|Year : 2015 | Volume
| Issue : 3 | Page : 130-137
CD44 as a diagnostic and prognostic marker in Egyptian patients with non-Hodgkin lymphoma
AA Hasnaa MD 1, AE Mohamed1, HS Eman1, MA Hanaa2
1 Department of Clinical Pathology, Sohag Faculty of Medicine, Sohag University, Sohag, Egypt
2 Department of Clinical Pathology, Ain Shams Faculty of Medicine, Ain Shans University, Cairo, Egypt
|Date of Submission||02-May-2015|
|Date of Acceptance||15-May-2015|
|Date of Web Publication||8-Sep-2015|
A A Hasnaa
Clinical Pathology Department, Sohag University Hospital, Sohag
Source of Support: None, Conflict of Interest: None
Background CD44 is an adhesion molecule that plays a role in lymphocyte homing; it is a multifunctional cell surface molecule involved in cell proliferation, angiogenesis, presentation of cytokines, chemokines, and growth factors.
Patients and methods This study was carried out on 50 newly diagnosed non-Hodgkin lymphoma patients (33 men and 17 women) and 20 healthy controls; the patients were divided into two groups: group I included 20 patients with stage I, II, and III non-Hodgkin lymphoma and group II included 30 patients with stage IV non-Hodgkin lymphoma. They ranged in age from 38 to 75 years; all patients were subjected to a clinical examination, a complete blood count, bone marrow aspiration, and immunophnotyping using monoclonal for CD44 in addition to a routine panel of different chronic lymphoproliferative disorders (CD19, CD5, CD10, CD20, CD22, CD23, SIgM, CD38, CD79b, FMC7, CD103, CD123, κ, and λ light chains).
Results CD19, CD20, CD22, CD23, κ/CD19, CD5/CD19, CD10, CD123, and CD103 expression levels showed no statistically significant differences between group I and group II, using the Mann-Whitney U-test, whereas other investigations showed significant differences; these differences were highly significant in terms of gated cell count, CD79b, SIgM, FMC7, λ/CD19, and CD44 expression levels. There was a positive correlation with significant differences between CD44 and FMC7, lymphocytic count, and LDH, a positive correlation with significant differences between CD44 and gated cell count and SIgM, and a highly statistically significant correlation between CD44 and λ/CD19, FMC7, lymphocytic count, and platelet count. Sixty percent of CD44-positive patients in group II had lymph node enlargement compared with only 15% of CD44-positive patients in group I.
Conclusion Strong CD44 expression appears to be a powerful prognostic indicator in adult non-Hodgkin's lymphoma, with no difference between male and female patients. It is associated with standard poor prognostic markers such as bone marrow infiltration.
Keywords: CD44 , marker, infiltrating, non-Hodgkin lymphoma
|How to cite this article:|
Hasnaa A A, Mohamed A E, Eman H S, Hanaa M A. CD44 as a diagnostic and prognostic marker in Egyptian patients with non-Hodgkin lymphoma. Egypt J Haematol 2015;40:130-7
|How to cite this URL:|
Hasnaa A A, Mohamed A E, Eman H S, Hanaa M A. CD44 as a diagnostic and prognostic marker in Egyptian patients with non-Hodgkin lymphoma. Egypt J Haematol [serial online] 2015 [cited 2020 Apr 2];40:130-7. Available from: http://www.ehj.eg.net/text.asp?2015/40/3/130/164727
| Background|| |
CD44 is an adhesion molecule that plays a role in lymphocyte homing, T-lymphocyte activation, promotion of adherence between fibroblasts, lymphocytes, and extracellular material, signal transmission, renewal of the composition of interstitial tissue, accommodation of drug absorption and drug sensitivity, and pseudopod formation and cell migration  . CD44 is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines, and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival  . All these biological properties are essential to the physiological activities of normal cells, but they are also associated with the pathologic activities of cancer cells. Experiments in animals have shown that targeting of CD44 by antibodies, antisense oligonucleotides, and CD44-soluble proteins markedly reduces the malignant activities of various neoplasms, highlighting the therapeutic potential of anti-CD44 agents  . The standard CD44 isoform is expressed on all types of mature blood cells, the majority of mononuclear bone marrow precursors, and all CD34+ HPC. The level of its expression varies according to the hematopoietic cell lineage and stage of differentiation. For example, it is high on monocytic cells, intermediate on polymorphonuclear cells and CD34+ HPC, and low on erythroid cells and platelets  . The human CD44 gene is located at the short arm of 11 chromosomes, containing at least 20 exons spanning some 50 kb of DNA  . CD44 variants (CD44v) are mainly expressed on epithelial cells, encoding amino acids with extensive glycosylation sites and chondroitic acid-binding sites  . CD44 is highly expressed in many tumors, and correlated with the tumor biological