Abstract

Recently published data has identified the type-1 membrane glycoprotein CD200 as a potential therapeutic target in the treatment of CLL. CD200 has been reported to be expressed at significantly higher levels in CLL compared to normal mature B cells. CD200 has an immunosuppressive function and it’s expression by CLL cells may be involved in disease progression by exerting an immunomodulatory effect facilitating escape from host immune responses. In order to further evaluate the clinical and laboratory utility of this antibody the expression of CD200 has been assessed in a range of specimens. We have assessed CD200 expression in 41 CLL patients (36 peripheral blood and 5 bone marrow) and 12 normal samples (5 peripheral blood, 5 bone marrow, 2 lymph node biopsies). Biological features were assessed in 24 known CLL patients (male to female ration 3:1, median age 70 years), 15 untreated and 9 post treatment. Patients were defined as being poor risk if they were ZAP-70+, had deletions of p53, ATM and/or unmutated IgVH genes. Using these criteria 50% of cases were defined as good risk, and 50% as poor risk. Leucocytes were isolated from the specimens by ammonium chloride lysis and an aliquot of 1x106 cells were analysed by 6 colour flow cytometry using the FACS Canto and analysed using FACS Diva software (Becton Dickinson, UK). The level of expression of CD200 above an isotype control was reported for Lymphocytes, Monocytes, Granulocytes, Mature B cells, Germinal centre B cells, Plasma cells and CLL cells. Mean fluorescence intensities were also recorded for each of these groups (table 1). This data shows that CD200 is expressed above control levels by the majority of CLL cells, expression levels were not significantly different between patients with poor and good risk disease (median mfi 1921 and 1677 respectively p=0.42). CD200 was not increased in normal CD5+ B-cells. The very weak expression seen in non-CLL leucocytes may be an artefact of the lower background fluorescence of the control antibody relative to the CD200. Expression levels of CD200 relative to control were to the same magnitude as CD52 and several fold higher than CD20 expression levels, suggesting that CD200 is potentially an excellent target for immunotherapy. In conclusion our data shows that CD200 expression is uniformly higher in CLL than in any other peripheral blood or bone marrow leucocytes. The level of expression appears to be unaffected by prognostic risk factors, suggesting that treatments which target CD200 would be applicable in all patients with CLL. In addition, the lack of CD200 expression on normal leucocytes, particularly on T-cells, is potentially important. The uniformly increased expression of this molecule in CLL also makes this a potential marker for diagnostic and minimal residual disease assays.

Table 1.
Cell TypeMedian %CD200+ (range)Mean MFI
CD200 Expression in CLL is consistently higher than in normal leucocytes 
CLL 96.60 (65.2–100) 2773.23 
Mature B cells 23.89 (6.06–36.75) 69.29 
Germinal Centre B cells 35.60 (2.7–48) 871.86 
Plasma Cells 42.50 (2.8–80.9) 1939.5 
T cells 0.25 (0.11–0.48) 7.73 
Monocytes 3.65 (0.23–6.31) 31.86 
Granulocytes 4.47 (0.13–13.25) 34.63 
Cell TypeMedian %CD200+ (range)Mean MFI
CD200 Expression in CLL is consistently higher than in normal leucocytes 
CLL 96.60 (65.2–100) 2773.23 
Mature B cells 23.89 (6.06–36.75) 69.29 
Germinal Centre B cells 35.60 (2.7–48) 871.86 
Plasma Cells 42.50 (2.8–80.9) 1939.5 
T cells 0.25 (0.11–0.48) 7.73 
Monocytes 3.65 (0.23–6.31) 31.86 
Granulocytes 4.47 (0.13–13.25) 34.63 

Disclosures: Dr Hillmen is in receipt of consultancy fees from Alexion Pharmaceuticals Inc.; Dr Hillmen’s research has been supported by an unrestricted educational grant from Alexion Pharmaceuticals Inc.; Dr Hillmen is in receipt of speaker fees from Alexion Pharmaceuticals Inc.

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