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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 43  |  Issue : 2  |  Page : 88-93

Relevance of CD49d and CD38 expressions as predictors of disease progression in chronic lymphocytic leukemia


1 Department of Clinical Pathology, National Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Human Cytogenetics, National Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
4 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission01-Dec-2017
Date of Acceptance02-Jan-2018
Date of Web Publication7-Aug-2018

Correspondence Address:
Amany H Abdelrahman
National Research Center, Cairo 12622
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_51_17

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  Abstract 


Background Chronic lymphocytic leukemia is a heterogeneous disease characterized by a highly variable clinical course. The majority of cases do not require therapy and show identical survival rate with their chronic lymphocytic leukemia-free counterparts of the same age. However, other patients may suffer from an aggressive course of the disease with early need for therapy and shorter overall survival.
Objective The aim of this study was to investigate the expression pattern and prognostic value of the adhesion molecules, CD49d and CD38, in chronic lymphocytic leukemia (CLL) patients. Moreover, we attempted to investigate their correlation with trisomy 12, which could help in the clarification of the distinctive clinical features of the trisomy 12+ CLL subset.
Patients and methods Seventy newly diagnosed chronic lymphocytic leukemia patients and 50 age-matched and sex-matched controls were included in this study. Patients were subjected to full history taking, clinical examination and abdominal ultrasonography. Laboratory investigations included complete blood count, cytogenetic analysis for the presence of trisomy 12, and flow cytometric assessment.
Results A significant positive correlation was detected between CD49d expression and CD38 expression. CD49d showed positive correlation with poor prognostic parameters that included organomegaly, high count of white blood cells, lymphocyte count and trisomy 12. CD38 showed a significant positive correlation with high white blood cell count, lymphocyte count, and splenomegaly. In addition, trisomy 12+ chronic lymphocytic leukemia patients showed significant higher lymphocyte count and splenomegaly.
Conclusion Our data confirm the suggested role of CD38 and CD49d as predictors for poor clinical outcome of CLL patients. Moreover, trisomy 12+ CLL cells exhibit a high CD49d expression that may account for the unique clinical characteristics of this group.

Keywords: chronic lymphocytic leukemia, CD49d, CD38, trisomy 12


How to cite this article:
Abdelrahman AH, Ibrahim MH, Hamza MT, Eid OM, Hassan EM, Kamel SA, Eid MM, Shahin RY, Sallam MT. Relevance of CD49d and CD38 expressions as predictors of disease progression in chronic lymphocytic leukemia. Egypt J Haematol 2018;43:88-93

How to cite this URL:
Abdelrahman AH, Ibrahim MH, Hamza MT, Eid OM, Hassan EM, Kamel SA, Eid MM, Shahin RY, Sallam MT. Relevance of CD49d and CD38 expressions as predictors of disease progression in chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2018 [cited 2018 Dec 11];43:88-93. Available from: http://www.ehj.eg.net/text.asp?2018/43/2/88/238768




  Introduction Top


Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by the accumulation of a neoplastic clone of cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. It is the most common leukemia in the Western world [1]. In Egypt, it accounts for 3.08% of lymphoid malignancies and 0.5% of all cancers, according to the National Cancer Institute hospital-based registry (2002–2010) [2].

The majority of cases do not require therapy until disease progression is encountered. Disease progression results from the microenvironmental alterations with subsequent inhibition of apoptosis and/or enhancement of neoplastic clone proliferation, which in turn leads to the acquisition of new genetic aberrations and hence disease progression [3],[4].

Adhesion molecules constitute a structural diverse group of ligands and cell membrane receptors belonging to the immunoglobulin-homing, selectin-homing, integrin-homing, and lymphocyte-homing receptor families. It has been speculated that their expression may explain the biological behavior, the pattern of infiltration and trafficking of the malignant cells, in addition to their potential role in treatment of the disease by targeted therapies [2],[3],

CD38 expression is a measure for cell division and growth in vivo. The aggressiveness of CD38+ cells appears to be combined by their ability to migrate and interact with the microenvironment. CD49d is an integrin that functionally acts as an adhesion structure for extracellular matrix components through its binding with vascular cell adhesion molecule-1 or fibronectin [5]. Previous preliminary study in the Italian population suggested that the high expression CD49d, alone or in combination with CD38, could be prognostic factors for short overall survival in CLL [6].

