Frequency analysis of HLA class I alleles in Iranian patients with progressive and non-progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a malignant disorder of B cell origin, with low incidence in Asian populations. In this study we investigated the HLA-class I A and B allele frequencies in 87 Iranian CLL patients and 64 healthy controls using sequence specific primer-polymerase chain reaction (SSP-PCR) technique. Our results showed increased frequencies of HLA-A11:01 (p=0.02) and HLA-B35:01 (p=0.002) alleles and HLA-A11:01/B35:01 haplotype (p=0.036) and decreased frequencies of HLA-A01:01 (p=0.02), HLA-A26:01 (p=0.03), HLA-B65:01 (p=0.03) and HLA-B53:01 (p<0.00001) alleles in CLL patients compared to the control group. Classification of the patients into non-progressive and progressive groups did not reveal significant differences for the frequency of any of the HLA-A and -B alleles or haplotypes between these two subtypes. Comparison between patients with immunoglobulin heavy chain variable region genes (IGHV) mutated (n=56) and unmutated (n=31) subtypes showed a significant increase in HLA-A32:01 (p=0.05) and HLA-A33:01 (p=0.05) alleles in IGHV unmutated patients compared to IGHV mutated patients. Similarly, a higher frequency of HLA-B52:01 (p=0.037) alleles was observed in CD38+ compared with CD38- patients. Our results obtained from an Iranian population indicate that CLL is associated with distinct HLA class I alleles and haplotypes some of which are linked to disease prognostic factors.Tehran University of Medical SciencesMinistry of Health and Medical Education of IranManuscrip

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia : association between CD38 expression and disease progression

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have hetero-geneous clinical courses, thus several biological parameters need to be added to the cur-rent clinical staging systems to predict disease outcome. Recent immunophenotypic stud-ies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. Objectives: To investigate the expression pat-tern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between in-dolent and progressive groups. Methods: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. Results: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/sIg+). The vast ma-jority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4%) and CD45RO (74/87, 83.9%) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and inten-sity of CD38 expression in progressive group (40.5%) compared to indolent (11.9%) pa-tients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. Conclusion: Our results obtained in an Asian ethnic popula-tion confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL.Tehran University of Medical Sciences and the Ministry of Health and Medical Education of Iran.Publishe

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. Objectives: To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. Methods: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. Results: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/sIg+). The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4%) and CD45RO (74/87, 83.9%) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and intensity of CD38 expression in progressive group (40.5%) compared to indolent (11.9%) patients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. Conclusion: Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL.Tehran University of Medical sciences, Tehran, IranPublishe

Comparative expression profile of orphan receptor tyrosine kinase ror1 in iranian patients with lymphoid and myeloid leukemias

It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia (CLL) and a subset of Acute Lymphoblastic Leukemia (ALL). In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia (AML) and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region (IGHV) gene mutated (n=55) and unmutated (n=29) and also indolent (n=42) and progressive (n=39) subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid (CLL and ALL), but not myeloid (AML) leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy.Tehran University of Medical Sciences and the Ministry of Health and Medical Education of Iran.Publishe

Alteration in Frequency and Function of CD4+CD25+ FOXP3+ Regulatory T cells in Patients with Immune Thrombocytopenic Purpura

Immune thrombocytopenic  purpura (ITP) is an autoimmune bleeding disorder characterized by production  of auto-antibodies against platelet antigens. It is obvious that regulatory T cells (Tregs) have a major role in controlling immune homeostasis and preventing autoimmunity. To investigate the frequency and functions of Tregs, twenty ITP patients and twenty age- and sex- matched healthy controls were recruited. The peripheral blood mononuclear cells were isolated and the proportion of Tregs was defined by flow cytometry method. The expression of immune-regulatory markers, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and glucocorticoid induced tumor necrosis  factor  receptor  (GITR)  were  also  assessed by  quantitative  Real-time polymerase chain reaction TaqMan method. For evaluation of Treg function, Tregs were enriched and their ability to inhibit proliferation of T cells was measured and levels of immune-regulatory cytokines IL-10 and Transforming growth factor beta (TGF-β) were also measured.Results showed that the frequency of Tregs  and  the  mean  fluorescence  intensity  of  forkhead  box  P3  (FOXP3)  protein  significantly decreased in ITP patients compared to those in healthy controls. In addition, there was a significant reduction  in relative expression of both  CTLA-4 and GITR  mRNA  in ITP  patients (p=0.02 and p=0.006, respectively). The suppressive function of Tregs also diminished in ITP patients compared to controls. Both  IL-10 and TGF-β  cytokines were produced  in lower amounts  in ITP  patients than controls. It  could  be  concluded  that  alteration  in  Treg  frequency and  functional  characteristics might  be responsible for loss of self-tolerance and subsequently destructive immune responses observed in ITP patients

