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.

Summary: Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%,P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2–3 and lack of CD20⁺PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients’ diagnosis and possible prognosis.

Long non-coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides. Due to modern genomic techniques, the involvement of lncRNAs in tumorigenesis has been revealed; however, information concerning lncRNA interplay in multiple myeloma (MM) and plasma cell leukemia (PCL) is virtually absent. Herein, we aimed to identify the lncRNAs involved in MM to PCL progression. We investigated representative datasets of MM and PCL patients using next-generation sequencing. In total, 13 deregulated lncRNAs (p < 0.00025) were identified; four of them were chosen for further validation in an independent set of MM and PCL patients by RT-qPCR. The obtained results proved the significant downregulation of lymphocyte antigen antisense RNA 1 (LY86-AS1) and VIM antisense RNA 1 (VIM-AS1) in PCL compared to MM. Importantly, these two lncRNAs could be involved in the progression of MM into PCL; thus, they could serve as promising novel biomarkers of MM progression.

Background: Tumor burden in multiple myeloma (MM) is routinely evaluated in the bone marrow, though its prognostic value is not proven. There is an increasing interest in liquid biopsies due to its minimally invasive nature and more comprehensive evaluation of tumor burden. Growing evidence supports the quantification of circulating plasma cells (cPCs) measured by multiparameter flow cytometry (MFC) as a powerful diagnostic biomarker suitable for risk stratification of newly diagnosed transplant eligible MM (Garces et al, EHA 2021). Nevertheless, there are virtually no data regarding prognostic impact of cPCs in MM patients ineligible for transplantation. Primary plasma cell leukemia (pPCL) is a rare and most aggressive monoclonal gammopathy with dismal outcomes defined by more than 20% of cPCs and/or absolute cPCs count of ≥2 x10 ⁹/L. Recently, there have been many efforts to redefine these criteria as lower number of cPCs probably portends equally poor prognosis. Aims: To evaluate prognostic significance of cPCs in a large cohort of transplant ineligible (Tx-ineligible) newly diagnosed MM patients and to define cut-offs for risk stratification. Moreover, to establish cut-off identifying ultra high risk MM patients mimicking the prognosis of pPCL and propose new definition of pPCL. Methods: Circulating PCs were measured by 8 color flow cytometry in 402 Tx-ineligible MM patients (including n=7 pPCL) diagnosed between 2012 and 2019 at University Hospitals Brno and Ostrava, Czech Republic. Patients were treated in real-world setting and the clinical analysis was performed retrospectively based on data from the Czech Registry of Monoclonal Gammopathies. Median follow-up was 20.5 months. The intermediate cutoff was identified using ROC analysis considering overall survival (OS). Moreover, data from the large published cohort of pPCL patients treated in real-world setting were used to find a new cut-off identifying these ultra-high-risk pPCL-like patients (Jurczyszyn et al. BJH,2018). Results: Circulating PCs were detected in peripheral blood (PB) of 303/402 (75%) patients. In 303 patients with detectable cPCs the median percentage was 0.06% with range 0.0008% - 79%. The median limit of detection of MFC technique was 0.006 (sensitivity 10e-5). Patients stratification into 3 subgroups according to quantification of cPCs (low: ≤ 0.2%; intermediate: 0.2 - 2% and high: >2.0%) resulted in significantly different OS (36.5 vs. 28.1 and 13.6 months; p < 0.0001) and progression free survival (PFS) (17.9 vs. 14.7 and 3.4 months; p < 0.0001). Patients with no detected cPCs (0%) did not separate from subgroup >0% to 0.05) suggesting that next-generation flow cytometry (NGF) with sensitivity 10e-6 is needed for the identification of this favorable prognostic group. In order to demonstrate that patients with more than 2% of cPCs have similarly poor outcome as pPCL patients, we compared those with 2% - 20% (n=15) vs. those with >20% (n=7) of cPCs. The outcomes were practically identical with median PFS of 3.1 vs. 4.2 months (p = 0.23) and median OS of 13.6 vs. 14.6 months (p=0.23). Next, we analyzed patient´s characteristics in association with the level of cPCs (low, intermediate and high) and we demonstrated that there was significantly higher proportion of patients with ISS III stage (38%, 52% and 82%), elevated LDH level (8%, 19% and 55%) and high risk cytogenetics (12%, 21% and 36%) hand in hand with increasing number of cPCs. CPCs were identified as the most powerful prognostic marker in univariate (HR=2.7 for OS and HR=6.3 for PFS; p=0.001) and multivariate analysis (HR=4.2 for OS and HR=5.8 for PFS; p<0.001), including ISS, LDH and FISH cytogenetics. Conclusion: The quantification of cPCs in PB of newly diagnosed MM is the most powerful prognostic factor as we demonstrated on a large cohort of transplant ineligible patients. We defined 2% of cPCs as a new cut-off for ultra high risk myeloma resembling behavior of primary PCL. We propose this 2% cut-off for redefinition of pPCL criteria that warrants further investigation in prospective setting. To identify subgroup with especially favorable outcome with no detectable cPCs, NGF with high sensitivity of 10e-6 is needed. Quantification of cPCs by MFC is easy, fast, affordable and worldwide available procedure providing highly relevant prognostic information that might be implemented into routine clinical practice.

