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Heparin-induced thrombocytopenia (HIT) is an immunologically-mediated complication, which usually follows heparin exposition, less frequently exposition to other drugs or even occurs spontaneously. The type of heparin, its dose and mode of application as well as the exposition time, major trauma or operation, and obesity represent the main risk factors for HIT. The probability of HIT correlates with so-called 4T-score. A confirmatory laboratory diagnostic should be exclusively reserved for patients with a medium to a high probability of HIT development (more than 3 points in 4T-score). The screening method is based on serological detection of antibodies against heparin-platelet factor-4 complexes; confirmation tests aim to identify the activation of platelets. The treatment of HIT requires an immediate interruption of heparin application and rigorous antithrombotic treatment with an alternative agent. Herein authors describe a clinical case of HIT manifested as an extreme urticarial reaction in the location of nadroparin application as well as thrombosis of deep subcutaneous veins in a polymorbid obese patient with an extensive and infected burn. Due to timely diagnosis and fondaparinux treatment, no more severe thrombotic events occurred in this patient.

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 &lt, 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 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. Figure 1 Figure 1. Disclosures Jurczyszyn: Janssen-Cilag, Amgen: Honoraria, Speakers Bureau. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Hajek: Janssen: Consultancy, Honoraria, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Aldehyde dehydrogenase (ALDH) is highly active in physiological stem cells as well as in tumor-initiating cells of some malignancies including multiple myeloma (MM). Finding higher activity of ALDH in some cell subsets in monoclonal gammopathies (MG) could identify potential source of myeloma-initiating cells (MICs). Methods: Bone marrow of 12 MM, 9 monoclonal gammopathy of undetermined significance (MGUS), and 10 healthy donors (HD) were analyzed by flow cytometry. ALDH activity of B-cells and plasma cells (PC) was analyzed using Aldefluor. Results: Similar changes of ALDH activity were found during B-cell development in HD and MG. Decreasing of ALDH activity from immature to naive B-cells was found. In postgerminal stages, the activity started to increase, and in PCs, the ALDH activity was the same as in immature B-cells. Increased ALDH activity of all PC subsets compared to naive B-cells was found in MM as well as in HD, while in MGUS, only CD19-PCs have higher ALDH activity. In HD, ALDH activity was higher in CD19+PCs compared with MG. Conclusions: Our results indicate that changes of ALDH activity are the natural phenomenon in B-cell development; thus, high ALDH activity as a single marker is not appropriate for MICs identification.

Background: Adaptive response to hypoxia is regulated by several mechanisms and transcription factors, including hypoxia-inducible factors (HIFs). Activation of HIF-1α is associated with increased expression of P-glycoprotein and multidrug resistance in cancer cells. In this retrospective study, we analyzed candidate single-nucleotide polymorphisms (SNPs) in HIF-1α and HIF-1β associated with risk of monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM). Patients and Methods: Genotypes of SNPs associated with hypoxia were determined in an independent cohort of monoclonal gammopathies (MG) (275 MM and 228 MGUS patients) and in 219 cancer-free controls by real-time PCR allelic discrimination. Results: When MM patients were compared to controls, protective role of CG genotype compared to CC in HIF-1β (rs2228099) for MM development was observed (OR=0.65; CI=0.45-0.95; p=0.026). Even after adjustment for patients’ age and body mass index (BMI), there were significantly lower odds (OR=0.55; p=0.045) of developing MM patients of CG genotype in comparison to CC genotype. Log-rank test confirmed association of GT haplotype (rs11549467, rs2057482) in HIF-1α with better overall survival [median 41.8 months; (CI=35.1-48.5)] for ‘none GT’ and median 93.8 months (CI= 31.3-156.4) for ‘at least one GT’ haplotype (p=0.0500). Further, significant associations between SNPs in MDR1 and outcome of MM were found in 110 MM patients that underwent bortezomib-based treatment. Conclusion: Our study showed a genetic predisposition for risk of MG development and/or outcome of MM patients; nevertheless, further studies are needed to confirm our initial analysis.