The aim of this study was to investigate the expression pattern and prognostic value of the adhesion molecules, CD49d and CD38, in CLL patients. Moreover, we attempted to investigate their correlation with trisomy 12, which could help in the clarification of the distinctive clinical features of the trisomy 12+ CLL subset.


  Patients and methods Top


Ethical statement

The study was approved by the ethical committee of the National Research Center, which is in accordance with ethical standards of the Declaration of Helsinki. All participants gave informed consent before their inclusion in the study.

Study design

This case–control study was conducted on CLL patients recruited from Internal Medicine Department of Ain Shams University Hospitals, between December 2015 and January 2017. The study included 70 newly diagnosed CLL patients (44 male patients and 26 female patients); their age ranged from 46–78 years. In addition, 50 age-matched and sex-matched healthy participants (29 male individuals and 21 female individuals), ranging in age from 45 to 70 years served as a control group. Patients on treatment were excluded from this study.

The diagnosis of CLL was established based on the WHO classification of hematolymphoid tumors [7]. CLL was diagnosed by the presence of at least 5000 monoclonal B lymphocytes/µl with a CLL immunophenotype in the peripheral blood (PB) for the duration of at least 3 months. Typically, CLL lymphocytes are small and mature looking with a scanty cytoplasm and a dense nucleus with partially aggregated chromatin. CLL cells coexpress CD5 and B-cell surface antigens CD19, CD20, and CD23. They express dim/weak to negative CD79b, surface IgM and they are negative for FMC7 [7].

All patients were subjected to full history taking, clinical examination with special emphasis on the presence of lymphadenopathy, splenomegaly and hepatomegaly, signs of anemia and thrombocytopenia, abdominal ultrasonography for assessing organomegaly, in addition to cytogenetic analysis of trisomy 12.

All participants were subjected to assessment of CD49d and CD38 expression level and complete blood count (CBC) analysis.

Sampling

Six ml venous blood sample was withdrawn from each participant under complete aseptic conditions and was divided into two EDTA vacutainers for CBC and flow cytometric analysis. As regards CLL patients, an additional 4 ml blood was collected in a heparinized vacutainer for cytogenetic analysis.

Laboratory investigations

  1. CBC analysis: CBC was performed on an automatic cell counter Sysmex XS500 (Sysmex, Bohemia, New York, USA) with examination of Leishman-stained smears.
  2. Cytogenetic analysis: trisomy 12 was detected by interphase fluorescence in-situ hybridization, using α satellite DNA probe CEP12 (Vysis, London, UK), directly labeled with Spectrum Organ to detect aneuploidy of chromosome 12. The heparinized PB samples were cultured using complete culture media without phytohemagglutinine, incubated at 37°C for 3 days, then harvested as described previously by Schlette et al. [8]. Fluorescence in-situ hybridization was carried out according to the manufacturer’s instructions, and examination of at least 200 interphase cells was conducted using Cytovision software (Leica, NJ, USA).
  3. Immunophenotyping by a flow cytometric immunophenotypic analysis was performed on the PB samples on the same day of their collection, using coulter EPICS-XL four-color flow cytometry (Coulter Diagnostic, Hialeah, Florida, USA).


A standard diagnostic panel of monoclonal antibodies (MoAbs) was used including CD5, CD19, CD10, CD20, CD11c, CD22, CD23, CD79b, CD25, CD103, CD52, FMC7, HLA-DR, SmIg, and κ and λ light chains (all supplied by Beckman Coulter, Fullerton, California, USA). All MoAbs are fluorescein isothiocyanate labeled, except CD5, CD10, CD79b, CD49d, CD38 and lambda light chain, which were labeled by phycoerythrin. Phycoerythrin-cyanine-5-labeled CD19 was used for gating on B-cells. Specific isotypic controls for fluorescein isothiocyanate conjugated and phycoerythrin-conjugated monoclonal antibodies were used.

Results were expressed as percentage of β cells expressing the tested MoAbs, wherein expression was considered positive if at least 30% of the cells expressed the MoAbs over the corresponding isotypic control [9].

