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.

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species–mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.

PURPOSE: Patients with multiple myeloma (MM) may show patchy bone marrow (BM) infiltration and extramedullary disease. Notwithstanding, quantification of plasma cells (PCs) continues to be performed in BM since the clinical translation of circulating tumor cells (CTCs) remains undefined. PATIENTS AND METHODS: CTCs were measured in peripheral blood (PB) of 374 patients with newly diagnosed MM enrolled in the GEM2012MENOS65 and GEM2014MAIN trials. Treatment included bortezomib, lenalidomide, and dexamethasone induction followed by autologous transplant, consolidation, and maintenance. Next-generation flow cytometry was used to evaluate CTCs in PB at diagnosis and measurable residual disease (MRD) in BM throughout treatment. RESULTS: CTCs were detected in 92% (344 of 374) of patients with newly diagnosed MM. The correlation between the percentages of CTCs and BM PCs was modest. Increasing logarithmic percentages of CTCs were associated with inferior progression-free survival (PFS). A cutoff of 0.01% CTCs showed an independent prognostic value (hazard ratio: 2.02; 95% CI, 1.3 to 3.1; P = .001) in multivariable PFS analysis including the International Staging System, lactate dehydrogenase levels, and cytogenetics. The combination of the four prognostic factors significantly improved risk stratification. Outcomes according to the percentage of CTCs and depth of response to treatment showed that patients with undetectable CTCs had exceptional PFS regardless of complete remission and MRD status. In all other cases with detectable CTCs, only achieving MRD negativity (and not complete remission) demonstrated a statistically significant increase in PFS. CONCLUSION: Evaluation of CTCs in PB outperformed quantification of BM PCs. The detection of ≥ 0.01% CTCs could be a new risk factor in novel staging systems for patients with transplant-eligible MM.

PURPOSE: Primary plasma cell leukemia (PCL) is the most aggressive monoclonal gammopathy. It was formerly characterized by ≥ 20% circulating plasma cells (CTCs) until 2021, when this threshold was decreased to ≥ 5%. We hypothesized that primary PCL is not a separate clinical entity, but rather that it represents ultra-high-risk multiple myeloma (MM) characterized by elevated CTC levels. METHODS: We assessed the levels of CTCs by multiparameter flow cytometry in 395 patients with newly diagnosed transplant-ineligible MM to establish a cutoff for CTCs that identifies the patients with ultra-high-risk PCL-like MM. We tested the cutoff on 185 transplant-eligible patients with MM and further validated on an independent cohort of 280 transplant-ineligible patients treated in the GEM-CLARIDEX trial. The largest published real-world cohort of patients with primary PCL was used for comparison of survival. Finally, we challenged the current 5% threshold for primary PCL diagnosis. RESULTS: Newly diagnosed transplant-ineligible patients with MM with 2%-20% CTCs had significantly shorter progression-free survival (3.1 v 15.6 months; P < .001) and overall survival (14.6 v 33.6 months; P = .023) than patients with < 2%. The 2% cutoff proved to be applicable also in transplant-eligible patients with MM and was successfully validated on an independent cohort of patients from the GEM-CLARIDEX trial. Most importantly, patients with 2%-20% CTCs had comparable dismal outcomes with primary PCL. Moreover, after revealing a low mean difference between flow cytometric and morphologic evaluation of CTCs, we showed that patients with 2%-5% CTCs have similar outcomes as those with 5%-20% CTCs. CONCLUSION: Our study uncovers that ≥ 2% CTCs is a biomarker of hidden primary PCL and supports the assessment of CTCs by flow cytometry during the diagnostic workup of MM.

Purpose: Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model. Experimental Design: We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment. Results: Patients with >0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P < 0.001). Presence of >20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio >20, >2 g/dL M-protein, and >0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN− nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient’ stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years. Conclusions: This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.