Introduction: Multiple myeloma (MM) is a plasma cell dyscrasia causing damage of multiple organs with fatal consequences for patients. Despite the success of modern therapies eliminating a vast bulk of the aberrant cells, surviving residual clones eventually lead to the relapse of the disease. Accumulation of genomic alterations during the stage of minimal residual disease (MRD) likely contributes to a selective grow advantage and survival under the drug pressure. Identification of specific mutations in MM patients with MRD can provide unique opportunities to target the residual plasma cell clones. Here we present the first whole exome sequencing (WES) analysis of 22 MM samples of patients with MRD that identified 814 mutated genes with 4% of genes previously implicated in the pathogenesis of MM. Methods: Aberrant plasma cells (A-PCs) and peripheral blood (PB) were collected from patients after signing informed consent form. Presence of MRD was assessed with EuroFlow protocol and A-PCs were sorted out from bone marrow according to their pathological immunophenotype based on the expression of antigens CD38, CD45, CD19 and CD56. DNA from A-PCs was isolated and amplified by Repli-g Single cell kit (QIAGEN). Sequencing libraries were prepared using SureSelect Human All Exon V6 Kit (Agilent Technologies) and sequenced by Macrogen Inc. on Illumina HiSeq 4000 platform with average coverage 50x and 2x 100bp read length. Sequencing data were processed using the Bcbio framework following the standard workflow for tumor-matched-normal studies. Specifically, reads were mapped to the human reference genome GRCh37, successively marking duplicates using Picard. Germline mutations were identified using GATK HaplotypeCaller, whereas somatic mutations were identified using MuTect2 reporting as significant variants observed in at least 5 reads and minimum allele frequency of 10%. Variants in homopolymer regions longer than 5 nucleotides were filtered out. The final set of calls were further characterised by assessing their functional impact using snpEff and by annotating each variant using data from 1000 Genomes Phase 3, ExAC, and ClinVar. We then used OncodriveCLUST to identify putative oncogenic genes, and later compared these results with a literature curated list of MM driver genes (Weaver & Tariman, 2017). Results: Our dataset comprises 22 samples from 21 patients (one patient was sampled in two time points) with MM MRD, who received bortezomib-based regimen (age 41-71, average 59 years, 11/22 males, 10/22 females). 8 patients reached complete response, 9 patients had very good partial response and 4 patients had partial response. In our analysis, we detected 1,014 tumour somatic variants (8-287 per sample, median 36), most of them being missense mutations (676/1014), splice site mutations (145/1014) and frameshift insertions (134/1014). The variants affected a total of 814 genes, 97 genes were shared in at least two samples. The most frequently mutated genes were KIAA1211 (11/22), the immunoglobulin gene IGLV3-1 (8/22), apoptotic chromatin condensation inducer ACIN1 (7/22) and CCR4-associated factor 3 CNOT3 (7/22). We also identified 32 genes known to be mutated in MM in 64% of our samples (14/22). We found mutations shared by at least 2 samples in KRAS (4/22), DIS3 (3/22), TRAF3 (3/22), NRAS (2/22), ANK2 (2/22), BRAF (2/22) and RBM15 (2/22). Further analysis with OncodriveCLUST identified 18 putative oncogenic genes (FDR < 0.1), including KRAS, DIS3, ACIN1. Conclusion: We presented the first whole exome study of MM MRD, providing a characterisation of the mutations observed in A-PCs. We overcame problem with low amount of A-PCs in this disease stage by using whole genome amplification and a highly customised bioinformatic analysis pipeline. Our study suggests that A-PCs are characterised by new MM MRD specific set of mutated genes, along with the presence of mutations in well-known multiple myeloma cancer driver genes. This offers a great potential for design of novel precise treatments targeting MRD after standard MM therapies. Supported by Ministry of Health of the Czech Republic (17-30089A, CZ-DRO-FNOs/2016) and Ministry of Education of the Czech Republic (SGS18/PřF/2017-2018) Disclosures Kryukov: JSC BIOCAD: Employment. Maisnar:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Hajek:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Research Funding.

