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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 2  |  Page : 42-46

Flow cytometric evaluation of CD200 as a tool for differentiation between chronic lymphocytic leukemia and mantle cell lymphoma


1 Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Clinical Hematology and Bone Marrow Transplant Unit, Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission23-Jan-2014
Date of Acceptance23-Jan-2014
Date of Web Publication30-Aug-2014

Correspondence Address:
Mohamed T.H. Sallam
Lecturer of Clinical Pathology, Faculty of Medicine, Ain Shams University, Abassya, 11556, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.139754

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  Abstract 

Background In the majority of cases, flow cytometry enables the differentiation of chronic lymphocytic leukemia (CLL) from mantle cell lymphoma (MCL). However, the diagnosis of CLL becomes challenging when CD23 is not expressed by the leukemic cells or in cases of MCL expressing CD23. CD200 is a membrane glycoprotein expressed on a subset of T and B lymphocytes. Its expression has been observed on human myeloma, plasma cells, and CLL cells.
Aim of the work We investigated the pattern of expression of CD200 in CLL and MCL patients, aiming to clarify its possible role in differentiating these often overlapping disorders.
Patients and methods This study was carried out on 30 patients with newly diagnosed CLL and 10 patients with MCL. Flow cytometric immunophenotyping was performed using the CD200 monoclonal antibody in addition to the standard panel of chronic lymphoproliferative diagnosis.
Results CD200 was expressed in 100% of CLL cases with moderate intensity compared with only 10% of MCL cases with low intensity.
Conclusion CD200 is a powerful addition to the routine flow cytometric panel in the differentiation between CLL and MCL with high sensitivity and specificity.

Keywords: CD200, chronic lymphocytic leukemia, flow cytometry, mantle cell lymphoma


How to cite this article:
El-Sewefy DA, Khattab DA, Sallam MT, Elsalakawy WA. Flow cytometric evaluation of CD200 as a tool for differentiation between chronic lymphocytic leukemia and mantle cell lymphoma. Egypt J Haematol 2014;39:42-6

How to cite this URL:
El-Sewefy DA, Khattab DA, Sallam MT, Elsalakawy WA. Flow cytometric evaluation of CD200 as a tool for differentiation between chronic lymphocytic leukemia and mantle cell lymphoma. Egypt J Haematol [serial online] 2014 [cited 2017 Dec 12];39:42-6. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/42/139754


  Introduction Top


Flow cytometric immunophenotyping is the preferred method for the characterization and classification of chronic lymphoproliferative disorders (CLPDs) [1] .

Chronic lymphocytic leukemia (CLL) is the most common CLPDs. It characteristically expresses CD5 and CD19 on the cells surface. The differential diagnosis of CD5/CD19 dual-positive CLPDs lies mainly between CLL and mantle cell lymphoma (MCL). MCL has a significantly poorer prognosis than CLL, often requiring more aggressive treatment; thus, it is of clinical importance to distinguish the two [2],[3],[4].

In the majority of cases, CLL is easily distinguished from MCL on immunophenotype alone as it usually expresses CD23, has weak cell surface expression of immunoglobulin (sIg), and lacks expression of FMC7. In contrast, MCL most commonly has the phenotype CD5+, CD23-, FMC7+, and sIg strong [5] . In addition, CD20 expression is strong in MCL and weak in CLL when compared with normal B lymphocytes [6] .

However, among CLL and MCL cases lies a gray zone where CLL cells may lack the expression of CD23 [7] or MCL cases expressing CD23 on the surface of their leukemic cells, making the diagnosis of these disorders challenging [8] . Thus, flow cytometry alone cannot be relied upon to differentiate these two disorders and demonstration of t(11;14)(q13;q32) remains the gold standard of MCL diagnosis [1] .

CD200 (previously referred to as OX2) is a membrane glycoprotein belonging to the immunoglobulin superfamily that seems to play an immunosuppressive role. It is expressed on a subset of T and all CD19+ B lymphocytes, but not on natural killer cells, monocytes, granulocytes, or platelets. Its expression has also been reported on human myeloma, plasma cells, and CLL cells [9] .

The aim of this study is to carry out a comprehensive flow cytometric immunophenotypic analysis of CLL and MCL using the CD200 monoclonal antibody to provide data on the pattern of expression of this marker, in an attempt to resolve the immunophenotypic overlap, and hence clarify the possible role of CD200 in the precise diagnosis of these overlapping disorders.


  Patients and methods Top


Patients

The present study was carried out on 40 newly diagnosed adult patients of CLPDs; they were classified into 30 patients with CLL and 10 patients with MCL who attended the Haematology/Oncology Unit of Ain Shams University hospitals during the period from September 2011 to August 2012. A written consent was obtained from all patients. An approval from the local ethical committee was obtained.