behavior including tumorigenesis, growth, metastasis, and prognosis. It is a reliable indicator of tumor load and disease activity, and also called metastasis-associated protein  . It was presumed that the over-expression of CD44v6 facilitated the invasion of the tumor as it escaped from recognition by the immune system; thus, they could invade the lymph node and metastasized more easily  . It was detected that serum CD44 levels were significantly higher in patients with Hodgkin's disease (HD), non-Hodgkin's lymphoma (NHL), and Burkitt's lymphoma (BL) than those in the control group. Serum sCD44 levels decreased significantly in HD and NHL patients who were in complete remission  . Expression of CD44 was significantly high in patients with HD and NHL who were in advanced stages of disease. A high serum CD44 level was also associated with high tumor tissue expression of CD44 in patients with HD and BL. Evaluation of the prognostic value of soluble CD44 in B-cell chronic lymphocytic leukemia (B-CLL) showed that serum levels of sCD44s and sCD44v6 were significantly elevated in these patients in comparison with normal individuals  . The anti-CD44 monoclonal antibodies can effectively suppress the proliferation and induce differentiation or apoptosis of leukemic cells in some subtypes. Studies on CD44 antibodies therapy are only in the phase of primary experiments and have not reached consensus; thus, further studies need to be carried out in this respect  . NHLs are a diverse group of blood cancers that include any kind of lymphoma, except HLs  . The 'Revised European American Lymphoma Classification' can be considered a comprehensive summary of known entities and an important next step toward a more biologically oriented grouping of malignant lymphomas  . The WHO classification has been updated again as a joint effort of the hematopathology societies, provisional borderline categories, the recognition of small clonal lymphoid populations, and the identification of diseases characterized by the involvement of specific anatomic sites or other clinical features such as age  . In about 10-20% of lymphomas, B-cell antigens are found on tumor cells that show no SmIg by IHC study or a low percentage of Ig-positive cells by flow cytometric analysis. Because SmIg is the antigen receptor expressed on normal B cells, its expression was absent in neoplastic cells  .
Aberrant expression of antigens or marked expansion of a subset of lymphocytes can also signal for clonality. Examples include expression of Bcl-2 by CD10+ B cells in follicular lymphoma, aberrant expression of T-cell-associated antigens (CD2, CD4, CD7, and CD8) on B cells, and clonal expansion of CD5+, CD19+ B cells in SLL and MCL  . Immuno-phenotyping can detect markers as CD38 expression was associated with bad prognosis, due to diffuse marrow infiltration, high peripheral blood lymphocytes and atypical morphology.  . ZAP-70 protein, a strong prognostic marker in B-CLL/SLL, can also be detected by IPT. Current data indicate that ZAP-70 expression by more than 20% of CLL/SLL cells is associated reliably with a poor prognosis  . Bcl-2 and Bcl-6 are two markers linked to germinal center B cells. Both markers have also been proved to have an effect on the prognosis of mature B-cell neoplasms  . The serum β-2 microglobulin level is useful in patients with CLL and other indolent lymphomas, where it has been used for prognostic purposes, as a surrogate measure of disease volume, and for monitoring response to therapy  .
| Patients and methods|| |
This study was carried out on 50 newly diagnosed NHL adult patients attending the hematological unit of the clinical pathology department, Ain Shams University, and 20 healthy controls. The candidate patients were recruited into the study on the basis of clinical, laboratory, bone marrow aspirate findings, and immunophenotypic criteria of NHL. Sometimes, lymph nodes biopsy and bone marrow trephine biopsy results were provided to confirm the diagnosis.
This study was approved by the research and ethical committee at the faculty of medicine, Sohag University. All patients were informed about the aim of the study and provided written consent. The patients included 33 men and 17 women, with a male to female ratio of 1.9 : 1.0; their ages ranged from 30 to 75 years, mean age 52.27 years. The patients were divided into two groups according to the stage of the disease on the basis of the Ann Arbor classification system.
Group I included 20 patients with stage I, II, and III NHL. There were 12 men and eight women, with a male to female ratio of 1.5 : 1. Their ages ranged from to 71 years, with a mean of 44.75 years.
Group II included 30 patients with stage IV NHL. There were 21 men and nine women, with a male to female ratio of 2.3 : 1. Their ages ranged from 38 to 75 years, with a mean of 59.8 years.
All patients were subjected to the following.