Introduction: Great progress achieved in treatment of multiple myeloma (MM) over the past decade changed overall perception of importance of minimal residual disease (MRD) assessment. Since new drugs induce deep responses, MRD must be evaluated using sensitive techniques, such as allele specific PCR (ASO-PCR), next-generation sequencing (NGS) or flow cytometry. MM is a genetically heterogeneous cancer of plasma cells characterized by multiple focal lesions in the bone marrow (BM). Hence, a single-site biopsy can create a sampling bias. In spite of this, BM samples are typically used for MRD analysis, but currently an alternative approach called liquid biopsies, which utilizes body fluids for analysis of various molecules and cells, is intensively studied. Cell-free DNA (cfDNA) as one type of the molecule which can be analyzed using liquid biopsy approach showed promising results previously. In our study, patient-specific, clonotypic rearrangement of immunoglobulin heavy chain (IgH) gene, identified in bone marrow samples, was used for qPCR analysis of cfDNA samples from peripheral blood. We demonstrate that dynamics and quantity of patient-specific, clonotypic IgH rearrangement found in cfDNA can predict the outcomes and response of MM patients. Methods: Total of 45 patients enrolled in the study. Samples of BM were collected at diagnosis, and CD138+ cell fraction was sorted using magnetic activated cell sorting. At diagnosis and at three-month intervals, samples of peripheral blood (PB) were collected for cfDNA extraction and analysis until a patient reached complete remission (CR). If CR was not reached, samples were collected for 24 months after diagnosis. Two more samples of PB were collected (CR+3, CR+6) if patients reached CR. Patient-specific VDJ rearrangement was identified using previously described PCR method from genomic DNA extracted from CD138+ cell fraction; based on the results, patient-specific primers and probes were designed for use in ASO-qPCR. Obtained data were evaluated by absolute and relative frequencies of categorical variables and median (minimum-maximum) of quantitative variables. Results: First, we assessed time to CR. Patients were classified according to the quantity of cfDNA measured at time of diagnosis into three groups: negative, PNQ (= positive non-quantifiable) and positive. As PNQ had a similar profile to negative-classified samples (in K-M plot), PNQ were grouped together with negative results except extremely high values (> 5, n = 2) which were reclassified from PNQ to positive group. The Kaplan-Meier estimates at 12 months were reported and supplemented by the 95% confidence interval derived using Greenwood formula. The results show that significantly higher number of patients classified as negative or PNQ with quantity < 5 have reached CR in contrast to patients classified as positive or PNQ with quantity > 5. The same trend applies to association of quantity of tumor-specific cfDNA with time to CR where Cox proportional-hazards model was adopted. Patients classified as negative or PNQ with quantity < 5 have significantly increased chance of achieving CR (2.7 times) in comparison to patients classified as positive or PNQ with quantity > 5. Conclusion: Our results demonstrate that MM patient-specific cfDNA fragments are released into the bloodstream and that patients either with no or very few DNA fragments have a higher chance of achieving better treatment response eventually. Work was supported by grant AZV 17-29343A Disclosures Hajek: Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Background. The number of CTC predicts risk of transformation in smoldering MM and survival in active MM. Growing evidence suggests that as the tumor progresses and the microenvironment becomes hypoxic, clonal plasma cells (PC) constantly invade new regions of the bone marrow (BM) through induced systemic recirculation. Of note, the frequency of CTCs is typically low and thus, it is conceivable that the dissemination of MM depends on few tumor cells with unique features that induce them to egress the BM and spread the disease through peripheral blood (PB). This hypothesis has not been yet demonstrated because the transcriptional profile of CTCs in MM has not been investigated. Aim. To identify gene regulatory networks related to MM dissemination by comparing the transcriptional profile of CTCs with patient-matched BM clonal PCs. Methods. We used FACS to isolate CTCs and BM clonal PCs of paired PB and BM samples from 34 patients: 24 newly diagnosed MM, 9 relapsed MM and 1 MGUS. Transcriptomes were analyzed using Affymetrix arrays (n =31) and the BD WTA Precise assay was used for single-cell