Lucie Říhová

Analysis of B-Cell Subpopulations in Monoclonal Gammopathies

BACKGROUND Multiple myeloma (MM) is characterized by accumulation of pathological plasma cells (PCs) in bone marrow (BM) as a result of deregulation of B-cell development. To clarify its pathophysiology it is necessary to investigate in detail the developmental stages of B-cells. MATERIALS AND METHODS Enumeration of total CD19-positive (CD19(+)) cells and their subpopulations together with PCs was done in peripheral blood (PB) and BM of newly diagnosed monoclonal gammopathy patients and control subjects. Representation of subsets was compared among groups and relationships between subset percentage and cytogenetic/biochemical findings were analyzed. RESULTS A lower number of total CD19(+) cells was found in MM, particularly in advanced stages of disease. Reduction of naive (P < .01) and transitional B-cells (P < .05) and increase of switched memory and switched CD27(-) B-cells and germinal center founder cells were detected in PB of MM compared with controls (P < .01). Similar results were found in BM. β2 microglobulin level in MM positively correlated with the number of PCs and negatively with percentage of naive B-cells (P < .05). CONCLUSION Our results provided a detailed phenotypic profile and enumeration of B and PC subpopulations in monoclonal gammopathy patients. A reduced number of B-cells and particularly a differentiation shift to more numerous antigen-stimulated forms was observed in MM. This might indicate a potential source of myeloma-initiating cells in one of these subpopulations.

Immunophenotyping in Multiple Myeloma and Others Monoclonal Gammopathies

Clonal plasma cell disorders (PCD) including mostly monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) are characterised by expansion of abnormal (clonal) plasma cells (PCs) producing monoclonal protein (M-protein, MIG). Although multiparametric flow cytometry (MFC) allows identification and characterisation of these neoplastic PCs, this approach is used in routine diagnostics of monoclonal gammopathies (MGs) complementarily, mostly in unusual cases. The technological development of flow cytometry (FC) in connection with new findings reveal the need for MFC in clinical analysis of MGs. The main applications of immunophenotypisation in MGs are (1) differential diagnosis, (2) determining the risk of progression in MGUS and asymptomatic MM (aMM), (3) detection of minimal residual disease in treated patients with MM, and (4) analysis of prognostic and/or predictive markers. MFC is also very useful also for research analyses focused on different aspects of B and plasma cell (PC) pathophysiology in term of MG development as well as in looking for potential myeloma-initiating cells. MFC thus should be included as a routine assay in monoclonal gammopathy patients. Clinical significance, usefulness and examples of MFC analyses in MGs are reviewed in this chapter.

A first Czech analysis of 1887 cases with monoclonal gammopathy of undetermined significance

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition with a risk of malignant conversion.

Activity of aldehyde dehydrogenase in B‐cell and plasma cell subsets of monoclonal gammopathy patients and healthy donors

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).

Whole Exome Sequencing of Aberrant Plasma Cells in a Patient with Multiple Myeloma Minimal Residual Disease

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.

Circulating plasma cells in monoclonal gammopathies

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.

Waldenström′s macroglobulinemia: Two malignant clones in a monoclonal disease? Molecular background and clinical reflection

Waldenström′s macroglobulinemia (WM) is a complex disease characterized by apparent morphological heterogeneity within the malignant clonal cells representing a continuum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells. At the molecular level, the neoplastic B cell–derived clone has undergone somatic hypermutation, but not isotype switching, and retains the capability of plasmacytic differentiation. Although by classical definition, WM is formed by monoclonal expansion, long‐lived clonal B lymphocytes are of heterogeneous origin. Even more, according to current opinion, plasma cells also conform certain population with pathogenic and clinical significance. In this article, we review the recent advances in the WM clonal architecture, briefly describe B‐cell development during which the molecular changes lead to the malignant transformation and mainly focus on differences between two principal B‐lineage clones, including analysis of their genome and transcriptome profiles, as well as immunophenotype features. We assume that the correct identification of a number of specific immunophenotypic molecular and expression alterations leading to proper aberrant clone detection can help to guide patient monitoring throughout treatment and successfully implement therapy strategies directed against both B‐ and plasma cell tumor WM clones.