CD200 Expression in Diagnostic and Prognostic Assessment of Mature B Cell Lymphophoproliferative Neoplasms

Background: Multiparameter flow cytometry is a useful tool for diagnostic evaluation of mature B-cell neoplasms (MBN). Recently, it has been shown that assessment of CD200 expression may improve the distinction between chronic lymphocytic leukemia (CLL; CD200 positive) and mantle cell lymphoma (MCL; CD200 negative), but any potential as a prognostic marker for CLL remains to be established. Materials and methods: This cross sectional study was conducted on sixty-seven patients newly diagnosed as having mature B-cell lymphoproliferative disorders Levels of CD 200 in lymphoma cells were assessed. Results: CD200 was consistently expressed in CLL and hairy cell leukemia B cells, but not in MCL cells. Heterogeneous expression was noted in other CD5 positive Non-Hodgkin lymphomas. High CD200 expression (≥50%) was associated with a higher CD5, 19 and CD23 expression, older age, higher TLC and absolute lymphocyte count, hepatomegaly, splenomegaly and a higher Rai stage. There were no significant correlations between CD200 expression and response to treatment. Conclusion: CD200 could be of high value in distinguishing CLL, MCL, and atypical CLL. CD200 expression can also be of prognostic and therapeutic value in CLL cases.


Introduction
The diagnosis of mature B cell lymphoproliferative disorders is based on immunophenotyping (IMP) (Van Dongen et al., 2012;Ivancević et al., 2014). Mantle cell lymphoma (MCL) is an aggressive disease in comparison to chronic lymphocytic leukemia (CLL) and requires intensive treatment. Sometimes MCL is misdiagnosed as CLL (Challagundla et al., 2014).
CLL is usually diagnosed by IMP being positive for CD23, having weak cell surface expression immunoglobulin (sIg), lacking expression of FMC and weak expression of CD20. MCL is usually CD23negative and CD20 strongly positive. However, in some cases, CD23 is expressed in MCL and is lacking in CLL leading to misdiagnosis (Barna et al., 2008).
A membrane glycoprotein expressed in all CD19+ B lymphocytes and some of the T is known as CD200. It is not expressed in natural killer cells, monocytes, granulocytes or platelets. This glycoprotein previously named OX2 is also expressed in myeloma plasma cells and acute myeloid leukemia (Moreaux et al., 2008).
Some studies have evaluated the role of CD200 expression in the differential diagnosis of CLL and other mature B cell lymphoproliferative disorders (Moreau et al 1997) Methods The diagnosis of mature B-cell neoplasm was established according to the WHO classification based on clinical data, and on morphologic, immunophenotypic, and genetic criteria whenever possible (Matutes and Polliack, 2000). All patients were subjected to complete history taking and Clinical examination.
Samples were considered positive for a marker if ≥ 20% of cells expressed that marker, except for CD38 positivity was considered ≥ 30% (Matutes and Polliack 2000).
The diagnosis of MCL was confirmed by immunohistochemical detection of cyclin D1 in tissue biopsies or detection of t(11;14) (q13;q32) by FISH or PCR.
Patients were followed up for at least 6 months. The Clinical Response to Treatment was monitored according to the criteria of response by the National Comprehensive Cancer Network (NCCN) (National Comprehensive Cancer Network 2016), and the European Society of Medical Oncology (ESMO) (Eichhorst et al., 2015)

Sample Collection
Two ml of PB collected on K-ethylene di amine tetra-acetic acid (K-EDTA) for CBC and preparation of Leishman-stained PB smears. One ml PB or BM sample on EDTA for immunophenotyping and flow cytometric detection of CD200. Bone marrow aspirates or PB samples were processed within 24 hours of collection, being preserved at room temperature.

Statistical Methods
Data was analyzed using IBM SPSS advanced statistics version 22 (SPSS Inc., Chicago, IL). Numerical data were expressed as mean and standard deviation or median and range as appropriate. Qualitative data were expressed as frequency and percentage. Chi-square test or Fisher's exact test was used to examine the relation between qualitative variables. For not normally distributed quantitative data, comparison between two groups was done using Mann-Whitney test (non-parametric t-test). Comparison between 3 groups was done using Kruskal-Wallis test (non-parametric ANOVA) then post-Hoc "Schefe test" was used for pair-wise comparison based on Kruskal-Wallis distribution. Spearman-rho method was used to test correlation between numerical variables. All tests were two-tailed. A p-value ≤ 0.05 was considered significant.

