The Use of Next Generation Flow in Multiple Myeloma Patients: A Preliminary Real-Life Multicenter MRD Harmonization Experience
Authors: Oliva Stefania , Saraci Elona , Trimarco Valentina , Chiarini Marco , Parrinello Nunziatina Laura , Zambello Renato , Cordone Iole , Villanova Tania , Masi Serena , Rossi Giovanni , Marino Sara , Triolo Anna Maria , Roccaro Aldo , Carella Angelo Michele , Buccisano Francesco , Belotti Angelo , Irno Consalvo Maria , Romano Alessandra , Tonini Alessia , Mercadante Silvio , Bruno Benedetto , Terragna Carolina , Zamagni Elena 
Published: December 2023

Introduction:
Minimal residual disease (MRD) detection represents a sensitive tool to appropriately measure response in MM. The major concern about MRD detection in real-world setting is the reproducibility of results among different laboratories possibly due to methodological discrepancies. Therefore, a harmonized approach, according to criteria of Next generation Flow (NGF)/ Next generation sequencing (NGS) established by IMWG (Kumar S et al. Lancet Oncol 2016), is warranted. The aim of this project was to create an “Italian MM MRD network” using standardized NGF and NGS-MRD approach; here we report preliminary results of the NGF part.
Methods:
The “NGF harmonization project” includes 7 Italian Laboratories willing to commit to EuroFlow protocols after an initial MRD survey: Brescia (L1), Catania (L2), Padova (L3), Roma 1 (L4), Roma 2 (L5), S. Giovanni Rotondo (L6) and Torino (L7). Standardization of all flow cytometers settings was performed by implementation of the EuroFlow Standard Operating Protocol (SOP) for instrument setup and compensation for BD FASCCanto II, BD FACSLyric, BC Navios and BC DxFlex flow cytometers (www.euroflow.org).
Bone marrow (BM) aspirates were collected from newly diagnosed MM patients treated with 4 cycles of Dara-VTD (Daratumumab, bortezomib, thalidomide, dexamethasone) induction followed by autologous stem cell transplantation (ASCT) in complete-response/very-good-partial-response at day 100 (+/- 15 days) after ASCT. Anonymized samples were split equally and shipped at room temperature to the participati ng laboratories and simultaneously analyzed.
Analyses were carried out following NGF methodology (Flores-Montero J et al. Leukemia 2017) by using a two-tube 8-color antibody panel for monoclonal plasma cells identification (Tube 1:CD27, CD138, CD38, CD56, CD45, CD19, CD117, CD81 and Tube 2: CD27, CD138, CD38, CD56, CD45, CD19, cytoplasmic κ and λ light-chain).
We aimed to acquire ≥ 3,000,000 events per tube for a sensitivity of at least 1 x 10 -5; a sample was considered MRD positive when ≥ 20 monoclonal plasma cells were detected. Intraclass Correlation Coefficient (ICC) was performed to evaluate degree of correlation and agreement between measurements; standard deviation (SD) and Coefficient of variation (CV) to measure variability of the dataset.
Results.
In stage 1 of the study, before starting MRD evaluation on freshsamples, 4 laboratories (L1, L2, L3, L7) performed a blinded analysis of the same 5 anonymous flow-cytometry files from a retrospective data repository of MM patients with/without MRD, to evaluate the inter-operator variability: 100% of the participants were concordant that samples #1, #2 and #4 were MRD positive. Sample #3 was considered MRD positive by 75% of participants whereas 75% considered sample #5 as MRD negative (ICC=0.91, 95% CI 0.69-0.98, p&lt;0.001).
In stage 2, a total of 7 samples have been distributed and processed after 48 hours from specimen collection, due to different shipping time in all laboratories. The inter-laboratory correlation study showed a concordance of 100 % for the sample #1 and sample #3 considered as MRD positive, 100% of the participants considered sample #5 and sample #6 as MRD negative, sample #2 was considered MRD positive by 71% of participants whereas sample #4 was considered MRD negative by 86 % of participants. Sample #7 was considered as not evaluable from all participants due to a high percentage of dead cells. (ICC=0.61, 95% CI 0.31-0.91, p&lt;0.001) (Fig. 1).
The highest difference in term of antigen expression of monoclonal plasma cells was recorded for CD45 and CD27. We obtained a median of 7 x 10 -6 of Limit of detection (LOD, range 6 x 10 -5 - 2 x 10 -6) and 1 x 10 -5 Limit of Quantification (LOQ, range 1 x 10 -4- 5 x 10 -6) for all samples. A centralized revision (in L7 lab) of all FCS files is ongoing to confirm our preliminary results; moreover, the study will be corroborated increasing the number of fresh samples.
Conclusion: our preliminary results demonstrate the importance of a harmonized NGF-MRD assessment to improve the accuracy and comparability of MM-MRD testing among different laboratories. Major discrepancies have been found in fresh samples vs the retrospective dataset suggesting an impact of the transportation time and sample processing on the concordance of our results.