Introduction: The clinical and prognostic utility of MRD monitoring in MM bone marrow (BM) by first generation (4-6-color) flow cytometry (flow-MRD), has been now demostrated for more than a decade. Thereby, flow-MRD is considered to be a well-suited technique for MRD monitoring in MM, due to its high applicability and specificity, and its broad availability in diagnostic laboratories. However, recent results have confirmed that 1st generation flow-MRD has a lower sensitivity than molecular techniques such as allele-specific oligonucleotyde (ASO)-PCR and next generation sequencing (NGS); in addition, the lack of standardization of conventional flow-MRD approaches, also has a negative impact on its reproducibility. Here we report on the validation of the next generation flow (NGF)-MRD approach developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for ultrasensitive and standardized detection of MRD in MM.

Methods: A total of 275 BM samples were included in the study: 1) a group of 31 normal/reactive and 63 diagnostic MM BM samples were evaluated to identify the most efficient candidate markers for the NGF panel, using a multivariate analysis-based approach; 2) next, a total of 181 BM samples from 15 healthy donors (HD), 36 MGUS, 15 MM and 3 solitary plasmacytoma (SP) patients studied at diagnosis, plus 112 MM follow-up samples, most of which (n=71) corresponded to BM samples from MM patients in very good partial response, complete remission (CR) or stringent CR were analyzed. These samples were evaluated by the EuroFlow-IMF NGF-MRD method. The method was based on a (standardized) lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of BM cells (≥5x106 cells/tube) and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of BM plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC): tube 1: CD138BV421 CD27BV510 CD38FITC CD56PE CD45PerCP Cy5.5 CD19PE Cy7 CD117APC CD81APC C750, and; tube 2: identical to tube 1 except for cytoplasmic (Cy) Immunoglobulin (Ig) kAPC/CyIglAPC C750). Results obtained with the NGF-MRD MM method in the 71 VGPR, CR and sCR samples, were then compared with a 2nd generation (routine) 8-color flow-MRD approach which involved a single 8-color combination staining for the same markers described above for tube 1, but in the absence of full optimization of the positions for the antibody-fluorochrome conjugates and no selection for treatment-independent antibody CD38 clones. In a subset of 16 of these 71 MRD samples in which enough material was available, comparison with NGS was also performed in parallel.

Results: In all MGUS, MM and SP cases analyzed at diagnosis, aPC showed aberrant phenotypes vs. nPC from HD BM, based on multivariate analysis of individual cells from each of the patients against a reference data base of normal/reactive PCs (100% applicability). For the MM MRD BM samples, a median of 9.8x106 (range: 2.4-15.3) events were acquired (tube 1 plus 2) vs. 1x106 (range: 0.03-5) events for the 2nd generation flow-MRD approach with an (estimated) 10 times lower limit of detection and 10 times lower limit of quantitation of 3x10-6 and 5x10-6 vs. 3x10-5 and 5x10-5 for the NGF-MRD vs. the 2nd generation flow-MRD approaches, respectively. This led to a higher rate of MRD+ samples with the NGF-MRD method: 14/48 (29%) cases that were flow-MRD-negative with the 2nd generation 8-color flow-MRD method became MRD+ (median percentage of residual aPC of 0.0007%; range: 0.0002 to 0.02%) (Figure 1A). In contrast, 4/38 (11%) samples were negative by NGF, while positive by 2nd generation flow-MRD; one of these four proved to be MRD-negative by cytoplasmic immunoglobulin light chain restriction analysis. Further comparison of NGF vs NGS showed 9 of the 16 samples evaluable were MRD-negative by NGS; from them, one third (3/9) were positive by NGF with a median number of residual aPCs of 0.005% (range: 0.0002-0.006%) (Figure 1B).

Conclusions: The EuroFlow-IMF NGF-MRD approach provides a fast, highly applicable, ultrasensitive, standardized and accurate approach for the assessment of MRD in BM samples from MM patients and overcomes the current limitations of both 1st and 2nd generation flow-MRD approaches; preliminary results showed higher sensitivity than NGS.


Paiva:Sanofi: Consultancy; Binding Site: Consultancy; EngMab AG: Research Funding; BD Bioscience: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Janssen-Cilag: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy. San Miguel:Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson & Johnson: Consultancy. van Dongen:Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics.; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge. Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics. Royalty income for EuroClonality-BIOMED-2 Consortium; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences)., Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL..

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