Statistical analysis

All of the statistical calculations were made using excel program and SPSS, version 18 program (SPSS Inc, Chicago, Illinois, USA). Qualitative data were presented as frequency and percentage. χ2-Test was used to compare groups. Quantitative parametric data were presented as mean and SD, whereas the nonparametric data were expressed as median and interquartile range. For comparison between two groups, Student’s t-test and Mann–Whitney test (for nonparametric data) were used. Correlation coefficients (r) were calculated using Pearson’s correlation analysis (for parametric data) and Spearman’s correlation analysis (for nonparametric data). An effect was considered statistically significant at P value less than 0.05.


  Results Top


The study was performed on 70 CLL patients and 50 age-matched and sex-matched healthy controls; the descriptive data of CLL patients are presented in [Table 1].
Table 1 Chronic lymphocytic leukemia patients’ clinical data

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As regards the immunophenotypic data of CLL patients, CD5, CD23, and CD52 were positive in all (100%) studied CLL patients. CD20, CD79b, and FMC7 were expressed on 69 (98.6%), 21 (33.3%), and nine (12.8%) cases, respectively. The CD38 was positive in 12 (17.1%), whereas CD49d was positive in 34 (48.6%) patients. In terms of the cytogenetic aberration of CLL patients, trisomy 12 was detected in 15 (21.4%) of 70 CLL patients ([Table 2]).
Table 2 Immunophenotypic and cytogenetic data of chronic lymphocytic leukemia patients

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Statistical comparison of the laboratory data between CLL patients and controls revealed that the total leukocyte count, lymphocytic count, and CD38 and CD49d expression were significantly higher; meanwhile, the platelet count was significantly lower in CLL patients as compared with the control group (P<0.001) ([Table 3]).
Table 3 Comparison between laboratory data of chronic lymphocytic leukemia patients and controls

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CLL patients were further divided into two subgroups according to CD49d and CD38 expression; as regards CD49+/CD38− and CD49+/CD38+ subgroups, upon comparison, no significant statistical difference was detected between the two groups (P>0.05) ([Table 4]).
Table 4 Comparison between chronic lymphocytic leukemia patient subgroups in terms of coexpression of CD49d and CD38

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As regards the presence of trisomy 12 in CLL patients, trisomy 12+ patients demonstrated higher lymphocyte count, CD49d expression (P<0.001) and splenomegaly (P<0.05) as compared with trisomy 12-negative cases ([Table 5]).
Table 5 Comparison between chronic lymphocytic leukemia patients with regard to trisomy 12

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In the correlation study presented in [Table 6], CD49d-positive expression showed a significant positive correlation with lymphadenopathy, splenomegaly, white blood cell (WBC) count, lymphocyte count, trisomy 12 (P<0.001), and hepatomegaly (P<0.05). Although CD38 showed a significant positive correlation with WBC count, lymphocytic count (P<0.001), and splenomegaly (P<0.05), both CD49d and CD38 showed a significant negative correlation with platelet count (P<0.05). Finally, a significant positive correlation was detected between CD38+ expression and CD49 expression (P<0.001).
Table 6 Correlation analysis between CD49d and CD38 expressions and unfavorable prognostic factors

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  Discussion Top


CLL is a common lymphoid malignancy characterized by extreme clinical heterogeneity; whereas the majority of patients are asymptomatic, having an indolent course and living for decades without the need for therapy, others experience an aggressive disease course that may lead to premature death [10].

As many available prognostic parameters are achievable for use in the early stage of CLL, progress will entail the identification of molecular features that offer insight into disease biology and have the possibility for therapeutic targeting [11].

Our study showed that the mean expression of CD49d and CD38 in CLL patients was significantly higher than in the control group. Our results were clarified by Buggins et al. [12] who reported the presence of a multimer complex involving CD38, CD49d, CD44, and matrix metalloproteinase-9 in the majority of CLL cases but it was absent in normal β cells; it plays a key role in tissue invasion and prosurvival signaling of malignant cells. Moreover, Brachet et al. [13] reported a high expression level of CD49d and CD38 in the CLL cells and recognized the association of the proliferating marker Ki-67 with only the CD49+/CD38+ subset of CLL cells.

