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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-06-1639.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Hematopathology, Biostatistics,
and Leukemia, The University of Texas M. D. Anderson Cancer
Center, Houston, TX.
CD20 is a 33- to 36-kDa transmembrane phosphoprotein involved in
the activation, proliferation, and differentiation of B lymphocytes. The predicted amino acid sequence of the CD20 suggests 4 transmembrane-spanning regions with both N- and C-termini located in
the cytoplasm. We demonstrate herein that significant levels of
circulating CD20 (cCD20) can be detected in the plasma of patients with
chronic lymphocytic leukemia (CLL) and that cCD20 interferes with the binding of rituximab, a humanized anti-CD20 monoclonal antibody, to CLL
cells. An enzyme-linked immunosorbent assay (ELISA) was developed to
measure circulating cCD20 levels in the plasma. We measured cCD20
levels in the plasma of 180 patients with CLL and correlated these
levels with clinical characteristics and outcome. Circulating CD20
levels correlated positively with CD20, also called B1 (Bp35), is a phosphoprotein
detected on the surface of B lymphocytes1-3 and believed
to play a major role in the regulation, activation, proliferation, and
differentiation of these cells.4-6 The gene for CD20 has
been mapped to human chromosome 11 at position q12-q13, centromeric to
the Bcl-1 locus, which is involved in the translocation
t(11;14)(q13;q32).7,8 The CD20 gene codes for a protein
that varies in molecular weight from 33 to 36 kDa, secondary to
alternative splicing at the 5' end and to differences in
phosphorylation.1 Higher levels of CD20 phosphorylation
have been reported in proliferating malignant B cells than in
nonproliferating B cells.1 Although the function of the
CD20 protein is not well defined, it has been suggested that CD20
regulates transmembrane calcium conductance.5 Activation of CD20 by binding to antibodies directed toward the extracellular portion of CD20 leads to tyrosine kinase pathway activation and modulation of cell cycle progression via interaction with src-related kinases.9-11 Sequence analysis of the CD20 molecule showed
4 transmembrane domains with N- and C-termini in the cytoplasm without
evidence of shedding.6 It has been reported, however, that
CD20 relocalizes into a detergent-insoluble membrane compartment upon
binding to antibodies.12 Binding of the humanized
monoclonal antibody rituximab to CD20+ cells leads to their
death via complement-dependent cellular cytotoxicity or
antibody-dependent cellular cytotoxicity.13-19 Use of
rituximab in the treatment of B-cell malignancies has led to an
improvement in outcome, and the use of this antibody is expanding
rapidly.13-19 The optimization of the dosing and
scheduling of rituximab could potentially increase its efficacy.
Several investigators have documented variations in intensity of CD20 expression on the surface of malignant B cells in different
lymphoproliferative diseases and have suggested that these variations
may influence binding and efficacy of rituximab
therapy.20-22 We investigated the possibility that
circulating CD20 was present in plasma; binding of circulating CD20 to
rituximab could potentially influence efficacy. We used
immunoprecipitation, Western blot, and novel immunoassays to
investigate the presence and levels of circulating CD20 (cCD20) in the
plasma of healthy individuals and patients with chronic lymphocytic
leukemia (CLL). Furthermore, we examined the correlation between plasma
levels of cCD20 and the clinical features of the disease and prognosis.
