ROLE AND RELEVANCE OF THE EUROFLOW ANTIBODY PANEL IN IMMUNOPHENOTYPING OF CHRONIC LYMPHOPROLIFERATIVE DISORDERS

Detection of phenotypically aberrant and clonal mature lymphocytes is the diagnostic hallmark of chronic lymphoproliferative disorders (CLPD). B-CLPD is the commonest of all the CLPD. In this study we evaluated the role of CD200 and CD43 new markers that have been introduced in the Euroflow panel in the separation between chronic lymphocytic leukemia (CLL) and all other mature B cell malignancies .These markers were also correlated with the markers used in the Matutes score like FMC7 and Surface membrane Immunoglobulin. Patient samples between October 2017-February 2018, peripheral blood or bone marrow aspirates of patients with suspected B-cell lymphoproliferative disorders were subjected to evaluation by flow cytometry. After washing, samples were stained by antibodies targeting the antigens CD45, CD19, CD5, CD10, CD20, CD23, CD43, CD79b, CD200, FMC7, CD25,CD103 ,CD11c ,sIgM, kappa and lambda . Immunophenotyping was performed using a BD FACS Canto flow cytometer. There were a total of 108 cases of B CLPD that were analysed by flow cytometry . Mean age (SD ) was 65 years. There were 68 males and 40 female patients . There were 71 cases of typical CLL , 7 cases of atypical CLL , 3 cases of Mantle cell lymphoma (MCL) , 5 cases of Follicular lymphoma ( FCL ) , 8 cases of Splenic lymphoma with villous lymphocytes (SLVL) , 6 cases of Hairy cell leukemia ( HCL) , 6 cases of unclassifiable B-NHL and 2 cases of lymphoplasmacytic lymphoma. All our cases of typical CLL showed bright expression of CD200 .It was also brightly expressed in all our atypical cases of CLL . CD43 was brightly positive in all our cases of typical CLL . The expression of this marker along with CD200 negativity was helpful in diagnosing of MCL.There were diagnostic difficulties in differentiating atypical CLL from B-NHL unclassifiable. The lack or dim expression FMC7 expression in all these cases along with absent or dim expression of sIgM were informative. Their use along with the panel by Euroflow is suggested to provide an accurate diagnosis.

Thy28 partially prevents apoptosis induction following engagement of membrane immunoglobulin in WEHI-231 B lymphoma cells

Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27Kip1. The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H2O2) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H2O2 activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27Kip1 in WEHI-231 B lymphoma cells.

Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells

Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL–derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.