Next Generation Flow (NGF) Cytometry for Minimal Residual Disease (MRD) Evaluation in Multiple Myeloma (MM) Patients with Sustained Complete Response (CR) after Frontline Therapy: Results of a Prospective Single-Center Analysis

MRD evaluation is included in the novel IMWG response criteria with a minimum sensitivity level for MRD^- that of 10^-5. However, several data suggest a sensitivity level of at least 10^- for MRD^- may be more appropriate. Such high sensitivity would be particularly relevant to identify, among MM patients who remain at sustained response for several years, those who may still be at risk of relapse due to persistent MRD and for whom closer monitoring could be beneficial. Thus, the primary endpoint of this prospective study was to evaluate the incidence of MRD^- in patients who remained in CR for ≥2-years after first line therapy, using NGF. Secondary endpoints included the detection level of MRD, the reproducibility of the method, and the correlation of MRD status with the hematopoietic bone marrow (BM) profile defined by NGF as well as with prognostic factors at diagnosis.

Fifty-two patients who were treated at the Department of Clinical Therapeutics (Athens, Greece) and were in sustained CR for ≥2-years (median period of CR: 58 months, range: 24-197 months) were evaluated for the presence of MRD using NGF, according to the EuroFlow guidelines. BM samples were labeled using two independent 8-color panels, both containing CD19-PEC7, CD27-BV510, CD38-FITC, CD45-PERCP, CD56-PE, CD138-BV421 and additionally CD81-APCC750 and CD117-APC for the surface tube or CyIgκ-APC and CyIgλ-APCC750 for the intracytoplasmic tube. A minimum of ten million events were recorded per patient-sample with a BD FACSCantoII and data analysis was conducted with Infinicyt software, allowing merging of the two panels and detection of MRD with a median sensitivity level of 2.2x10^-6 (range, 2.1x10^-6 - 2.8x10^-6). Two independent experts evaluated the results for reproducibility assessment. Using the same markers, we identified the major BM cell subsets (erythroid and myeloid progenitors, erythroblasts, T, NK and NKT cells, naïve B cells, memory B cells and B-cell precursors, neutrophils, monocytes, basophils, eosinophils and mast cells) and analyzed the hematopoietic profile of each patient.

Our analysis revealed 30 (58%) MRD^- and 22 (42%) MRD⁺ patients, mostly at the level of 10^-5 (n=7/22, 32%) and 10^-6 (n=5/22, 23%). The reproducibility of the method was 98%, as there was only one disagreement between the two independent researchers (one patient who was MRD⁺ at the level of 10^-6) and MRD levels were significantly correlated (r=0.892, p<0.001). The most informative markers for MRD detection were primarily CD19 and CD45 (all MRD⁺ cases were CD19^-CD45^dim/-) and secondarily CD27 (17/21 MRD⁺ cases were CD27^-/dim). NGF-based quality control confirmed the presence of B-cell precursors, erythroblasts and mast cells in all samples analyzed, thus excluding potential false-negative results due to hemodilution. The analysis of the major BM subsets revealed that the presence of MRD correlated with higher percentages of erythroblasts (4.26% of total BM cells in MRD⁺ vs. 2.7% in MRD^-, p=0.03) and of monocytes (5.22% in MRD⁺ vs. 3.87% in MRD^-, p=0.04) in contrast to MRD^- samples where neutrophils predominated. Moreover, MRD⁺ samples were characterized by increased activation of the lymphoid lineage and contained higher percentages of T, NK and NK-T cells, though these differences did not attain statistical significance.

The MRD status was not associated with any of the prognostic clinical parameters at diagnosis (age, ISS, cytogenetic profile or LDH), although the majority of MRD⁺ had ISS-1 disease (58%). Three (10%) MRD^- patients and 3 (14%) MRD⁺ had high-risk cytogenetics. Nineteen (61%) MRD^- patients and 13 (62%) MRD⁺ had received ASCT. There were no differences regarding frontline therapy among MRD^- and MRD⁺ patients (PI- vs IMiDs- vs PI+IMiD-based therapies).

We demonstrated that the NGF method by EuroFlow can be standardized, and provide a detection MRD level of 2.1x10^-6 with high reproducibility rate. We also showed that 40% of MM patients in prolonged CR have persistent MRD⁺. Interestingly, such patients displayed more erythroblasts and monocytes together with increased T, NK and NK-T cell numbers. By contrast, no significant clinical differences were observed between MRD⁺ and MRD^- patients with ≥2-years in CR. Thus, NGF-based MRD assessment is warranted to discriminate independently of treatment or disease risk, patients reaching sustained CR that could have higher risk of relapse due to persistent MRD and may benefit from closer monitoring.