Patient and specimens
Western blot analysis of plasma
Immunoprecipitation of CD20 Mouse anti-CD20 (catalog no. SC-7733, lot no. K011; Santa Cruz Biotechnology, Santa Cruz, CA) was attached to protein G beads using the immunoprecipitation kit from Pierce (catalog no. 45210; Rockford, IL) as recommended by the manufacturer. Plasma (200 µL) from each subject was mixed with 10 µL of 0.1 M dithiothreitol (DDT) and then incubated with protein G-conjugated antibody. The conjugated protein G-cCD20 was washed extensively with 25 mM Tris (tris(hydroxymethyl)aminomethane), 0.5 M NaCl buffer (pH 7.2) and then eluted from the column using 30 µL ImmunoPure solution, pH 2.8 (Pierce). The eluted protein (5 µL) was resolved by 9.5% SDS-polyacrylamide gel, stained with 0.25% Coomassie blue R-250 (Sigma-Aldrich, St Louis, MO), and destained as recommended by the manufacturer.Plasma CD20 enzyme-linked immunosorbent assay We used an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory to measure plasma CD20 levels. Briefly, a 96-well polystyrene microplate was coated with 100 µL rabbit antigoat immunoglobulin at a concentration of 1 µg/mL in 0.05 M carbonate buffer (pH 9.5). Plates were incubated for 6 hours at room temperature and then washed with PBS containing 0.01% Tween 20. Each well was blocked with 250 µL of 2% BSA in PBS containing 0.1% Tween 20 for 2 hours at 37°C. Goat anti-CD20 (1 µL) was then added to each well, and the microplate was incubated overnight at 4°C. After a wash in PBS containing 0.1% Tween 20, 100 µL plasma was added to each of the wells, which were then incubated for 3 hours with constant shaking at room temperature. The plates were again washed in PBS containing 0.1% Tween 20 and then incubated for 3 hours with 20 µL horseradish peroxidase-conjugated humanized anti-CD20 antibody (rituximab) at a dilution of 1:400 in 2% BSA, 0.1% Tween 20. Horseradish peroxidase-enzyme conjugation of the rituximab was carried out by using standard technique. The plates were incubated for another 3 hours. The wells were then washed 6 more times with PBS containing 0.1% Tween 20. Substrate was added (100 µL) to develop the color. The plates were incubated with constant shaking for 15 to 30 minutes. The reaction was stopped with 50 µL of 2 N HCl, and the plates were read at the 450 nm wavelength. Serial dilution of the known number of molecules of synthetic CD20 peptide (catalog no. SC-7703; Santa Cruz Biotechnology) was used to generate a standard curve. For confirmation of specificity of the detected CD20, we used the same CD20 peptide or the CD20 protein precipitated from BJAB cell line in blocking experiments. Adding an increasing amount of CD20 from BJAB cells or peptide demonstrated increasing blocking of the cCD20 detection by the ELISA.Human histocompatability class I ELISA An established ELISA assay for the human leukocyte antigen (HLA) class I was used to analyze HLA-1 in the supernatants of tissue culture. Briefly, rabbit antimouse immunoglobulin (Sigma-Aldrich) was used for capturing the monoclonal anti-HLA class I antibodies (clone W6; Sigma). Plates were washed with PBS containing 0.01% Tween 20; 100 µL of 1:50 diluted medium in PBS containing 0.01% Tween 20 was added to each well. Horseradish peroxidase-labeled anti- 2-microglobulin (Dako, Glostrup, Denmark)
was used for detection. The standard curve was prepared using purified
HLA class I protein (B7 Calabratoer; SangStat Medical). Substrate was
added, and plate color intensity was evaluated at 450 nm within 15 minutes.
Cell culture and quantification of cCD20 Cells from patients with CLL were maintained in RPMI 1640 medium containing 10% fetal calf serum at 5% CO2. Phorbol 12-myristate 13-acetate (PMA) (Sigma) was used to induce shedding. PMA was added to duplicate cultures at a concentration of 10 nM/L.Competition experiments and blocking of rituximab binding We performed several mixing studies to test the ability of circulating cCD20 to bind to rituximab and the effects of that binding on cells. We added 1 µg rituximab to 1 million cells isolated from patients with CLL or Raji cells. Flow cytometry and phycoerythrin (PE)-labeled anti-CD20 antibodies were used to monitor rituximab's effect on the CD20 binding sites on the surface of cells. The anti-Fc fragment of mouse immunoglobulin (Ig; Jackson Immuno Research Laboratories, West Grove, PA) was used to detect rituximab on the surface of cells and anti-CD19 antibody to detect B cells. Rituximab binding to cells was also analyzed after cells were mixed with 100, 300, and 500 µL of plasma or after a similar amount of buffered saline. If plasma obtained from a different patient was added to CLL cells or Raji cells, we heated the plasma for 30 minutes at 56°C to inactivate the complement pathway.Statistical analysis The correlations between cCD20 and other covariates were calculated using Spearman rank correlation coefficients. The Wilcoxon rank sum test or Kruskal-Wallis test was used to compare cCD20 levels among categorical variables such as Rai stage and sex. Probabilities of survival were estimated by the method of Kaplan and Meier.24Unadjusted between-group comparisons of survival were made using the log-rank test.25 All scatter plots were smoothed using the Loess method of Cleveland, with predictive variables transformed as appropriate on the basis of these plots.26 The Cox proportional hazard regression model was used to assess the ability of clinical characteristics in predicting survival, with goodness of fit assessed by Martingale residual plots and likelihood ratio statistics.27 The cut point was chosen from univariate analysis using RPART.28 This coincided with the largest change in the relative risk of death suggested by a Martingale residual plot. The procedure of RPART works as follows: It starts from a cut point that best splits the data into 2 groups. This process is then applied separately to each subgroup and continued recursively until either the subgroups reach a minimum size or no further improvement can be made. Multivariate Cox proportional hazard models were then fitted to evaluate these predictors simultaneously. Recursive partitioning was implemented to find cut points for cCD20 or Rai staging based on Martingale residuals. All statistical analyses were carried out using Splus29 (Insightful, Seattle, WA) and CART (Classification and Regression Tree) packages (Statcon, Witzenhausen, Denmark).30 P values less than .05 were considered statistically significant.