Clinical sheet details in terms of assessment of history and clinical examination, with a focus on the presence of fever, weight loss, pallor, petechiae, infections, organomegaly, and lymphadenopathy.
Complete blood count (CBC) was performed on a Coulter Max-M cell counter (Coulter, Electronics, Hialeah, Florida, USA), with examination of PB smears stained with Leishman stain. Special attention was paid to lymphocytic count and morphology. The following parameters were assessed: red blood cell count, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, and platelet counts.
Bone marrow aspiration with examination of Leishman-stained smears for assessment of bone marrow lymphocytes percentage as well as morphology.
Flow cytometric immunophenotyping was performed on bone marrow or peripheral blood samples of cases in the leukemic phase using monoclonal CD44 in addition to a routine panel of different chronic lymphoproliferative disorders (CD19, CD5, CD10, CD20, CD22, CD23, SIgM, CD38, CD79b, FMC7, CD103, CD123, κ, and λ light chains). Flow cytometry was performed on a Coulter Epics XL 3-color flow cytometer (Coulter Electronics). Cells were considered positive for a certain marker when it expressed by 20% or more.
Two milliliters of venous blood was aseptically collected from each patient and dispensed into a tube containing K-EDTA to be used for CBC and spreading of PB smear for Leishman staining and differential leukocyte counting.
Bone marrow (BM) aspiration was performed for all patients and several BM smears were spread for examination by Leishman stain. One milliliter of BM aspirate was drawn into a sterile K-EDTA vacutainer tube for flowcytometry.
Reagents used in a flow cytometry study
FITC Mouse IgG1 and PE Mouse IgG1 were fluorescence controls used for human cells. Mouse IgG1 fluorescence controls were used to estimate the amount of nonspecific binding as well as binding mediated by interaction with Fc receptors using a BD FACS brand flowcytometer. FITC Mouse IgG1 used in this study was provided as a bottle concentration of 100 µg in 2 ml of PBS. PE Mouse IgG1: used in this study was provided as bottle concentration of 100 µg in 2 ml of PBS. An isotype-matched negative control (appropriate for the MoAbs used) was used in all cases to assess the background fluorescence intensity (nonspecific binding of MoAbs).
A panel of fluorescein isothiocyanate/phycoerythrin-conjugated monoclonal antibodies to B-lineage markers (CD19, CD10, and CD20), T-lineage markers (CD5), and a routine panel of different chronic lymphoproliferative disorders (CD22, CD23, CD38, SIgM, CD79b, FMC7, CD103, CD123, κ, and λ light chains) were used, in addition to FITC-labeled monoclonal antibodies to CD44 supplied by BD Pharmingen (San Diego, CA, USA).
NaCl 8.5 mmol/l, NaHPO 4 -(anhydrous) 1.07 mmol/l, and NaH 2 PO 4 -2H 2 O 0.39 mmol/l to complete to one litter by distilled water, pH 7.4, stored at 4°C, and used as long as no turbidity.
Reagent provided: NH 4 Cl 1.5 mmol/l, KHCO 3 100 mmol/l, and tetrasodium EDTA 10 mmol/l; lysing solution 10× concentrate was provided as 100 ml of the proprietary buffered solution containing less than 15% formaldehyde and less than 50% diethylene glycol, made up to 1 liter with distilled water, pH adjusted at 7.2. Lysing solution was used for lysing red blood cells following direct immunofluorescence staining of human peripheral blood cells with monoclonal antibodies before flow cytometric analysis.
Storage and handling
The reagents were stored at 2-8°C and used before the expiration date. The reagent was protected from direct light during storage and incubation with cells. The reagent vial was kept dry.
BD Falcon Tubes was stored in their original foil pouch at 2-25°C; to avoid potential condensation, the pouch was opened only after it had reached room temperature and the pouch was resealed carefully immediately after removing a tube.
The tubes were used within 1 h after removal from the foil pouch and before the expiration date.
The EDTA anticoagulated BM sample was analyzed on the same day of collection and diluted 1 : 3 with PBS. The final cell count suspension was adjusted between 5 and 10 × 10 9 /l to ensure that the cell count was within the linear range.
Procedure of surface marker staining
- For each sample, a set of tubes was prepared for all the MoAbs to be used including one for the isotype control. Fifty microliter of the diluted marrow sample was dispensed into each tube. Smearing of blood down the side of the tube was avoided.
Five microliter of each MoAbs was added to the test tube.
Five microliter of the appropriate isotype-matched negative control was added to the control tube.
- The tubes were vortexed and then incubated for 15 min in the dark at room temperature (20-25°C).
- Two milliliter of PBS was added, as a wash buffer, to each tube and mixed well; the tubes were centrifuged at 3000 rpm for 5 min, then the supernatant was discarded, and the wash was repeated twice.
