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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 2  |  Page : 564-568

Role of CD305 and CD38 in Chronic Lymphocytic Leukemia Clinical Relevance


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
3 Clinical Pathology Department, Menoufia Chest Hospital, Menoufia, Egypt

Date of Submission18-Nov-2017
Date of Acceptance23-Feb-2018
Date of Web Publication27-Aug-2018

Correspondence Address:
Iman A Ahmedy
Faculty of Medicine, El Menoufia University, Yassin Abd El Ghaffar Street Shebin El-Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_775_17

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  Abstract 


Objective
This work aimed to study CD305 and CD38 expression in chronic lymphocytic leukemia (CLL) patients and its relation to clinical and prognostic criteria of the disease.
Background
CD305 is an inhibitor of B-cell receptor mediated signaling, and has been reported to be of lower expression in high risk CLL patients. Studies addressing CD305 in CLL and its relation to biological variables and standard prognostic factors had been so scarce yet .On the other hand, CD38 expression, a well established prognostic factor, in CLL, is associated with an aggressive clinical behavior.
Patient and methods
Our study included 54 patients. Forty CLL consecutive patients and 14 age-matched and sex-matched apparently healthy controls. Clinical examination and laboratory investigations were done for all of the studied groups.
Results
CD305 showed a positive correlation with lymphocyte doubling time. However it showed a negative correlation with; β2microglobulin, lactate dehydrogenase, modified ray staging and Coombs' test. CD38 expression showed a positive significant correlation with modified ray staging, white blood cells, β2microglobulin, lactate dehydrogenase and Coomb's test however it showed a negative correlation with CD305 and lymphocyte doubling time. 55% of patients did not receive chemotherapy were CD305 positive. The remaining 45% of patients received chemotherapy and were CD38+.
Conclusion
CD305 and CD38 expression can be used as simple reliable, inexpensive independent prognostic factors in CLL patients and they can even predict at presentation patients who will initiate chemotherapy early.

Keywords: B-cell, flow cytometry, immunophenotyping, leukemia, lymphocytic


How to cite this article:
Ahmedy IA, Alhassanin SA, Moussa NS. Role of CD305 and CD38 in Chronic Lymphocytic Leukemia Clinical Relevance. Menoufia Med J 2018;31:564-8

How to cite this URL:
Ahmedy IA, Alhassanin SA, Moussa NS. Role of CD305 and CD38 in Chronic Lymphocytic Leukemia Clinical Relevance. Menoufia Med J [serial online] 2018 [cited 2018 Nov 19];31:564-8. Available from: http://www.mmj.eg.net/text.asp?2018/31/2/564/239771




  Introduction Top


In its classification of hematopoietic neoplasias, the WHO described Chronic Lymphocytic Leukemia (CLL), as leukemic, lymphocytic lymphoma, being only distinguishable from small lymphocytic lymphoma by its leukemic appearance [1]. Most patients affected by chronic lymphocytic leukemia (CLL) are diagnosed by flow cytometry. Several immunophenotypic markers have been identified to be a significant and independent prognostic variables; especially from retrospective cohorts. Detection of these markers was found to be inexpensive and feasible in most laboratories but only few have been validated by independent series. Leukocyte associated immunoglobulin like receptor 1 (known as LAIR1, LAIR-1 or CD305), is an inhibitor of B-cell receptor mediated signaling, and has been reported to be of lower expression in high risk CLL patients. Studies addressing CD305 in CLL and its relation to biological variables and standard prognostic factors had been so scarce yet [2].

CD38 expression by CLL cells and its association with a more aggressive clinical behavior of the disease was first reported in 1994 and confirmed by several subsequent studies. Consequently, many clinical centers considered determination of the percentage of CD38 expressing cells within a clone a part of their routine workup for CLL patients, and it is generally accepted that CD38+ patients will have a rapidly progressive and aggressive disease, require earlier and more frequent treatments, and ultimately die sooner [3].

The aim of this work was to study CD305 and CD38 expression in CLL patients and its correlation with the clinical and prognostic criteria of the disease.


  Subjects and Methods Top


This is a prospective case–control study that included 54 patients. Forty CLL consecutive patients presented to Clinical pathology and Oncology Department, Menoufia University in the period between September 2014 to July 2016 with their age ranging from 41 to 83 years old and 14 age-matched and sex-matched apparently healthy controls.

