Karthick Muthu Raja

Review of phenotypic markers used in flow cytometric analysis of MGUS and MM, and applicability of flow cytometry in other plasma cell disorders.

Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification and monitoring of disease in monoclonal gammopathies. The clinical sensitivity of flow cytometry is comparable with advanced molecular methods. Clinical application of flow cytometry in monoclonal gammopathies has various dimensions, such as differential diagnosis of malignant plasma cell disorder from reactive plasmacytosis, identifying the progression risk in monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic multiple myeloma (MM), and minimal residual disease detection. Flow cytometry‐based clonality assessment with immunophenotyping encourages and enables the most stringent method of diagnosis and follow‐up. The objective of this review is to update the malignant plasma cells phenotypic profile of MGUS and MM. The most comprehensive antigens, such as CD19, CD27, CD28, CD45, CD56 and CD117, play a significant role in the characterization of normal and malignant plasma cells. Several research groups described the putative phenotype of myeloma cell progenitors, but no remarkable suggestion could be made because of disparity. This review also focuses on the association of malignant phenotypic markers and chromosomal aberrations that identify the specific prognostic features in monoclonal gammopathies.

Increased T Regulatory Cells Are Associated with Adverse Clinical Features and Predict Progression in Multiple Myeloma

Background Regulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions. Methods We analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD). Results We found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naïve (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (>10 mg/dL), decreased normal plasma cell (≤5%) count and IgA myeloma subtype. We also showed that MM patients with ≥5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pre-treatment. Conclusions Our study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression.

Functionally Suppressive CD8 T Regulatory Cells Are Increased in Patients with Multiple Myeloma: A Cause for Immune Impairment

Background Multiple myeloma (MM) is a plasma cell malignancy frequently associated with impaired immune cell numbers and functions. In MM, several studies have previously shown that CD4 regulatory T (Treg) cells hamper effector T cell functions and enhance immune dysfunction. In this study, we aimed to prove the presence of functionally suppressive Treg cells expressing CD8 phenotype (CD8 Treg cells) in MM. To the best of our knowledge, this has not been reported previously in MM. Methods We analyzed CD8 Treg cells and their transcription factor FoxP3 from 64 newly diagnosed MM patients using flow cytometry and real time-polymerase chain reaction (RT-PCR). RNA profile of cytokines in CD8 Treg cells was also assessed using RT-PCR. CD8 Treg cells from 5 MM patients and 5 healthy donors were functionally evaluated using proliferation assays. Results CD8 Treg cells (CD8+CD25hi+) were significantly elevated in MM patients (P<0.0001), and their transcription factor FoxP3 expression was also higher in MM (P<0.0001) compared to healthy donors which was evidenced by flow cytometry and RT-PCR analyses. CD8 Treg cells negatively correlated with total lymphocyte count (P = 0.016). Functional studies revealed that CD8 Treg cells isolated from MM patients and healthy donors inhibited proliferation of CD4 T cells in a concentration dependent manner. In the presence of CD8 Treg cells in proliferation assays, level of IFN-γ was decreased but not IL-10. CD4 T cells from MM patients secreted abnormal level of IL-10 compared to healthy donors (P = 0.01) in proliferation assays without CD8 Treg cells. RNA profile of cytokines from CD8 Treg cells did not differ significantly between MM patients and healthy donors. Conclusions These findings show the presence of increased number of functionally suppressive CD8 Treg cells in MM patients. We believe that these suppressive CD8 Treg cells might enhance immune impairment and disease progression in MM.

Induction by lenalidomide and dexamethasone combination increases regulatory cells of patients with previously untreated multiple myeloma

The introduction of novel drugs (thalidomide, lenalidomide [immunomodulatory drugs; IMiDs] and bortezomib) optimistically changed the outcome of patients with multiple myeloma (MM) in terms of progressionfree and overall survival when compared to conventional chemotherapies.

Flow cytometry-based enumeration and functional characterization of CD8 T regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment.

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In this study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len‐dex) treatment in MM patients.

Contribution of regulatory T cells to immunosuppression and disease progression in multiple myeloma patients.

Multiple myeloma (MM) patients exhibit consistent degrees of immune dysfunction. Regulatory T cells contribute to the establishment of an immunosuppressive status in MM patients, hence favoring disease progression.

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.

Regulatory Cells and Multiple Myeloma

Large evidence is available in hematological malignancies and solid tumors for elevated level of various regulatory and suppressor cells which impede anti-tumor responses. Therefore, targeting the regulatory cells could be a useful strategy to enhance the anti-tumor immunity. Approaches of depletion or inhibition of regulatory cells showed countable benefits in pre-clinical and clinical studies of some cancers including renal cell carcinoma, metastatic melanoma and colorectal carcinoma. Targeting regulatory T cells in a non-specific approach might cause detrimental autoimmune toxicities which is the key issue. Further studies are necessary to identify tumor associated regulatory cells which will enhance the depletion of specific regulatory cells but not the global population of regulatory cells.