- One and half ml of the lysing solution was added to each tube, mixed well by vortex, and then the tubes were incubated for 10 min in the dark at room temperature.
The cells were washed once with 2 ml PBS, with centrifugation, and the supernatant was discarded. Cells were suspended in 500 µl PBS and processed by the flowcytometery (FCM). If the tubes were not processed within 2 h, 0.5 ml of fixative (4 g paraformaldehyde in 100 ml PBS with 0.1% Na azide, pH 7.4) was added and the tubes were kept at 4°C until analyzed within 24 h.
Sample processing on FCM
A minimum of 5000 events were studied. Gating was performed on the small mononuclear cells, which were predominantly lymphocyte cell populations on the basis of their forward and side scatter properties.
The positivity was expressed as a percentage with a cut-off of more than 20% over the corresponding isotypic control. For CD44 marker expression, a value of 20% expression was used to categorize the patients into a negative group (<20%) and a positive group (>20%).
Data were analyzed using IBM-SPSS, version 19 (IBM, Chicago, Illinois, USA).
Qualitative data were described as number and percentage. Quantitative data were described as mean ± SD for parametric results.
Comparison between two groups of quantitative nonparametric data was performed using the Mann-Whitney U-test (Z-value).
The Pearson correlation test was used to compare two quantitative variables. The value of r is explained as follows:
r < 0.2→negligible correlation.
r = 0.2-0.4→weak correlation.
r = 0.4-0.7→moderate correlation.
r = 0.7-1→strong correlation.
r positive→positive correlation.
r negative→negative correlation.
For all these tests, the level of significance or the probability of being by chance (P) value was calculated for all parameters and was evaluated as follows:
No significance, P ≥ 0.05.
Significance, P < 0.05.
High significance, P < 0.01.
Linear regression analysis was used to examine the role of CD44-positive cells as risk factors between patients in different groups.
| Result|| |
This study included 50 patients divided into two groups: group I included 20 stage I, II, and III NHL patients and group II included 30 stage IV NHL patients; controls were 20 apparently healthy individuals. Groups I and II included patients attending the hematological unit of the clinical pathology department, Ain Shams University; this study was approved by the Sohag Faculty Committee for Research Ethics.
To investigate the role of CD44 in staging NHL patients, bone marrow samples were collected from the two patient groups (stage I, II, III, and IV) as well as the normal controls. Blood samples were then subjected to CBC, chemistry tests, and bone marrow sample for a flow cytometric full panel assay for patients only and CD44 for controls and patient groups.
The patients were 33 men and 17 women, with a male to female ratio of 1.9 : 1.0. Their ages ranged from 30 to 75 years, with a mean of 52.27 years, as shown in [Figure 1].
From [Table 1] we can see that 60% of CD44-positive patients in group II had lymph node enlargement, whereas only 15% of CD44-positive patients in group I had lymph node enlargement.
|Table 1 Comparison between group I and group II in lymph node enlargement|
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From [Table 2] we can see that CD19, CD20, CD22, CD23, κ/CD19, CD5/CD19, CD10, CD123, and CD103 expression levels showed no statistically significant differences between group I and group II, using the Mann-Whitney U-test, whereas other investigations showed significant differences; these differences were highly significant for gated cell count, CD79b, SIgM, FMC7, λ/CD19, and CD44 expression levels.
|Table 2 Comparison between group I and group II in gated cell count, CD19, CD20, CD22, CD23, CD79b, sIgM, k/CD19, l/CD19, FMC7, CD5/CD19, CD10, CD38, CD123, CD103, and CD44 using the Mann– Whitney U-test|
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From [Table 3] we can see that hemoglobin and lactate dehydrogenase enzyme levels showed no statistically significant differences between group I and group II, using the Mann-Whitney U-test, whereas other investigations showed significant differences; these differences were highly significant for age of the patient and white blood cell count.
|Table 3 Comparison between group I and group II in white blood cell count, age of the patients, hemoglobin level, and lactate dihydrogenase enzyme level using the Mann-Whitney U-test|
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[Table 4] shows that the ratio of CD44 expression was low in group I and the control groups, with no statistically significant difference.
|Table 4 Comparison between groups I, II and the control group in CD44 expression levels using the Mann-Whitney U-test|
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[Table 4] shows that the ratio of CD44 expression was higher in group II than in the control groups, with a highly statistically significant difference.