The patients were classified into two groups; group I included 40 newly diagnosed CLL patients 19 female and 21 male, either discovered accidentally during routine laboratory investigations or presented with fatigue and fever with/without lymphadenopathy with/without organomegaly. All patients were subjected to complete history and clinical examination, abdominal, pelvic and chest computed tomography scan for detection of internal organs or lymph node involvement. Cases with persistent lymphocytosis of at least 5 × 109/l in the peripheral blood with blood film showing characteristically small, mature looking lymphocytes and smudge cells were further investigated to confirm diagnosis of CLL and to assess the tumor burden and prognosis by repetition of complete blood count and blood film, immunophenotypic scoring system (CD5, CD23, FMC7, CD22 and surface membrane immunoglobulin), estimation of lactate dehydrogenase (LDH) and β2 microglobulin levels, flow cytometric detection of CD305, CD19 and CD38 in peripheral blood. Group II included 14 age-matched and sex-matched apparently healthy controls. We took the consent of all the involved subjects and the approval of the local ethics committee.

Whole peripheral blood samples were collected, at the time of presentation and diagnosis, in an EDTA anticoagulant and processed within 24 h of collection.

Directly labeled monoclonal antibodies against the following antigens; CD305 FITC antibodies, CD19 PE and FITC antibodies, and CD38 PE antibodies (Abcam, Cambridge, Massachusetts, USA) were used. The isotype controls for surface antigens were simultaneously stained. To study the surface antigen expression, 20 μl of antibody was added to 100 μl of sample and incubated for 15 min After addition of 2 ml lysing solution (1: 10 dilution; BD Biosciences, San Diego, California, USA), the samples were incubated for another 15 min Then samples were centrifuged at 400g for 5 min The supernatant was discarded and the remaining pellet was washed twice with PBS and then resuspended in 500 μl PBS. Immunophenotyping expression was measured by (BD Biosciences, San Diego, USA) flowcytometer. We gated on CD19+/5 +. cells The cut-off value for CD 305 positivity was 30% [2].

Serum levels of β2M were determined using Microparticle Enzyme Immunoassay technology on (Cobas 6000 Analyzer; Roche Diagnostics, Indianapolis, Indiana, USA). Serum levels of LDH were determined using (Beckman AU480 Chemistry Analyser, Carlsbad, California, USA) and complete blood count were determined using (Sysmex XN1000; Kobe, Japan).

Results were collected, tabulated, statistically analyzed by IBM personal computer and statistical package for the social sciences computer program (version 16; SPSS Inc., Chicago, Illinois, USA). Descriptive data were expressed in percentage and quantitative data as mean ± SD and median. We used Mann–Whitney U-test to compare variables of not normally distributed data between two groups, Spearman correlation test to assess the relationship between variables in the same group. Receiver operating characteristic was also used to compare diagnostic tests by drawing a curve for each, the area under a curve represents the overall accuracy of a test; the larger the area, the better the test. The more close the curve to the upper left corner, the greatest the sensitivity and specificity. Binary Logistic regression was used to predict independent prognostic factors. P value considered nonsignificant difference if P value more than 0.05, significant difference if P value up to 0.05 and highly significant difference if P value less than 0.001.


  Results Top


In this study we found out that CD305 expression was significantly higher in patients compared to control group (mean ± SD of 38.80 ± 32.37 and 2.03 ± 1.07 respectively, P < 0.001) and CD38 expression was significantly higher in patients compared to control group (mean ± SD of 41.73 ± 31.32 and 2.01 ± 1.67 respectively, P < 0.001). Moreover a comparison between CLL patients and control group was done and showed a significant decrease in hemoglobin concentration and a highly significant decrease in platelet counts in CLL patients in relation to control, whereas white blood cells (WBCs), lymphocyte counts, LDH, β2 microglobulin, and CD305 and CD38 showed a highly significant increase in CLL patients in relation to control group (P < 0.001).

In this study, CD305 expression showed a highly significant positive correlation with both hemoglobin level and platelets count (P < 0.001). It also showed a positive correlation with lymphocyte doubling time (LDT); 23 out of 40 (57.5%) patients had LDT more than 12 months and were CD305 positive with mean value 60.83 ± 25.14 (P < 0.001) [Table 1].
Table 1: Correlation between CD305, CD38 with different parameters in cases group (n=40)

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However it showed a highly significant negative correlation with each of the following; WBCs, lymphocytes, β2 microglobulin, LDH (P < 0.001). It also showed a significant decrease with positive Coomb's test (17/ 40 patients) CD305 mean± SD value 14.94 ± 18.27 (P < 0.001) [Table 1].

