Next Generation Flow and Next Generation Sequencing for Measurable Residual Disease Assessment in Multiple Myeloma Patients: A Real-Life Italian Multicenter Harmonization Experience.

The NGF inter-operator retrospective study (Stage 1) showed high inter-center concordance in monoclonal plasma cell detection.

• Intraclass correlation coefficient (ICC) = 0.90, p < 0.001
• Seven laboratories participated in the NGF workgroup
• The EuroFlow Standard-Operating-Protocol was implemented to reach minimum 1 × 10-5 sensitivity
• This stage involved retrospective analysis of samples across operators

The NGF inter-laboratory correlation study (Stage 2) using in-vivo samples showed only moderate concordance.

• ICC = 0.63, p < 0.001 for inter-laboratory correlation in in-vivo samples
• Greater variability was observed compared to the inter-operator stage
• Median limit-of-detection (LOD) reached was 8 × 10-6
• Median limit-of-quantification (LOQ) reached was 2 × 10-5
• Greater variability was also observed in the analysis of other bone marrow cell populations

The NGS workgroup achieved 100% concordance among centers for clonotype identification in the first quality-control round.

• Four laboratories participated in the NGS workgroup
• A targeted amplicon-based approach was employed to detect clonotypic IGH/IGK gene rearrangements
• Clonotypes identified in diagnostic samples were subsequently used to track MRD in mock samples
• QC1 focused on finding a shared strategy for clonotype identification and achieved 100% concordance

NGS MRD quantification concordance in mock samples improved from the second to the third quality-control round.

• Concordance in QC2 (mock sample MRD quantification) was 81%
• Concordance in QC3 (mock sample MRD quantification) improved to 91%
• The 10-5 sensitivity level was successfully reached in most tested dilutions: 19/20 (95%) in QC2 and 19/23 (83%) in QC3

Reproducibility across laboratories is a major concern in MRD evaluation for multiple myeloma, as discrepancies among results make comparability impractical.

• MRD level is described as one of the most important features correlating depths of response and long-term outcomes in MM
• MRD evaluation is currently considered the gold standard tool for assessing treatment response
• The Italian MM-MRD network project was established specifically to address inter-laboratory harmonization
• Both NGF and NGS approaches were evaluated across multiple centers in newly diagnosed MM patients