The significant positive correlation observed between CD49d expression and splenomegaly and hepatomegaly, in addition to the positive correlation between CD38 expression and splenomegaly, is in agreement with previous studies by Ibrahim et al. [10] and Gattei et al. [6]. These authors found that CD49d expression was associated with advanced CLL stage, and showed that the molecular complex between CD49d and CD38 triggers a signaling cascade, ultimately preventing apoptosis and hence implicated in the aggressive disease behavior. Moreover, Zucchetto et al. [14] suggested that CD49d and CD38 may act synergistically to confer an aggressive clinical course. It is identified that CD49d requires Ca2+ in order to promote homing and migration of lymphocytes. Furthermore, CD38 functions as a cyclic ADP-ribose that influences mobilization of Ca2+. Consequently, their coexpression could facilitate the homing of CLL cells to tissues with a more favorable environmental milieu, which in turn promotes rapid growth and longer survival to leukemic β cells.

The significant positive correlation detected between both CD49d and CD38 expressions and high WBC count, high lymphocytic count, and low platelet count, is in agreement with studies of Uzay et al. [15] and Gattei et al. [6], who observed the association between high CD49d expression and poor clinical outcome and advanced CLL stage. However, the study conducted by Ghia et al. [16] reported the association of CD38+ expression with high lymphocytic count and low platelet count, which may have resulted from bone marrow infiltration or may be related to an autoimmune phenomenon.

Our results demonstrated that trisomy 12+ patients had lymphadenopathy, splenomegaly and higher CD49d expression as compared with trisomy 12− patients. This is in agreement of Zucchetto et al. [17], who reported the presence of trisomy 12 in about 90% of CD49d+ patients, and deduced that overexpression of CD49d may clarify the specific tropism of trisomy 12+ cells toward lymph nodes and the unique clinical characteristics of this CLL subset, in which massive enlargement of lymph nodes is frequently recognized and final transformation to Richter’s syndrome is more expected than in other cytogenetic abnormalities. This finding could be explained by Dal Bo et al. [18], who demonstrated that the binding of CLL cells on stromal cells of microenvironmental niches occurs mainly through CD49d (α chain of α4β1 integrin heterodimer), wherein CD49d-driven interactions play a role in controlling β lymphocyte development, chemokine-induced transendothelial migration of mature β cells during their recirculation and homing, and antigen-specific β-cell differentiation within the germinal centers of secondary lymphoid organs. β Cells that express β-cell receptor (BCR) with high affinity for the antigen are saved from apoptosis by interaction with follicular dendritic cells through the α4β1/vascular cell adhesion molecule-1 axis. This inside–outside stimulation of the α4β1 integrin is BCR-controlled through the sequential activation of intracellular protein tyrosine kinases LYN, SYK, PI3K, BTK, PLCγ2, IP3R, and PKC. Upon BCR stimulation, α4β1 is released from the cytoskeletal constraint by Ca2+-mediated BCR-dependent calpain activation and then mobilized to the lipid rafts; this process results in the formation of α4β1 clusters that may become tethered to the actin cytoskeleton, eventually enhanceing α4β1 avidity and adhesion. In this model, β cells that expressed BCR with high affinity for the presented antigen were preserved in the germinal center by integrin-mediated signals, whereas those β cells that expressed BCR with low affinity for the presented antigen failed to have sufficient integrin-mediated signals and were more prone to apoptosis.

Many studies reported that CD49d is a surface marker whose expression can be easily investigated, is highly reproducible, is stable over time and is stable in frozen samples. Moreover, it can be easily evaluated by flow cytometric analysis, which is a less expensive and widely used technique. CD49d determination should be recommended for the prognostic assessment of CLL patients at diagnosis; in addition, it may have therapeutic implications, as anti-CD49 antibodies could hinder the migration and adhesion of leukemic cells and overcome the apoptotic resistance. Therefore, the combination between CD49d and the established immunotherapy or chemotherapy protocols give a future promise in CLL therapies.


  Conclusion Top


Our data confirm the suggested role of CD38 and CD49d as predictors for the poor clinical outcome of CLL patients. Moreover, trisomy 12+ CLL cells exhibit a high CD49d expression that may account for the unique clinical characteristics of this group.

Acknowledgements

National Research Center, Egypt partially funded this work through a project (number of the project was P100501).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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