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.

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p <0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p <0.0001) and a shorter survival in MM patients with active disease requiring treatment (p ≤ 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM.

Transforming growth factor-beta (TGF-beta) is a cytokine known to participate in several processes related to the development of chronic kidney disease (CKD), including tubular degeneration. This is thought to occur mainly through apoptosis and epithelial-to-mesenchymal transition (EMT) of tubule epithelial cells, which give rise to a reduction of the tubular compartment and a scarring-like, fibrotic healing process of the interstitial compartment. In vivo blockade of TGF-beta action has been shown to reduce CKD-associated tubular damage. However, a direct action of TGF-beta on tubule cells is controversial as the underlying mechanism. On the one hand, TGF-beta is known to induce EMT of tubular cells, although its incidence in vivo can hardly explain the extent of the damage. On the other hand, a few publications have reported that TGF-beta induces a mild degree of apoptosis in cultured tubular cells. This most likely reflects the consequence of the cell-cycle arrest rather than a direct pro-apoptotic effect of TGF-beta. The implications of these observations are analyzed in the pathological context, where normal tubular cells do not normally proliferate, but they might divide for repair purposes. Furthermore, renal fibrosis, a TGF-beta-mediated event, is integrated as a potential, indirect effect contributing to tubule deletion.