Plasma cell leukemia (PCL) is a rare dis-ease and possibly the most aggressive form of monoclonal gammopathy. It is classified into two forms - primary PCL that occurs without a previously identifiable multiple myeloma stage, and secondary PCL that develops from previously dia-gnosed multiple myeloma. These two forms have different cytogenetic and molecular profiles, but both forms have an aggressive clinical course. Combinations of different therapeutic approaches includ-ing autologous stem cell transplantation and currently proteasome inhibitors and immunomodulatory drugs are used to treat PCL. Current dia-gnostic criteria, developed in the 1970s, may underestimate PCL prevalence; thus, prospective re-evaluation is be-ing considered. The aim of this study is to review all available information about PCL with an emphasis on dia-gnostics, treatment, and circulat-ing plasma cells features. Although PCL is rare, it is quite a severe dis-ease. Current treatments us-ing the latest therapeutics have prolonged patient survival. However, due to the low incidence of PCL, information about the dis-ease is very limited and comes mostly from small retrospective studies. Further studies of PCL are needed, because new information could increase in patient survival and our understand-ing of its pathogenesis. Key words plasma cell leukemia - multiple myeloma - plasma cells - cytogenetics - treatment This work was supported by grant NV18-03-00203. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submited: 2. 11. 2018 Accepted: 18. 11. 2018.

Introduction: Release of the aberrant plasma cells (PC) from the bone marrow (BM) and their presence in the peripheral blood (PB) is a maker of disease progression and worse survival in multiple myeloma (MM) (Nowakowski et al., 2005). Circulating plasma cells (cPCs) are able to survive without homing microenvironment, evade the original tumor and colonize other bone marrow niche. Detailed analysis of various surface proteins showed that cPCs display decreased levels of integrins, adhesion molecules N-CAM (CD56) and the stem cell factor receptor (Paiva et al., 2013). Comprehensive analysis of the genome-wide gene expression profiling that could provide deeper insight into the expression patterns of cPCs of MM is still lacking. Aims: To identify differentially expressed genes in paired samples of aberrant plasma cells from BM and PB and to describe potential biomarkers of cPCs in MM. Material and methods: Ten patients with multiple myeloma (seven new diagnoses and three relapses) have been included in the study after signing the informed consent form. Paired samples of aberrant plasma cells from bone marrow and peripheral blood were obtained from each patient. Aberrant plasma cells (aPCs) were sorted according to the immunophenotype as CD45^dim/CD38⁺/CD19^-/CD56^-/+ cells. Gene expression profiling (GEP) was performed on paired samples using Affymetrix GeneChip Human Gene ST 1.0 array. RMA normalized data at gene level were analyzed using Wilcoxon paired test with Benjamini-Hochberg multiple testing correction. Results: The median infiltration of aberrant PC in the BM was 27.5% (range 1.1 - 93%) and 1.2% (range 0.19 - 2.8%) for cPCs in the PB. The median level of M-protein was 32.35 g/l (range 18.6 - 62.2 g/l). GEP analysis of paired BM and PB samples revealed 1001 significantly changed genes in cPCs (adjusted p-value<0.05). Gene ontology analysis did not reveal any significantly affected pathways. Nevertheless, two genes upregulated in cPCs, ADGRE5 and CD44, can be suggested as biologically relevant potential biomarkers of cPCs (Figure 1). Conclusion: The infiltration of aPCs in the bone marrow does not correlate with the amount of cPCs (p=0.16). Among differentially expressed genes, two surface markers upregulated in cPCs are of particular interest: CD44 and ADGRE5 (CD97). The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. Moreover, CD44 contribute to lenalidomide resistance in multiple myeloma (Bjorklund et al., 2014). CD97 is encoded by ADGRE5 gene and belongs to the EGF-TM7 subgroup of adhesion G-protein-coupled receptors. The expression of CD97 has been linked to invasive behavior in thyroid and colorectal cancer. Moreover, higher CD97 expression levels have been detected in 54% (208/385) of primary AML samples based on flow cytometric analysis (Wobus et al., 2015). Nevertheless, neither ADGRE5 nor CD97 expression were described in plasma cell dyscrasia previously. Thus, despite non-systemic changes of gene expression at the whole transcriptome level, cPCs in MM likely represent distinct biological entity with specific expression profile underlying advanced PC malignant transformation. To confirm the results, flow cytometric analysis on the bigger cohort will be performed. Acknowledgment: This study was supported by Institutional Development Plan of University of Ostrava (IRP201550) and The Ministry of Education, Youth and Sports (Specific university research of the Faculty of Medicine, University of Ostrava) project no. SGS03/LF/2015-2016, Ministry of Health Czech Republic RVO-FNOs/2014/17P and RVO-FNOs/2016/21.