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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Author contributions and disclosures: M.Z. contributed to the research by fluorescence activated cell sorting, DNA processing and amplification, bioinformatic and following data analysis and wrote the manuscript, G. S. designed and led the bioinformatic analysis, T.Š. designed the research, consulted results and wrote the manuscript, V. F. performed the pathway analysis, Z. CH., K. G. and L.B. contributed to bone marrow preparation and DNA processing, T.

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.

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.

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