Discussion and Conclusions Acknowledgements and References Correlating FMC7 and CD20 expression in B cell malignancies Jamie Arberry1, Toby A. Eyre2,3, Wale Atoyebi2, Kevin Leyden2, Ashley Cooper2, Chris S. Hatton2 1. Oxford University Medical School, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom 2. Department of Haematology, Churchill Hospital, Oxford, OX3 7LE, United Kingdom 3. Early phase clinical trial unit, Churchill Hospital, Oxford, OX3 7LE, United Kingdom 4. Department of Haematology, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom Aims and Objectives Methods To compare CD20 and FMC7 expression in different subtypes of B cell lymphoproliferative disorder (B-LPD) We collected flow cytometry data from the peripheral blood, bone marrow, pleural fluid or cerebrospinal fluid of 419 patients with CLL or NHL. We gated the relevant populations and calculated the mean fluorescence intensity (MFI) of CD20 and FMC7. These were labelled with PerCP-cy5.5 (clone L27), and FITC respectively (Becton Dickinson). We calculated Spearman’s rank correlation coefficient for all CLL and NHL-subtype samples, to assess the strength of the CD20-FMC7 association. Introduction CD20 is an important therapeutic target of B cell malignancies and the monoclonal antibody FMC7 detects a conformational epitope on the CD20 molecule: The presence of this FMC7 antigen has been confirmed in studies where it was strongly expressed on CD20-transfected non-B cells1,2. The relationship between the expression of CD20 and FMC7 has been noted in three previous studies of B-LPD using flow cytometry. One of these assessed samples using chi squared and Fisher exact tests3, and the other two analysed their samples with scatter graph correlation1,4, but with significantly fewer samples. Only one of these studies has noted a difference in CD20-FMC7 correlation between chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) subtypes. Therefore, we compared CD20 and FMC7 expression using scatter plots in different subtypes of B-LPD. Figure 1 Flow cytometry data from a case with FMC7 positivity, demonstrating gating of appropriate population, and calculation of the MFI from a histogram Results Figure 3 Comparison of CD20 MFI to FMC7 MFI in all patients with a B cell clone that wasn’t typical CLL The majority of the cases we collected were CLL (n=251) and the remainder consisted of other forms of B cell NHL. We have considerably more cases of LPL/MZL than previously reported (Table 1). Most of the samples used for flow cytometry were peripheral blood samples (Table 1), which is the most relevant and applicable sample type for studying lymphoproliferative disorders. We assessed the relationship between CD20 and FMC7 MFI in a variety of different cases: Across all cases, we found a strong correlation (Figure 2, Table 2). Across CLL cases compared to non-CLL NHL cases, we found the relationship was more pronounced among the non-CLL NHL patients (Figure 3, Table 2). A relationship still existed among the samples from the CLL patients (Figure 4, Table 2), but it was much less evident due to the lack of FMC7 positivity. Disease Number (%) CLL 251 (59.9) Non-CLL B cell disorders 168 (40.1) - Mantle Cell Lymphoma/Atypical CLL 47 (11.2) - Follicular Lymphoma 9 (2.1) - Hairy Cell Leukaemia 13 (3.1) - Lymphoplasmacytic/marginal zone lymphoma 55 (13.1) - Splenic lymphoma with villous lymphocytes 22 (5.3) - Other Sample Type Peripheral Blood 352 (84.0) Bone Marrow 54 (12.9) Pleural Fluid 10 (2.4) Cerebrospinal Fluid 3 (0.7) Figure 4 Comparison of CD20 MFI to FMC7 MFI in all patients with typical CLL Table 1 Diagnosis of B cell lymphoproliferative disorder and type of sample used for flow cytometry Figure 2 Comparison of CD20 MFI to FMC7 MFI in all patients with a B cell lymphoproliferative disorder Across each subtype separately, in order to determine how the relationship between CD20 and FMC7 varies (Table 2). Some subtypes (e.g. follicular lymphoma, lymphoplasmacytic lymphoma/marginal zone lymphoma and mantle cell lymphoma/atypical CLL) demonstrated a much stronger correlation than others (hairy cell leukaemia and splenic lymphoma with villous lymphocytes). Disease Spearman’s rank correlation coefficient (p value) All samples 0.832 (p<0.0001) CLL 0.640 (p<0.0001) Non-CLL B cell disorders 0.821 (p<0.001) - Mantle Cell Lymphoma/Atypical CLL 0.807 (p<0.001) - Follicular Lymphoma 1.000 (p<0.001) - Hairy Cell Leukaemia 0.604 (p=0.032) - Lymphoplasmacytic/marginal zone lymphoma - Splenic lymphoma with villous lymphocytes 0.521 (p=0.014) Discussion and Conclusions 1. Serke, S., Schwaner, I., Yordanova, M., Szczepek, A. & Huhn, D. Monoclonal antibody FMC7 detects a conformational epitope on the CD20 molecule: evidence from phenotyping after rituxan therapy and transfectant cell analyses. Cytometry 46, 98-104 (2001). 2. Deans, J. P. & Polyak, M. J. FMC7 is an epitope of CD20. Blood 111, 2492; author reply 2493-2494, doi:10.1182/blood-2007-11-126243 (2008). 3. Delgado, J. et al. Diagnostic significance of CD20 and FMC7 expression in B-cell disorders. Am J Clin Pathol 120, 754-759, doi:10.1309/FNGC-YEMJ-E3MA-E5L2 (2003). 4. Hübl, W., Iturraspe, J. & Braylan, R. C. FMC7 antigen expression on normal and malignant B-cells can be predicted by expression of CD20. Cytometry 34, 71-74 (1998). Acknowledgements and References Table 2 Strength of association between FMC7 and CD20 for each B-LPD Our data clearly demonstrates a correlation between FMC7 and CD20. It shows that FMC7 becomes more readily apparent at higher levels of CD20 expression, explaining why the relationship is much stronger in lymphomas where populations of B cells have higher fluorescence intensities. The association is much weaker in CLL because the MFI of CD20 tends to be relatively low. Such data may have some relevance with regards to the modern era of WHO classification of lymphoproliferative disorders, which wasn’t as clear cut when other studies regarding the CD20-FMC7 relationship were carried out. It could also have further implications with regards to treatment of these conditions with biological therapies.