moment of diagnosis. Methods: We studied 76 patients with MM in complete response. All of them had received therapeutic regimens including anti-CD38, 19 patients had additionally received autologous hematopoietic stem cell transplantation (aHSCT). A minimum of 10 million nucleated cells from the bone marrow aspirate were analyzed, excluding debris and doublets, with the following panel: CD38+CD229 FITC, CD138 BV-421, CD45 V500, CD19 PE-Cy7, CD56 PE, CD117 BV-605, CD81 BV-711, CD28 BV-786, CD27 PerCPcy5.5, cy-kappa APC and cy-lambda APC-H7. FACS-Lyric Cytometer (Becton Dickinson, CA) Analysis strategy: 1.Plasma cells were selected attending to the expression of CD38+CD229, CD138 and cy-Kappa or cy-Lambda; once the quality of the sample had been verified (using internal parameters). 2.The pathological population was differentiated from the normal one by comparing the distribution of plasma cells according to the immunoglobulin light chain expressed (Figure 1). The parameters with the highest discriminant power were automatically calculated by a principal component analysis (PCA). As a validation of the technique, we performed serial dilution of abnormal plasma cells (obtained by FACS sorting) in healthy bone marrow samples and we evaluated the concordance between the expected and the detected cells. Results: The described strategy allows the analysis of MRD in a simple and objective way in patients treated with anti-CD38 schemes, avoiding the need to merge results from 2 different tubes. The calculated detection and quantification thresholds were similar to the standardized 8-color panels. EMR was detectable in 61% of the patients (min: 0.00059%; max: 0.29%) and the presence of different phenotypic subclones was confirmed in 23% of them. Markers with a significance ≥10% in the PCA showed an increased discrimination power, improving the separation of pathological clusters from normal plasma cells In some patients, up to 6 different markers were necessary to obtain the best differentiation of the aberrant population. In the overall series, the most discriminative markers were CD19, CD56, CD117, CD28, CD27 and FSC (Table 1). Residual populations frequently showed aberrant expression of CD28, compared to diagnosis. The remaining differences were not significant. Summary/Conclusion: -The described methodology is simple and allows the objective identification of residual populations in a single tube, no need to merge files or calculate data for the analysis. -All the selected markers are necessary to maximize the discriminative potential of the technique. -Larger studies are required in order to find out if the increase in CD28 expression or the appearance of different subclones in the residual populations may be related to chemoresistance and clonal selection phenomena.Figure 1. Table 1. - SIGNIFICANCE IN THE PCA* (kappa or lambda) MOST DISCRIMINATIVE MARKER** min mean max cases (%) CD19 5,65 17,32 29,39 92,3 CD56 8,8 18,88 29,66 80,5 CD28 0,83 8,51 13,76 40,1 CD27 1,6 8,08 16,24 36,3 CD117 1,6 7,1 13,35 35,9 CD81 0,85 10,5 15,4 26,7 FSC 1,56 10,79 34,44 26,1 SSC 0,26 7,57 31,4 20,2 CD45 0,09 7,45 11,7 16,1 CD38 + 229 0,06 3,9 10,24 4,5 *PCA: principal components analysis**singnificance ≥10 Keywords: Multiple myeloma, Minimal residual disease (MRD), Flow cytometry