Results
The Present study included consecutive 67 Patients newly diagnosed Mature B-Cell neoplasms (MBN) who presented to the outpatient Clinic of National Cancer Institute, Cairo University during the period from March 2014 to December 2014. Another 20 healthy age matched normal volunteers as control subjects were also studied. All patients were followed up for a minimum period of 6 months after receiving medical treatment.

CD200 expression in the different disease groups
All CLL and HCL patients showed positive CD200 expression ( Figure.1aandb) whereas all MCL patients were negative for CD 200 (Figure 2). Among other groups of Non-Hodgkin Lymphoma, three patients (30%) were positive for CD200 (one patient with Low grade NHL, and two patients with mature B-Cell NHL with κ chain restriction).
The mean CD 200 Expression was 78.9% ( ± 20.74) in CLL, 2.65% (± 4.20) in MCL , 39.6 ( ± 23.55) in HCL and 21.9% (± 33.8)in other NHL group. There was a high statistical significant difference between the CLL patients and other types of NHL (p <0.001). The difference was between CLL on one side and MCL and NHL on the other side; no significant difference was encountered between the other groups.

Correlation Studies
Correlation study was done between the expression CD CD200 in Mature B Cell Lymphophoproliferative Neoplasms (p=0.169) (Table 3 and 4).

The relation of treatment response and CD 200 expression
Positive CD 200 expression in the study group was associated with 66.7% (36/54) of complete or partial remission and 46.2% (6/13) with Stationary or Progressive disease, with no significant difference between the two groups. The mean CD200 was 58.5±36.2 and 50.5±39.3 respectively with no significant statistical difference. Among CLL patients 68.4% (26/43) showed complete or partial response to treatment while 31.6% (12/43) showed no response. No patient with MCL showed a response to treatment. The majority of HCL (75%, 6/9) responded to treatment while 25%(3/9) did not respond. Most of the other B-Cell NHL responded to treatment (70%, 7/10) patients while 30% (3/10) showed no response in the form of stationary or progressive disease (Table 2).

CLL with unusual immunophenotyping (IMP)
CLL patients showed unusual IMP in 51.16% (22/43) of cases and all were positive for CD200. CD79b+ positive was present in 34.88% (15/43) of 200 and various Hematological and immunophenotyping data of patients. There was a fair positive correlation between the expression of CD 200 and total Leucocytic count with(r =0.512, p <0.001), and between the expression of CD200 and percent of peripheral blood lymphocytes (r =0.405, p =0.001). A week positive correlation between the expression of CD 200 and hemoglobin percent, CD5 expression and CD5, 19 coexpression with (r =0.248 ,p = 0.046), (r =0.282,p =0.021),(r =0.341, p=0.006) respectively. A good positive correlation was found between the expression of CD 200 and CD 23 expression with (r=0.646, p<0.001).

Comparison between CD 200 positive and negative groups
CD 200 positivity was significantly correlated with splenomegaly (P= 0.011), anemia (p= 0.023) and CD23% (P= <0.001). There was borderline relation to male gender (P=0.054).The response to treatment was more evident in CD200 expression (36/42, 66.7% vs 6/13, 46.2%) however this did not reach statistical significance    cases, CD23-negative in 3 cases (6.98%) and two (4.65%) of them were atypical CLL cases (score 3on the Matutes scoring system) CD22 bright B was present in one case (2.33%), FMC7+ positive in 4 cases (9.3%) and bright Surface membrane immunoglobulin (Sm Ig ) in one case (2.33%). There were three cases among the above showing 2 unusual IMP. CD79b+ was associated with FMC7+ in one case. The other 2 cases had CD23-negative, one combined with CD22 bright and the other with bright Sm Ig.

Response to treatment and different prognostic factors
Analysis of the response to treatment and different prognostic factors was done in the whole group (67 patients). This included clinical, hematological and immunophenotyping . They did not show any significant statiscal difference within any of them.
The high CD 200 group had significant male predominance, splenomegaly and hepatomegaly (p= >0.001, 0.02, 0.05 respectively).They also had more advanced stage in both the Rai and the Binet staging system (p = 0.05 and 0.003). The initial response to treatment was good but was not significant. (p= 0.45). After a median follow up of 30 month, this group was associated with earlier relapse ( three vs 25 month, p =0007) and on significant difference in survival (81% vs 83%, p= 0.34).