Pearson correlation showed that there were negative correlations between CD44 and CD19, CD10, CD20, CD22, CD23, κ/CD19, CD38, and CD123. However, the difference was nonsignificant whereas other investigations showed a positive correlation with significant differences between CD44 and gated cell count and SIgM, and a highly statistically significant correlation between CD44 and λ/CD19, FMC7, lymphocytic count, and platelet count ([Figure 2] and [Figure 3]).
The most significant factors in differentiating group I from group II within the above factors are age of the patients, level of FMC7 expression, platelet count, and level of CD44 expression. The level of CD44 expression was much higher in group II than in group I, with a highly significant difference, followed by platelet count, age of the patients, and level of FMC7 expression, both of which were higher in group II, with significant differences; other factors showed no statistically significant differences.
[Figure 4] shows flow cytometry analysis for patients negative for CD44 and [Figure 5] for a patient positive for CD44.
|Figure 4 Flow cytometric analysis indicates negativity for CD44 with low expression. The left histogram shows gated cells (87.4%) and the right histogram shows CD44 expression (3.58%).|
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|Figure 5 Flow cytometric analysis shows a case with high CD44 expression. The left histogram shows gated cells (69.6%) and the right histogram shows CD44 expression (72.4%).|
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| Discussion|| |
The cell adhesion molecule CD44 is involved in a variety of important biological events such as embryogenesis, hematopoiesis, lymphocyte homing, lymphocyte activation, inflammatory reactions, and tumor dissemination  .
The present study aimed to address a potential role for CD44 expression in the staging of NHL and to identify different risk factors that upgrade NHL and can be considered as a risk factor for distant metastasis and infiltration of the disease.
In terms of patients' sex, in both groups, there was an increase in the number of the diseased patients in males than in females; this is in agreement with previous studies carried out by Abbott  , who confirmed a male predominance among NHL patients. In terms of the age of the patients, there was a steady increase in the age of patients with stage IV NHL, who were generally older than patients of the other stages, in agreement with previous studies carried out by Peveling-Oberhag et al.  . However, lactate dehydrogenase (LDH) enzyme levels showed no difference between both groups as the LDH levels were high in both groups. The results of our study are in agreement with those reported previously in another study carried out by Ruder et al.  , who reported that LDH levels increase in all stages of NHL. CD44 overexpression can cause gene toxic damage following enhanced DNA repair and lead to a poor prognosis in malignant lymphoma  . In a previous study carried out by Tacyildiz et al.  , it was found that serum CD44 levels were significantly higher in patients with HD, NHL, and BL than those in the control group. Serum CD44 levels decreased significantly in HD and NHL patients who were in complete remission  . In our study, on comparing the results of CD44 expression in both groups with the control group, it was found that the values of CD44 expression were low in group I and in the control group, whereas the values of CD44 expression were the highest in group II compared with the values of the control group and group I, in agreement with Sackstein and Robert  , in our study CD44 was highly expressed in group II than I so it was related to the tumor metastasis. Other investigated markers including gated cell count, CD79b, sIgM, λ/CD19, FMC7, CD38, and CD44 showed significant differences, and these differences were highly significant for gated cell count, CD79b, SIgM, FMC7, λ/CD19, and CD44 expression levels, in partial agreement with previous studies carried out by Knowles and Sun , . In group I, there was a negative correlation between CD44 and CD19, CD23, CD10, and hemoglobin level; however this correlation was nonsignificant, whereas other investigated markers such as age of the patients, CD20, CD22, CD79b, sIgM, κ/CD19, λ/CD19, CD5/CD19, CD38, CD123, and CD103 showed a positive correlation with CD44, but this correlation was nonsignificant, in partial agreement with a previous study carried out by Bailey and Anand  . However, other factors such as age of the patients, gated cell count, CD23, CD79b, sIgM, λ/CD19, FMC7, CD5/CD19, CD10, CD103, white blood cell count, hemoglobin level, and lactate dihydrogenase enzyme level showed a positive correlation, which is in partial agreement with a study carried out by Ryckman et al.  that suggests that the release of CD44 from lymphoma tissue may be associated with cell proliferation.
The significant positive correlation in group II according to our study was detected between CD44, gated cell count, and SIgM; also, a highly significant correlation was detected between CD44 and λ/CD19, FMC7, lymphocytic count, and platelet count in group II.
Our study proves that the most significant factors in differentiating group I from group II among the factors studied were age of the patients, level of FMC7 expression, platelet count, and level of CD 44 expression, in partial agreement with previous studies carried out by Bailey and Anand  and Ryckman et al.  .
| Conclusion|| |
Strong CD 44 expression appears to be a powerful prognostic indicator in adult NHL, with no difference between male and female patients. It is associated with standard poor prognostic markers such as bone marrow infiltration.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]