Detection of CD20 in plasma The presence of circulating CD20 in the plasma was investigated using Western blot and immunoprecipitation. As expected, CD20 was detected by Western blot in cells from patients with CLL but not in a myeloid cell line (HL60) (Figure 1A). Plasma from healthy individuals showed low levels of cCD20 on Western blot, whereas plasma of patients with CLL had high levels of CD20 (Figure 1A). The cCD20 bands detected in plasma corresponded to the 35 kDa CD20 detected in the CLL cells. Additional bands seen in cell samples from patients with CLL as well as in some plasma samples may correspond to the previously reported phosphorylated and alternatively spliced CD20 protein. Immunoprecipitation also showed high levels of CD20 in plasma from patients with CLL (Figure 1B). Figure 1B shows CD20 protein precipitated from 33 µL plasma from different patients with CLL. The plasma samples were denatured and then immunoprecipitated and visualized by Coomassie stain (Figure 1B).
Detection of cCD20 in plasma by ELISA We developed an ELISA assay for detection and quantification of cCD20 in plasma and compared the levels of cCD20 detected by the ELISA assay with those detected by Western blot (Figure 1A). The intensity of the CD20 bands on the Western blot correlated with the cCD20 levels detected by ELISA (Figure 1) (Spearman, p < .0001). Dilutions and measurements of diluted samples showed almost identical values (z = 0.35 and P = .7, sign test). The specificity of the assay for CD20 was confirmed by competition experiments. CD20 isolated from the BJAB cell line or a CD20 peptide was able to block the detection of signal by ELISA when an increasing amount of CD20 was added to the wells for the detection of CD20 in a plasma sample (Figure 2).
Levels of cCD20 in the plasma of patients with CLL were significantly
higher than those detected in the 31 healthy individuals tested
(P < .0001, Kruskal-Wallis test) (Figure
3). The cCD20 levels in patients with CLL
varied widely, from 52.89 to 15 740 nM (median, 776.9 nM). In
contrast, the levels of cCD20 in the plasma of the 31 healthy
individuals varied only from 123.55 to 547.10 nM (median, 470 nM).
Additional testing demonstrated that higher levels of cCD20 could be
detected in some healthy individuals who have a viral infection.
To test whether the detected cCD20 was the result of active shedding or
breakdown of cells, we assayed levels of cCD20 in supernatants of
cultured CLL cells at various time points. Peripheral blood cells from
3 different patients with CLL were cultured with and without PMA (used
as a shedding agent), and samples from the supernatants of these
cultures were collected at various time points. As shown in Figure
4, there was no increase in cCD20 in the
supernatants with time, even after 148 hours of culture. Cells cultured
with PMA also showed no increase in cCD20, confirming that cCD20 does
not shed from cells. As a control, levels of the HLA class I molecule,
which is known to shed from cells, were analyzed before and after
exposure to PMA.31 There was no significant increase in
HLA levels with time in supernatants from cells cultured without PMA,
but the supernatants from the same cells cultured with PMA showed
significant increase in the levels of HLA class I protein, confirming
the shedding of the HLA class I molecule (Figure 4A).