RNA sequencing (scRNAseq, n =3). Data was analyzed using Gene Set Enrichment Analysis (GSEA) and Limma for bulk and Seurat for scRNAseq data. The prognostic value of deregulated genes (FDR 0.5) was investigated using a Cox-regression model in the CoMMpass dataset (n =553, IA11 release). The role of specific deregulated genes was evaluated by shRNA knockdown and blocking using a monoclonal antibody (mAb). Results. Transcriptomic profiling of patient matched CTCs and BM clonal PCs revealed a high correlation in gene expression (r =0.93; p =10-16). Only 45 genes emerged as significantly deregulated in CTCs, and GSEA unveiled biological functions related to inflammatory and interferon response (e.g. CCL5), signaling by IL-6/JAK/STAT3, IL-2/STAT5 and TNF via NFKB (CD44), the epithelial mesenchymal transition (EMP3), mitotic spindle and G2M checkpoints (TOP2A), or E2F targets (BIRC5). A high correlation in gene expression was also observed by scRNAseq (r =0.9; p =10-16), with only 31 genes (e.g. MALAT1, B2M, RHOH, ENAM or DUSP5) differentially expressed (adj.p Conclusions. This is the first study analyzing the transcriptional profile of CTCs in MM. Our results reveal that gene expression of CTCs is almost identical to that of patient-matched BM clonal PCs, except for a few genes that are involved in interferon and inflammatory response, hypoxia, cell cycle and migration. Importantly, some of these genes are related to more aggressive disease and therefore, may represent novel therapeutic targets to overcome disease dissemination. Figure. Figure. Disclosures Rios: Amgen, Celgene, Janssen, and Takeda: Consultancy. Martinez-Lopez:Pfizer: Research Funding; Vivia: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Research Funding; Novartis: Research Funding. Hajek:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding. San-Miguel:Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria; Roche: Honoraria.

Multiple myeloma is a plasma cell dyscrasia. It is the second most common hematological malignancy which is characterized by proliferation of clonal plasma cells producing harmful monoclonal immunoglobulin. Despite treatment modalities greatly evolved during the last decade, small amount of aberrant residual cells reside in patients after therapy and can cause relapse of the disease. Characterization of the residual, resistant clones can help to reveal important therapeutic targets for application of effective and precious treatment. We use CD38, CD45, CD56 and CD19 sorted aberrant plasma cells to perform next generation sequencing of their exome. Among the 213 genes in which at least one variant was present, the most interesting was found gene NRAS, one of the most often mutated gene in multiple myeloma, and homologs of 88 gene panel previously used for multiple myeloma sequencing among which was a gene previously identified as gene meaningful in bortezomib resistance. Nevertheless, the results of next generation exome sequencing need to be interpreted with caution, since they rely on bioinformatical analysis, which is still being optimized. The results of next generation sequencing will also have to be confirmed by Sanger sequencing. Final results supported by larger cohort of patients will be published soon.Key words: multiple myeloma - minimal residual disease - exome - next generation sequencing.

Background Monoclonal gammopathies are characterized by presence of clonal plasma cells in the bone marrow, although peripheral blood circulating plasma cells can be found in a significant proportion of patients. The number of circulating plasma cells is an independent prognostic marker associated with shorter survival, but it can also help to predict early relapse. The reason and mechanism of plasma cell expansion from the bone marrow to enter peripheral blood is still not entirely clear, but possible changes in the expression of adhesion molecules are probably involved. Multiparametric flow cytometry allows simple and exact enumeration of circulating plasma cells in different types of cell suspensions, even in their low quantity. The phenotype profile and confirmation of clonality regarding to their bone marrow clonal counterparts should be verified as well. There is no uniform method used in clinical laboratories for circulating plasma cells analyses at this moment. Aim Review is focused on use of multiparametric flow cytometry for circulating plasma cells analysis in peripheral blood. It is comparing possibilities of their detection by different methods and on clinical relevance of that assessment. The standardization of analyses is the main goal. Conclusion 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.