Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic significance by Taghi Manshouri, Kim-anh Do, Xuemei Wang, Francis J. Giles, Susan M. O'Brien, Helen Saffer, Deborah Thomas, Iman Jilani, Hagop M. Kantarjian, Michael J. Keating, and Maher Albitar Blood Volume 101(7):2507-2513 April 1, 2003 ©2003 by American Society of Hematology

Western blot and immunoprecipitation demonstrating significant levels of cCD20 in the plasma of patients with CLL.(Panel A) Western blot showing detectable CD20 protein in plasma from patients with CLL. The plasma CD20 is similar in molecular weight to CD20... Western blot and immunoprecipitation demonstrating significant levels of cCD20 in the plasma of patients with CLL.(Panel A) Western blot showing detectable CD20 protein in plasma from patients with CLL. The plasma CD20 is similar in molecular weight to CD20 detected in cell lysates from BJAB cell line (BJ) and cells from a patient with CLL (C). As expected, no CD20 is detectable in lysate from the HL60 cell line (HL). Plasma from a healthy control individual (N) shows lower levels of CD20. Immunoprecipitation products from BJAB (B) and HL60 (H) cell lysates are also shown. Precipitation from beads and antibody without plasma or cell lysate is also shown as a negative control (NC). The molecular weight marker (M) is shown. One microliter of plasma is used in lanes that contain plasma. The levels of cCD20 detected in the plasma by ELISA are shown on the bottom. (Panel B) Coomassie blue-stained gel showing immunoprecipitation of CD20 from plasma of patients with CLL. The CD20 from cell lysates from BJAB (B) and CLL (C) are shown as positive control and from HL60 (H) as a negative control. Equal amounts of plasma (33 μL) from each sample were denatured before immunoprecipitation (abbreviation as in Panel A). Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Inhibition assay confirming the specificity of the ELISA assay in detecting CD20.Detection of CD20 in 2 plasma samples from patients with CLL was diminished when increasing amounts of purified CD20 were added to the detecting antibody (horseradish peroxidas... Inhibition assay confirming the specificity of the ELISA assay in detecting CD20.Detection of CD20 in 2 plasma samples from patients with CLL was diminished when increasing amounts of purified CD20 were added to the detecting antibody (horseradish peroxidase-labeled rituximab). Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Significantly higher levels of cCD20 were detected in patients with CLL than in healthy control individuals.Mean 1.00 and 1.96 standard errors (*SE) are shown. Significantly higher levels of cCD20 were detected in patients with CLL than in healthy control individuals.Mean 1.00 and 1.96 standard errors (*SE) are shown. Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

No evidence of ex vivo CD20 shedding No evidence of ex vivo CD20 shedding.(A) Supernatant from cultured cells of patients with CLL showed no significant increase with time regardless of whether the cells were cultured without or with a shedding agent (phorbol 12-myristate 13-acetate [PMA]). No evidence of ex vivo CD20 shedding.(A) Supernatant from cultured cells of patients with CLL showed no significant increase with time regardless of whether the cells were cultured without or with a shedding agent (phorbol 12-myristate 13-acetate [PMA]). In contrast, in HLA class I, levels increased significantly in the supernatant from cells cultured with PMA. (B) Similar levels of cCD20 were detected whether plasma samples were separated from cells immediately after collection (0) or after a prolonged period at room temperature (1, 2, 4, 8, 16, 24, 36, and 48 hours). Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Circulating CD20 levels correlate with clinical stages of CLL Circulating CD20 levels correlate with clinical stages of CLL.Levels of cCD20 correlated with Rai (A) and Binet (B) staging. Circulating CD20 levels correlate with clinical stages of CLL.Levels of cCD20 correlated with Rai (A) and Binet (B) staging. Mean 1.00 and 1.96 standard errors (*SE) are shown. Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Circulating CD20 levels correlate with survival Circulating CD20 levels correlate with survival.Kaplan-Meier curve showing significantly shorter survival times in patients with high levels of cCD20 (P = .01, log-rank test). Circulating CD20 levels correlate with survival.Kaplan-Meier curve showing significantly shorter survival times in patients with high levels of cCD20 (P = .01, log-rank test). Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Circulating CD20 is independent of Rai staging in predicting survival Circulating CD20 is independent of Rai staging in predicting survival.Classification tree showing that patients with advanced Rai stage disease separated into 2 groups: one with high cCD20 levels and shorter survival, the other with low cCD20 levels and rel... Circulating CD20 is independent of Rai staging in predicting survival.Classification tree showing that patients with advanced Rai stage disease separated into 2 groups: one with high cCD20 levels and shorter survival, the other with low cCD20 levels and relatively longer survival. The number in each node signifies the number of deaths per number of patients. RR is the relative risk (of death). Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology

Plasma cCD20 sequesters rituximab Plasma cCD20 sequesters rituximab.(A) Mixing study results demonstrating the capability of plasma cCD20 to compete for rituximab and prevent it from binding to CLL cells. Plasma cCD20 sequesters rituximab.(A) Mixing study results demonstrating the capability of plasma cCD20 to compete for rituximab and prevent it from binding to CLL cells. The top row shows an analysis of CD20 on the surface of CLL cells before and after rituximab was added alone or along with plasma. The lower row shows detection of the rituximab on the surface of CLL cells using the PE-labeled anti-Fc fragment of the mouse Ig. (B) Mixing study results using Raji cells and plasma from a patient with CLL and plasma from a healthy control individual. There is greater inhibition of rituximab binding to the Raji cells using plasma from the CLL patient with high cCD20 compared with plasma from the healthy control individual. Taghi Manshouri et al. Blood 2003;101:2507-2513 ©2003 by American Society of Hematology