To investigate the possibility that the cCD20 detected in samples was the result of ex vivo lysis, we measured cCD20 in samples from 3 patients with CLL and high lymphocyte counts at various time points after collection. Peripheral blood samples were collected in EDTA and kept at room temperature. Circulating CD20 was analyzed in aliquots taken at various time points after collection. Figure 4B shows a representative example from a patient with high white blood cell count; there was no significant increase in the plasma cCD20 level after 48 hours at room temperature. High levels of cCD20 correlate with advanced stage of CLL Correlation of plasma cCD20 levels with various characteristics and stage of disease in the 180 patients with CLL yielded mixed results. The patients were representative of typical populations of patients with CLL seen at M. D. Anderson Cancer Center (Table 1) and included 116 (64.4%) men. Thirty-two (17.8%) patients had Rai stage 0 disease, 81 (45.0%) had stage I-II disease, and 60 (33.3%) had stage III-IV disease. The median age was 61 years, and the median level of2-microglobulin was 288 nM (3.4 mg/L). There
was no significant difference in cCD20 levels between men and women
(P = .66). Circulating CD20 levels were highly correlated with 2-microglobulin levels, platelet count, percentage
of cells expressing CD19+/CD38+, and hemoglobin
level (Table 2). Circulating CD20 levels
did not correlate significantly with white blood cell count, lymphocyte count, or age. Levels of cCD20 correlated with both Rai and Binet stages. When cases were grouped by stages as Rai 0, Rai I-II, or Rai
III-IV, patients with higher Rai stage had significantly higher levels
of cCD20 (P = .01, Kruskal-Wallis test) (Figure 5A). When 2 groups were used, patients
with Rai stage 0-II had significantly lower cCD20 levels than those
with Rai stage III-IV disease (P = .01).
Similar results were obtained when Binet staging was used
(P = .004) (Figure 5B). No correlation was found between cCD20 levels and presence of hepatosplenomegaly or number of sites of
lymphadenopathy.
Survival analysis The univariate Cox proportional hazards model was used to test for variables that predict survival time in this patient group. As shown in Table 3, expression of CD38, hemoglobin level, platelet count,2-microglobulin
(2M) levels, and Rai staging were all predictors of
survival. When cCD20 was analyzed as a continuous variable in the Cox
regression model, it was a strong predictor of survival
(P = .002). To test for the linearity (goodness of fit) of
cCD20 in predicting survival, we examined Martingale residual plots
using log transformation. When the logarithm of cCD20 was used to refit
the univariate Cox model, cCD20 was only marginally significant
(P = .08) when used as a continuous variable. In contrast, when the cCD20 range was dichotomized using a cut point of 1875 nM/L,
Kaplan-Meier plots demonstrated 2 groups of patients with significantly
different survival. Patients with cCD20 levels more than 1875 nM/L had
significantly shorter survival than those with cCD20 level less than or
equal to 1875 nM/L (P = .01) (Figure 6). Median survival time in the patients
with high cCD20 levels was approximately 18 months, while the median
survival in those with lower levels had not been reached at the time of
analysis (Figure 6). The cut point of 1875 nM was reached using
recursive partitioning procedures, a standard computer-based analysis
that finds the best breakpoint for separating 2 groups. Multivariate analysis showed that the shorter survival in patients with cCD20 levels
more than 1875 nM/L was independent of Rai or Binet stage or hemoglobin
level. Specifically, a multivariate model including cCD20 and Rai stage
indicated an increased relative risk in patients with high cCD20 level
(relative risk = 3.51, P = .01) (Figure 7). Similar results were observed when
either Binet stage or hemoglobin level was included in the multivariate
model instead of Rai stage. However, further validation with a larger
number of patients is needed if this cut point were to be used for
stratifying patients for therapy.
Plasma cCD20 blocks rituximab binding to CLL cells To test the significance of the levels of cCD20 and their role in rituximab therapy, we performed ex vivo mixing experiments. Increasing amounts of plasma were added to a mixture of CLL cells and rituximab. The efficacy of rituximab binding to CLL cells was evaluated by its ability to mask surface CD20 detection by conventional flow cytometry. When 1 µL rituximab was added to 1 million cells, without the addition of plasma, rituximab completely occupied all the CD20 antigen sites and the B cells became negative for CD20 (Figure 8A). The rituximab on the surface of the CLL cells could be detected using PE-labeled antimouse immunoglobulin (Figure 8). The addition of 100, 300, or 500 µL of patients' plasma substantially blocked rituximab binding to cells. In other words, as shown in Figure 8, plasma cCD20 competed with cell surface CD20 for rituximab binding and significantly diminished the binding of rituximab to the surface of CLL cells. As the amount of plasma was increased, the binding of rituximab to CLL cells was further diminished (Figure 8A). This was demonstrated in samples from several patients, although the level of competition varied depending on the level of cCD20. The same effects were noted when the Raji cell line, which expresses high level of CD20, was used. The inhibition of binding of rituximab was significantly higher when plasma from patients with CLL was used as compared with plasma from healthy individuals (Figure 8B). Plasma from a patient with CD20 pre-B acute lymphoblastic leukemia
with a very low level of cCD20 did not inhibit rituximab binding to
Raji cells.