Methods

Patients were classified into CLL and MCL subgroups according to the WHO classification of mature B-cell neoplasms [4] . Routine diagnostic workup of cases includes a clinical examination, complete blood counts on a Coulter LH 750 Cell Counter (Coulter Electronics, Hialeah, Florida, USA), bone marrow examination, and lymph node biopsy. The diagnosis of MCL was confirmed by the demonstration of Bcl-1 expression by immunohistochemistry (Dako, Carpinteria, California, USA) or t(11;14) by fluorescence in-situ hybridization.

Flow cytometry

A total of 100 μl of EDTA or heparin anticoagulated peripheral blood or bone marrow aspirate specimens were washed using PBS (Sigma, St. Louis, Missouri, USA). Samples were resuspended in 1 ml PBS and 50 μl aliquots were pipetted into each of the test and control tubes. Samples were then incubated for 15 min with fluorochrome-conjugated antibodies protected from light. These antibodies included CD200, CD19, CD5, CD10, CD11c, CD20, CD22, CD23, CD38, CD79b, FMC7, SmIg, and κ and λ light chains (all supplied by Beckman Coulter, Fullerton, California, USA; except CD200, which was supplied by R&D Systems, Minneapolis, Minnesota, USA). All antibodies were fluorescein isothiocyanate labeled, except CD19, CD10, CD11c, CD38, CD79b, and CD200, 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. Red blood cells were then lysed with ammonium chloride lysis buffer, washed with PBS, and resuspended in PBS. Tubes were vortexed and then analyzed using a Coulter Epics XL Flow Cytometer (Coulter Electronics).

Statistical analysis

Statistical analysis of the data was carried out using the SPSS 15.0 software package (SPSS Inc., Chicago, Illinois, USA) under a Windows XP operating system. Categorical data parameters were presented in the form of frequency and percentage and analyzed for group differences using the χ2-test or the Fisher exact test (χ2 value) according to the nature of the data. Continuous data parameters were analyzed for normality using the Shapiro-Wilk test; accordingly, central tendency of the data was presented in the form of mean for normally distributed data or median for a nonparameteric distribution. Comparative analysis was carried out using the Mann-Whitney U-test (Z value) for two independent samples, the Wilcoxon signed rank test (Z value) for paired samples, and analysis of variance in association with Fisher's least significant difference for multiple samples. Groups were assumed to differ significantly when the P value was less than 0.05 and highly significant when the P value was less than 0.001. Nonsignificant difference was assumed if the P value was greater than or equal to 0.05. Graphic presentation of data was performed using MS Excel 2007 software.


  Results Top


Patients

To investigate the value of CD200 expression as a basis to exclude MCL, this study included 30 patients with CLL and 10 patients with MCL fulfilling the criteria described. A male predominance was noted among patients with MCL and with CLL/small lymphocytic lymphoma (SLL). In the CLL group, the mean age in years was 57.7 ± 8.1 compared with 62.4 ± 9.9 in MCL (P = 0.143).

Patients' diagnosis and confirmatory studies

The diagnosis of cases was made on the basis of the WHO classification of hematologic neoplasm guidelines [4] . For MCL patients, the morphology was of small cell type in 80% of cases whereas the other 20% were the blastoid variant. No immunophenotypic differences were detected between these two morphologic variants of MCL. All MCL cases included in the study had documented evidence of the t(11;14) by conventional karyotyping or fluorescence in-situ hybridization studies and/or had positive immunohistochemical results for cyclin D1 on neoplastic cells [Figure 1] and [Figure 2].
Figure 1: Typical case of mantle cell lymphoma. (a) Morphology in peripheral blood (PB) smears (×1000); (b) H & E-stained trephine biopsy sections (×100); (c) positive t(11;14) by the fluorescence in-situ hybridization technique; (d) cyclin D1 nuclear staining by immunohistochemistry on bone marrow biopsy sections (×400).

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Figure 2: Morphologic variants of mantle cell lymphoma (×1000). (a) Small cell type; (b) blastoid type.

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Flow cytometric studies

The lymphocytes in the CLL group expressed pan B-cell markers, CD19 (100.0%), CD20 (90.0%), and CD22 (47.0%). CD79b was negative in 63%; positive cases showed low expression. In the MCL group, the pan B-cell markers were expressed in 100% of cases. No statistically significant differences were detected between the two groups [Table 1] and [Figure 3].
Table 1: Statistical comparative study of immunophenotyping data between chronic lymphocytic leukemia and mantle cell lymphoma groups

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Figure 3: Comparative study of immunophenotyping data in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).

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In the CLL group, CD23 was expressed in 100% of cases and FMC7 was not expressed in any of our CLL cases compared with the MCL group, where CD23 was expressed in 20% of cases and FMC7 was expressed in 90% (P < 0.001) [Table 1] and [Figure 3].

Predominant κ surface light chain restriction was observed in 75.3% of our CLL cases compared with 67.3% λ light chain restriction in our MCL cases (P = 0.015) [Table 1].

