Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.

Key Points• MRD assessment by sequencing is prognostic of TTP and OS in multiple myeloma patients.• Among patients in complete response, MRD assessment by sequencing enables identification of 2 distinct subgroups with different TTP.We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy.Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJ H , IGH-DJ H , and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allelespecific oligonucleotide polymerase chain reaction (ASO-PCR).

Monoclonal B-cell lymphocytosis (MBL)indicates the presence of less than 5 ؋ 10 9 /L circulating monoclonal B cells in otherwise healthy subjects. Recently, it has been reported that circulating chronic lymphocytic leukemia (CLL)-like B cells can be detected using 4-or 5-multicolor flow cytometry in 5% to 7% of adults with normal lymphocyte counts. We investigated the frequency of circulating monoclonal B cells in 608 healthy subjects older than 40 years with normal blood counts, using a highly sensitive 8-color flow cytometry approach and systematic screening for total PB leukocyte count higher than 5 ؋ 10 6 . We show that the frequency of PB monoclonal B cells is markedly higher than previously reported (12% for CLL-like B cells, found at frequencies of 0.17 ؎ 0.13 ؋ 10 9 cells/L), the incidence progressively increasing with age. Most cases (62%) showed clonal B-cell levels below the maximum sensitivity of the techniques described by others (< 0.01%), supporting the notion that detection of MBL may largely depend on the sensitivity of the flow cytometry approach used.

We have analyzed the applicability, sensitivity and prognostic value of allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) as a method for minimal residual disease (MRD) assessment in patients with multiple myeloma (MM), comparing the results with those of multiparameter flow cytometry (MFC). A total of 170 patients enrolled in three consecutive Spanish trials achieving at least partial response after treatment were included. Lack of clonality detection (n=31), unsuccessful sequencing (n=17) and suboptimal ASO performance (n=51) limited the applicability of PCR to 42% of cases. MRD was finally investigated in 103 patients (including 32 previously studied) with persistent disease identified by PCR and MFC in 54% and 46% of cases, respectively. A significant correlation in MRD quantitation by both the techniques was noted (r=0.881, P<0.001), being reflective of treatment intensity. Patients with <10(-4) residual tumor cells showed longer progression-free survival (PFS) compared with the rest (not reached (NR) vs 31 months, P=0.002), with similar results observed with MFC. Among complete responders (n=62), PCR discriminated two risk groups with different PFS (49 vs 26 months, P=0.001) and overall survival (NR vs 60 months, P=0.008). Thus, although less applicable than MFC, ASO RQ-PCR is a powerful technique to assess treatment efficacy and risk stratification in MM.