Multiparameter flow cytometry (MFC) has become standard in the management of patients with plasma cell (PC) dyscrasias, and could be considered mandatory in specific areas of routine clinical practice. It plays a significant role during the differential diagnostic work-up because of its fast and conclusive readout of PC clonality, and simultaneously provides prognostic information in most monoclonal gammopathies. Recent advances in the treatment and outcomes of multiple myeloma led to the implementation of new response criteria, including minimal residual disease (MRD) status as one of the most relevant clinical endpoints with the potential to act as surrogate for survival. Recent technical progress led to the development of next-generation flow (NGF) cytometry that represents a validated, highly sensitive, cost-effective and widely available technique for standardized MRD evaluation, which also could be used for the detection of circulating tumor cells. Here we review current applications of MFC and NGF in most PC disorders including the less frequent solitary plasmocytoma, light-chain amyloidosis or Waldenström macroglobulinemia.

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Our study suggests a possible prognostic impact of UCA1 expression levels on MM patients.

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.

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Objectives Progress in multiple myeloma treatment allows patients to achieve deeper responses, for which the assessment of minimal residual disease (MRD) is critical. Typically, bone marrow samples are used for this purpose; however, this approach is site‐limited. Liquid biopsy represents a minimally invasive and more comprehensive technique that is not site‐limited, but equally challenging. Methods While majority of current data comes from short‐term studies, we present a long‐term study on blood‐based MRD monitoring using tumor‐specific cell‐free DNA detection by ASO‐qPCR. One hundred and twelve patients were enrolled into the study, but long‐term sampling and analysis were feasible only in 45 patients. Results We found a significant correlation of quantity of tumor‐specific cell‐free DNA levels with clinically meaningful events [induction therapy (P = .004); ASCT (P = .012)]. Moreover, length of cfDNA fragments is associated with better treatment response of patients. Conclusions These results support the concept of tumor‐specific cell‐free DNA as a prognostic marker.

Background: Multiple myeloma (MM) is characterised by proliferation of malignant plasma cells (PCs) usually in bone marrow (BM). The mechanisms of extramedullary (EM) spread in MM are poorly understood. It probably involves possible changes in expression of adhesive molecules. An increase incidence of aggressive and mostly resistant EM relapses was reported in the era of new treatment approaches. Finding a marker allowing predict a risk of EM relapse could help to improve treatment strategies and prolong patient life. Aim: Phenotype profile of PCs in BM and tumor tissue (TU) samples of EM relapses and comparison with "intramedullary" (IM) relapses of MM. Material and methods: There were analysed 40 patients with confirmed EM relaps and 17 patients with IM relaps (without EM spread). Analysis of 57 BM and 34 TU was done by polychromatic flow cytometry (FC). Phenotype of CD38+CD138+ PC was analysed using CD19, CD27, CD28, CD44, CD56, CD81, CD117 and nestin. PCs were considered positive for given marker when its expression on PCs was higher than 25 %. Results: There were detected CD38+CD138+ PCs in 95.0 % (38/40) of EM-BM samples (due to sample dilution), in 100 % IM-BM samples and in 88.2 % (30/34) of analysed TU samples (probably acquired not from tumor site). PC infiltration was statistically significantly higher in TU then in EM-BM [median of infiltration 51.7 % (range 0.0-98.9) vs. 1.5 % (0.01-68.9); p<0.001]; IM-BM has higher PC infiltration then EM-BM [5.7 % (0.7-66.1) vs. 1.5 % (0.0-68.9); p<0.05]. Although TU PCs have relatively similar phenotype like EM-BM PCs, there was found significant increase in median of expression of CD81 on abnormal PCs (A-PCs) [2.6 % (0.0-96.9) in EM-BM vs. 11.2 % (0.0-99.2) in TU; p<0.02] and also in number of CD81+ cases (positivity) [20 % (4/20) of EM-BM vs. 41.2 % (7/17) of TU]. Moreover, no presence of CD19+ normal PCs was found in TU even if mixture of CD19+ normal and CD19- A-PCs was detected in EM-BM. TU PCs we almost negative for C27 and CD117 (median of expression 2.6 and 0.1 %; positivity 17.9 and 14.3 %; respectively); slightly positive for CD28 (median of expression 2.6 %; positivity 33.3 %) and usually positive for CD56 and nestin (median of expression 97.8 and 45.7 %; positivity 69.0 and 57.9 %; respectively). There was found significant increase of CD44 and nestin expression in EM-BM when compared to IM-BM [median of expression 91.8 (3.4-99.9) vs. 25.6 (1.4-99.8) %; p<0.04 and 52.7 (0-100) vs. 0.2 (0-99.4) %; p<0.01; respectively], other markers were expressed similarly. Conclusion: Almost all EM PCs (BM and/or TU) usually express nestin, a marker of stem/progenitor cells, and CD44, a glycoprotein involved in cell-cell interactions, cell adhesion and migration. Adhesion molecule CD56, which loss is considered as a marker of extramedullary spread, was not changed in different MM cases (EM vs. IM). Flow cytometry analysis of EM PCs allowed to identify a phenotype profile of A-PCs (CD19-CD27-CD56+CD81+CD117-CD44+nestin+) related to possible extramedullary involvement in MM. Disclosures Hájek: TAKEDA: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Research Funding.

Multiparametric flow cytometry is a very sensitive method for detection of circulating plasma cells, so using a standardized approach can lead to determination and implementation of the flow cytometry diagnostic threshold in plasma cell leukemia suspicious cases as well as in prognostication of monoclonal gammopathies patients. Moreover, analysis of plasma cells phenotypic profile could probably clarify their future behaviour.Key words: monoclonal gammopathies - circulating plasma cells - plasma cell leukemia - flow cytometry.