On the other hand CD38 expression showed a highly significant positive correlation with WBCs, lymphocytes, β2 microglobulin, LDH and a significant increase with Coomb's test (17/ 40 patients) CD38 mean ± SD value 66.88 ± 17.88 (P< 0.001). However it showed a negative correlation with hemoglobin and platelets. It also showed a significant decrease with LDT >12months (23 /40 patients) CD38 mean± SD value 19.35 ± 20.54 (P < 0.001) [Table 1].

CD305 expression was significantly higher in Mod Rai stage 1 (low risk) than Mod Rai stage 3 (high risk) (mean ± SD 72.80 ± 14.41 versus 13.0 ± 13.01 respectively, P < 0.001). However, CD38 expression was significantly lower in Mod Rai stage 1 than Mod Rai stage 3 (mean ± SD 8.20 ± 4.94 versus 67.29 ± 12.41respectively, P < 0.001). Regarding cytotoxic treatment we found out that CD305 expression showed a highly significant decrease with patients who initiated chemotherapy 18/ 40 patients) during the study, CD305 mean ± SD value was 13.50 ± 15.51 (P < 0.001) [Table 2]. While CD38 expression showed a highly significant increase with those patients (mean± SD value was 63.67 ± 23.69) (P < 0.001) [Table 2].
Table 2: Relation between both CD305 and CD38 and chemotherapy treatment in cases group (n=40)

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We found that CD305 at a cut off value of up to 34 can predict the cases that will need chemotherapy with a sensitivity of 94.44% and specificity of 77.27% while CD38 at a cut off value of more than 40 can predict the cases that will need chemotherapy with a sensitivity of 88.89% and specificity of 81.82% [Table 3] and [Figure 1]. The current study depicted that CD 305 was significantly inversely correlated with CD 38 (P < 0.001) [Table 1], [Figure 2]. Finally, when we applied Binary Logistic regression we found that CD305 and CD38 are the factors that most independently can predict leukemia (P = 0.021, OR = 13.63 and P = 0.009, OR =2.464 respectively) [Table 4].
Table 3: Agreement (sensitivity, specificity and accuracy) for CD305 and CD38 to predict chemotherapy cases

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Figure 1: Receiver operating characteristic curve for CD305 and CD38 to predict chemotherapy cases.

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Figure 2: Flowcytometric picture showed positive expression of CD 305 and negative expression of CD 38

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Table 4: Binary Logistic regression to predict independent prognostic factors

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  Discussion Top


Chronic lymphocytic leukemia (CLL) has a clinical course which is highly variable. Some patients had a normal expectancy of life, while others need chemotherapy. Several clinical and biological variables when assessed at presentation can predict outcome.[2] The aim of this study was to evaluate CD305 expression in peripheral blood of newly diagnosed CLL patients and to correlate them with the clinical and prognostic criteria of the disease. Interestingly we found out that CD305 expression showed a highly significant positive correlation with hemoglobin level, platelets count and LDT >12 months. Conversely CD305 decrease significantly with advanced modified Rai staging which means that CD305 positive patients are found in earlier clinical stages (stage 1). Similarly, Rawstron et al. found that CD305 positive patients were presented in early stages than in advanced stages (stage 2 and 3).[4] Moreover Poggi et al, confirmed that its expression is lower in high risk patient.[5] As CD305 inhibit B-cell receptor (BCR)-mediated signaling and control kinase pathways of cell proliferation.[4]

Our study showed that CD 305 decreased significantly with positive Coombs' test. Similar results were obtained by Perbellini et al. [2], they found that a positive Coombs' test was detected more frequently in LAIR1 patients. It was reported that CD 305 inhibits the activation of immunological cells using two immunoreceptor tyrosine-based inhibitory motifs located in the cytoplasmic tail of the receptor.[4] Our study showed a significant decrease in CD305 expression and initiation of cytotoxic treatment similarly Perbellini et al. [2], found a significantly lower proportion of LAIR1+ patients who initiated cytotoxic treatment during follow-up compared to LAIR1 patients.