CD200 was expressed by 100% of our CLL cases compared with 10% of MCL cases. The median percent expression was 90.65% (range 47.2-99.2%), with an MFI 8.865 (range 2.18-20.6) in CLL compared with 6.87% (range 1.1-37.2%), with an median fluorescence intensity (MFI) 1.775 (range 1.24-3.45) in MCL cases (P < 0.001) [Table 2].
Table 2: Percent positivity of the CD200 marker in the chronic lymphocytic leukemia and mantle cell lymphoma groups

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The cut-off value of CD200 percent expression on malignant clone cells in both groups was 30.43%, where it had 100% sensitivity, 90% specificity, 96.77% positive predictive value, and 100% negative predictive value [Table 3] and [Figure 4] and [Figure 5].
Table 3: Analytical criteria of CD200 (%)

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Figure 4: Diagnostic performance of CD200 expression in chronic lymphocytic leukemia and mantle cell lymphoma.

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Figure 5: Cut-off value of CD200 (%) in chronic lymphocytic leukemia and mantle cell lymphoma.

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  Discussion Top


CLL and MCL are CD5+ mature B-cell neoplasms that generally differ significantly in terms of prognosis. CLL tends to follow an indolent course, with median survival ranging from 95 to 293 months depending on immunoglobulin gene mutational status. In contrast, MCL becomes more clinically aggressive and treatment-refractory over time [10] , with a median survival of 36 to 60 months. Therefore, differentiation of these entities has important prognostic and therapeutic implications [4],[11] .

The characteristic immunophenotypic features of CLL are aberrant expression of CD5, together with CD23 expression and the negativity of CD79b and FMC7. This is in contrast to MCL, who also express CD5 and lack CD23 expression and strongly express FMC7 [12] .

The most common useful markers in their differentiation are CD23 and FMC7 [8],[13],[14],[15]. MCL is generally CD23- and FMC7+, with CLL showing the opposite pattern. When aberrant phenotypes occur, flow cytometric differentiation of CLL from MCL can be difficult [14] . In our study, all of our CLL cases were positive for CD23 whereas only 20% of our MCL expressed CD23. Previous reports of CD23 positivity in CLL ranged from 86.6 to 100% of cases [7],[16],[17]. In a review by Schlette et al. [18] , CD23 expression was identified in up to 45% of MCL cases. Similarly, Barna et al. [7] reported CD23 expression in six (43%) of 14 MCL cases.

FMC7 was not expressed by any of our CLL cases compared with 90% positivity among MCL cases. Previous studies reported FMC7 positivity of 0-12% in CLL cases [17],[19],[20],[21] and 90-100% in MCL [17],[22] .

CD79b is another useful marker that can help distinguish CLL from other B-cell neoplasms as most cases of CLL are CD79b negative or show low expression and the majority of other types of B-cell neoplasms are CD79b positive [5] . This is consistent with our study, where none of our CLL cases showed CD79b expression. The frequency of CD79b expression in CLL has been reported to vary from 5 to 45% [5],[19],[22],[23]. However, CD79b was expressed in every patient with MCL we studied.

Other phenotypes, such as CD20 bright /sIg dim or CD20 dim /sIg bright , may also enable the differentiation of CLL and MCL cases. CD20 showed a highly significant increase in the percent expression and MFI in the MCL group compared with the CLL group (P = 0.001). This is consistent with previous reports [21],[24],[25].

In terms of light chain restriction, our study found κ light chain predominance in the CLL group and λ chain predominance in the MCL group (P = 0.001). This is in agreement with previous reports that l light chain restriction is more commonly reported than κ light chain restriction in MCL compared with CLL [26],[27],[28],[29] .

In this study, we investigated the expression of CD200 on neoplastic cells of patients with CLL and MCL. CD200 was expressed in 100% CLL cases with moderate intensity compared with 10% in MCL cases with low intensity (P = 0.001). CD200 positivity showed a positive predictive value of 96.77%, a negative predictive value of 100%, a sensitivity of 100%, and a specificity of 90%.

We have confirmed previous reports that CD200 is expressed uniformly in CLL, whereas its expression is not detected in MCL. Alapat et al. [30] examined CD200 expression on 102 CLL and 58 MCL cases. CD200 was expressed on all of their CLL cases whereas none of their MCL expressed CD200. Another group examined CD200 expression on paraffin-embedded bone marrow and lymph node sections in 23 CLL and 44 MCL cases. Again, all CLL cells were CD200+ whereas all MCL cells were negative [14] .

Thus, in our series, there is a clear-cut difference between MCL cells, in which CD200 is not expressed versus CLL, with high expression as reported by other authors. Therefore, the inclusion of CD200 in flow cytometry and immunohistochemistry routine panels could be very useful in distinguishing between these two entities, in particular, in patients with a prevalent leukemic expression and inconclusive phenotypes.

However, if the treatment strategy, in CLL, is targeted against CD200 itself (anti-CD200 antibodies) would it decrease the activity of the disease? It seems reasonable to achieve this in the near future, whether using anti-CD200 alone or in combination with other B-cell cytotoxins, cancer vaccines, or other immunostimulatory therapies.


  Acknowledgements Top


 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


This article has been cited by
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International Journal of Laboratory Hematology. 2017;
[Pubmed] | [DOI]



 

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