Discussion
In this study, we investigated whether the use of CD200 as a novel routine marker could be helpful to improve the utility of flow cytometry in differentiating between B lymphoproliferative neoplasms, particularly between CLL cases with typical or atypical immunophenotype and MCL cases and the possible prognostic significance of this marker in CLL patients and others.
CD200 expression managed to differentiate CLL cases including those with unusual immunophenotype (22/43, 51.2%) from MCL. It was consistently positive in all CLL and negative in all MCL. The median expression of CD200 expression in CLL and MCL was 87 and 1.3 respectively. This is consistent with other studies (Sandes et al 2013;Eldesoukey et al., 2012;Rodrigues et al 2016), supporting the important diagnostic role of CD200 especially for differentiating CLL cases with Matutes score less than or equal to 3 from MCL being an easy and not expensive method when compared to MCL confirmation by cyclin D1immunostaining on tissue biopsy, and/or detection of t (11;14) on MCL which are expensive, time consuming and not available in all centers .
In our study, all patients with hairy cell leukemia were CD200 positive with the highest intensity of expression among all our studied cases. These findings were consistent with previous reports (Van Dongen et al., 2012;Sandes et al 2013) suggesting this over expression could play a role in HCL survival, proliferation and the inhibition of anti-tumor immune response.
The role of CD 200 in the distinction of classical HCL, HCL variant and SMZL couldn't be demonstrated as we had no case of HCL variant in our study and due to small sample size of SMZL.
Among the six patients in CD5 positive B-Cell NHL group (non-CLL and non MCL-non PLL) CD200 expression was heterogeneous. Comparing CD200 expression between MCL and CD5 positive B-Cell NHL  Table 5. CD200 Expression Above and Below 50% in the CLL Group group showed no significant difference. These results were in agreement with Sandes et al., (2013) who suggested that the absence of CD 200 in the CD5 positive NHL group cannot be considered an exclusive finding to differentiate them from MCL. In this study CD200 expression showed good positive correlation with CD23 and weak positive correlation with CD5 and19 co-expression. There was fair correlation with the total leukocyte count and the peripheral blood lymphocytes and weak correlation with the haemoglobin . CD200 was not correlated with the clinical presentation of the patients however it showed higher percent of male gender, anemia and splenomegaly. In contrast to our results, El Desouky et al., (2012) found no correlation between CD200 expressions in other B-Cell neoplasms and clinical or laboratory data which could be attributed to larger sample size in our study.
We attempted to classify the CLL patients group based on CD200 expression into a high (≥50%) expression group and a low (< 50%) expression group and compare the clinical response between both groups. The high CD200 expression was associated with higher mean age, TLC, peripheral blood lymphocytes and absolute lymphocytic count. There was higher mean CD5, 19 coexpression and CD23 expression. It was also accompanied with significant splenomegaly, hepatomegaly (p= 0.02, 0.05 respectively). It was also associated with advanced stage on both Rai and the Binet staging system (p = 0.05 and 0.003). However the initial good response was accompanied by significant shorter time to treatment but did not have a statistical impact on the overall survival. This is in accordance with Wang et al., 2014 andWong et al., 2012 that revealed a possible impact of CD200 on prognosis and treatment in CLL patients. Also Miao et al., 2016 showed that the level of CD200 greatly affected time to progression ( two vs 28months, p= 0.0008) but did not affect the overall survival (p= 0.2379).
The study was limited by the small number of patients and short follow up. Further studies should include survival analysis to determine the proper prognosis of these patients.
We conclude that High CD200 ≥50 was associated with advanced stage and earlier time to progression. The study recommends the addition of CD200 as a routine investigatory marker in the flowcytometry panel of chronic lymphoproliferative disorders to distinguish between their subtypes especially MCL and atypical CLL with unusual IMP. Blockade of CD200 should be explored in the treatment of CLL and HCL as it may improve prognosis.

Disclosure and conflict of interest
The study did not receive any fund. There was no conflict of interest.

Compliance with Ethical Standards
The study was approved by the institutional review board and the ethical committee. All the patients had to give an informed consent before being recruited.
This was in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.  CD 200 Expression in CLL patients had a mean ± SD of 78.9 ± 20.74 , In MCL a mean ± SD of 2.65 ± 4.20, In HCL a mean ± SD of 39.6 ± 23.55 and in other NHL group a mean ±SD of 21.9 ± 33.8 with a highly significant statistical difference ( P < 0.001).This difference was observed between the CLL patients and those with MCL and other types of NHL (p <0.001) while no significant difference was found between MCL patients and other NHL as regards CD200 expression.