On the other hand, CD38 expression has a positive significant correlation with β2 microglobulin and a negative significant correlation with hemoglobin level while no significant correlation with both age and sex of patients. Similar results were obtained by Thornton et al. [6] and Ibrahim et al. [7], We also found that CD38 expression has a positive significant relationship with modified ray staging (Mod Rai stage 3) where CD38 expression is associated with more advanced clinical stage of the disease; similarly there was a positive significant relationship between CD38 expression and both WBCs and lymphocytes. These results were in accordance with those obtained by Del Poeta et al. [8]. While disaccord with those obtained by Ibrahim et al. [7], who found no significant correlation between CD38 and Rai staging, WBC count and number of circulating lymphocytes. An inverse relation between CD38 and LDT was found with 72% of CD38+ patient had a LDT less than 12 which was similar to Del Poeta et al. [8].

Our study also showed that 63.67% of CD38+ patients showed a positive significant increase with those who received chemotherapy. These results were in agreement with those of Del Poeta et al. [8] and Durig et al. [9], who revealed that CD38-positive patients were characterized by an aggressive clinical course and an advanced stage of the disease. They also showed poor chemotherapy response, short time to initiation of first treatment, and shorter survival. This is due to the ability of CD38 to shift the balance on the favor of survival/proliferation versus apoptosis.[9] Furthermore, we revealed an inverse relation between the expression of CD305 and CD38.[2] Owing to its action on the BCR activation pathway, CD305 is very attractive, since drugs targeting BCR signaling (e.g., Bruton tyrosine kinase inhibitors) have recently shown impressive activity in patients with CLL. [10]

After we correlated CD305 and Cd38 with many of the standard prognostic factors in CLL and from our multivariate analysis, we can conclude that CD305 and CD38 expression can be used as simple reliable, inexpensive independent prognostic factors in CLL patients and they can even predict at presentation patients who will initiate chemotherapy early.


  Conclusion Top


CD305 and CD38 expression can be used as simple reliable, inexpensive independent prognostic factors in CLL patients and they can even predict at presentation patients who will initiate chemotherapy early.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Machulla HK, Muller LP, Schaaf A, Kujat G, Schonermarck U, Langner J. Association of chronic lymphocytic leukemia with specific alleles of the HLA-DR4:DR53:DQ8 haplotype in German patients. Int J Cancer 2001; 92:203–207.  Back to cited text no. 1
    
2.
Perbellini O, Falisi E, Giaretta I, Boscaro E, Novella E, Facco M, et al. Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia. Haematologica 2014; 99:881–887.  Back to cited text no. 2
    
3.
Malavasi F, Deaglio S, Damle R, Cutrona G, Ferrarini M, Chiorazzi N. CD38 and chronic lymphocytic leukemia: a decade later. Blood 2011; 118:3470–3478.  Back to cited text no. 3
    
4.
Rawstron A, Shingles J, de Tute R, Bennett F, Jack AS, Hillmen P. Chronic lymphocytic leukaemia (CLL) and CLL-type monoclonal B-cell lymphocytosis (MBL) show differential expression of molecules involved in lymphoid tissue homing. Cytometry B Clin Cytom 2010; 78B:S42-S6.  Back to cited text no. 4
    
5.
Poggi A, Catellani S, Bruzzone A, Caligaris-Cappio F, Gobbi M, Zocchi MR. Lack of the leukocyte-associated Ig-likereceptor-1 expression in high-risk chronic lymphocytic leukaemia results in the absence of a negative signal regulating kinase activation and cell division. Leukemia. 2008; 22(5):980-8.  Back to cited text no. 5
    
6.
Thornton PD, Fernandez C, Giustolisi GM, Morilla R, Atkinson S, A'Hern RP, et al. CD38 expression as a prognostic indicator in chronic lymphocytic leukaemia. Hematol J 2004; 5:145–151.  Back to cited text no. 6
    
7.
Ibrahim S, Keating M, Do KA, O'Brien S, Huh YO, Jilani I, et al. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001; 98:181–186.  Back to cited text no. 7
    
8.
Del Poeta G, Maurillo L, Venditti A, Buccisano F, Epiceno AM, Capelli G, et al. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 2001; 98:2633–2639.  Back to cited text no. 8
    
9.
Durig J, Naschar M, Schmucker U, Renzing-Kohler K, Holter T, Huttmann A, et al. CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia. Leukemia 2002; 16:30–35.  Back to cited text no. 9
    
10.
Byrd J, Furman R, Coutre S, Flinn I, Burger J, Blum K, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med. 2013; 369(1):32-42.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

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



 

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