CD20 is an important molecule in the maturation and proliferation of CD20+ B cells.7 The reported differences in intensity of surface CD20 expression between various B-cell malignancies suggest that CD20 may play a role in the biology of these malignancies and in their clinical behavior.13-15 The sequence of the CD20 protein indicates that this molecule is tightly bound to the cell surface membrane.7 However, some very tightly attached cellular molecules such as DNA can be detected in circulation.32 The data presented here suggest that CD20 protein is detectable at significant levels in the circulation of patients with CLL. CD20 is a hydrophobic protein and likely circulates with other proteins as cell membrane fragments or large membrane complexes. We were able to immunoprecipitate CD20 in circulation only after denaturing, which supports the hypothesis that it circulates in large complexes or as fragments of cell membrane. Results from the Western blot suggest that the full-length CD20 protein, rather than a cleaved immunoreactive fragment of the protein, is in circulation. We chose to use the term circulating CD20 (cCD20) rather than soluble CD20 to reflect our hypothesis that the circulating CD20 is a part of a membrane complex or fragment. The ex vivo data, which show that CD20 does not shed from cells activated by PMA, support the hypothesis that the cCD20 originates from cell breakdown. The variation in plasma CD20 level among patients with CLL likely reflects variations in tumor mass, rate of cell proliferation, and rate of cell turnover as well as the activity and capability of the reticuloendothelial system to remove products of the breakdown of leukemic cells. Furthermore, we have observed variation in levels among healthy individuals, and our preliminary data suggest that individuals with minor viral infection may have higher levels than other healthy individuals (data not shown). Plasma, rather than serum, was used for measuring cCD20 to circumvent the possibility that the clotting process may damage circulating cells and influence the levels of cCD20. The finding that levels of cCD20 were similar in freshly separated plasma and in plasma separated after samples were kept 24 to 48 hours at room temperature supports the concept that the detected cCD20 is not the result of ex vivo cell lysis. Furthermore, the lack of correlation between cCD20 levels and white blood cell count supports the hypothesis that cCD20 levels are influenced by the rate of turnover of malignant cells and not related solely to tumor mass. Levels of cCD20 could have an impact on patient care and
prognosis. Circulating CD20 levels correlated positively with Rai and
Binet stage and Measurement of cCD20 and rituximab levels may help in designing more effective dosing and scheduling for rituximab therapy. The presence of circulating antigen that binds to rituximab raises the possibility of formation of immune complexes. These immune complexes may have other implications for therapy and result in side effects. The concept of circulating antigens can be expanded to other antibody-based therapies that are currently used in treating hematologic malignancies, including alemtuzumab (anti-CD52)33-35 and gemtuzumabozogamicin (Mylotarg; anti-CD33).36 The presence of soluble HER-2 antigens has been reported in patients with breast cancer,37 but their clinical relevance in patients receiving anti-HER-2 therapy has not yet been determined.38 In summary, cCD20 can be detected at high levels in some patients with CLL, and its level is prognostically important. Circulating CD20 may have a significant impact on the effectiveness of therapy by anti-CD20 antibodies such as rituximab. Further studies are needed to elucidate the effect of cCD20 on the pharmacokinetics and pharmacodynamics of therapies based on the use of anti-CD20 antibodies.
Submitted June 4, 2002; accepted November 13, 2002.
Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-06-1639.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maher Albitar, Departments of Hematopathology and Leukemia, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 72, Houston, TX 77030-4095; e-mail: malbitar{at}mdanderson.org.
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© 2003 by The American Society of Hematology.
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M. J. Keating, N. Chiorazzi, B. Messmer, R. N. Damle, S. L. Allen, K. R. Rai, M. Ferrarini, and T. J. Kipps Biology and Treatment of Chronic Lymphocytic Leukemia Hematology, January 1, 2003; 2003(1): 153 - 175. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
,
and 
antibodies. Molecular studies included analysis of
immunoglobulin and T-cell receptor genes as well as Bcl-1 and Bcl-2
rearrangements. The diagnostic criteria for CLL were based on the
history and physical examination findings recommended by the National
Cancer Institute (NCI)-CLL Working Group.