(A) Immunoprecipated (IP) ROR1 from CLL cell lysates probed with serum from 4 non-progressive (NP) and 4 progressive (P) CLL patients as well as 4 control donors. Bands of 105 and 64 kDa could be seen. The blots were also probed with a goat anti-human ROR1 antibody, anti-CD20 MAb and an isotype control MAb. (B) CLL cells immunoprecipitated using anti-CD20 and isotype control MAbs. No ROR1 bands could be detected. (C) Patients’ sera were adsorbed with a pool of CLL cell lysate (n = 10). The 105 and 64 kDa bands disappeared or were significantly reduced. (D) Patients’ sera were absorbed with a pool of normal PBMC lysate (n = 5). The 105 kDa and 64 kDa ROR1 bands did not disappear.

<p>(A) Immunoprecipated (IP) ROR1 from CLL cell lysates probed with serum from 4 non-progressive (NP) and 4 progressive (P) CLL patients as well as 4 control donors. Bands of 105 and 64 kDa could be seen. The blots were also probed with a goat anti-human ROR1 antibody, anti-CD20 MAb and an isotype control MAb. (B) CLL cells immunoprecipitated using anti-CD20 and isotype control MAbs. No ROR1 bands could be detected. (C) Patients’ sera were adsorbed with a pool of CLL cell lysate (n = 10). The 105 and 64 kDa bands disappeared or were significantly reduced. (D) Patients’ sera were absorbed with a pool of normal PBMC lysate (n = 5). The 105 kDa and 64 kDa ROR1 bands did not disappear.</p

(A) Serum concentrations (ng/ml) of anti-ROR1 antibodies (ELISA) against a full length ROR1 protein and (B) a ROR1 KNG protein in CLL patients (n = 23): (●) non-progressive (n = 15) and (x) progressive (n = 8) CLL patients and (○) control donors (n = 20). Dotted line represents mean+2SD of controls. ***p<0.0001. (C) Reactivity of sera from CLL patients with a recombinant CEA protein. The results are shown as OD values at 450 nm for 1:50 and 1:100 serum dilutions. (D) Relation between conc. of antibodies against a full length ROR1 protein and ROR1 KNG protein in 23 CLL patients.

<p>(A) Serum concentrations (ng/ml) of anti-ROR1 antibodies (ELISA) against a full length ROR1 protein and (B) a ROR1 KNG protein in CLL patients (n = 23): (●) non-progressive (n = 15) and (x) progressive (n = 8) CLL patients and (○) control donors (n = 20). Dotted line represents mean+2SD of controls. ***p<0.0001. (C) Reactivity of sera from CLL patients with a recombinant CEA protein. The results are shown as OD values at 450 nm for 1:50 and 1:100 serum dilutions. (D) Relation between conc. of antibodies against a full length ROR1 protein and ROR1 KNG protein in 23 CLL patients.</p

(A) Representative experiments showing antibodies against a recombinant full length ROR1 protein (105 kDa) in 3 non-progressive (NP) and 3 progressive (P) CLL patients. (B) Representative experiments showing antibodies against a ROR1 KNG protein (37 kDa) in 3 non-progressive (NP) and 3 progressive (P) CLL patients.

<p>A goat anti-ROR1 antibody and an isotype control MAb served as positive and negative controls respectively.</p

(A) Cytotoxicity (%) of CLL cells (a pool of 10 CLL patients) induced by the IgG fraction alone (10 μg/ml) (without complement or effector cells) from sera of 5 non-progressive (●) and 5 progressive (x) CLL patients and 10 control donors (○). (B) Cytotoxicity (%) of diluted sera of CLL patients 5104 and 5028 (red and blue lines respectively) and pooled IgG of five control donors (green lines). (C) Representative experiments showing antibodies against a recombinant full length ROR1 protein (105 kDa) in 3 non-progressive (NP) CLL patients before and after adsorption of anti-ROR1 antibodies using a pool of CLL cell lysate (n = 10). (D) Cytotoxicity (%) of CLL cells induced by the IgG fraction (10 μg/ml) before and after adsorption of anti-ROR1 antibodies in 5 non-progressive (●) and 5 progressive (x) CLL patients. (E) Cytotoxicity (%) of normal PBMC (a pool of 10 normal donors) induced by the IgG fraction alone (10 μg/ml) from 5 non-progressive (●) and 5 progressive (x) CLL patients as we

<p>P-values (asterics) refer to comparison between CLL and control donors. *p<0.05, ***p<0.0001.</p