Original Study

Residual Cancer in Patients With Chronic Lymphocytic Leukemia After Therapy Show Increased Expression of Surface

Antigen CD52 Detected Using Quantitative* Fluorescence Cytometry

Michaela Pevna,1 Michael Doubek,2 Petr Coupek,1 Olga Stehlikova,2 Martin Klabusay1

Abstract The quantitative determination of the expression of CD20 and CD52 antigens in chronic lymphocytic leukemia (CLL) is important for treatment with monoclonal antibodies (mAbs). Patients with CLL in complete or partial remission have a higher level of CD52 antigen expression compared with patients with CLL untreated, in progression, or diagnosed with small lymphocytic (SLL). Our results support the possible signifi- cance of consolidation. Background: and alemtuzumab, mAbs used in recent years to treat CLL, are directed against antigens CD20 and CD52. CD20 is not highly expressed by CLL tumor cells, and rituximab does not have significant effec- tiveness in CLL unless combined with chemotherapy. Alemtuzumab targets CD52, which is much more highly expressed, and is currently the most effective agent used alone for CLL. Variability in expression of both antigens among these patients might be related to different individual therapeutic responses to mAb therapy. Patients and Methods: A total 95 patients diagnosed with CLL and/or SLL were divided into 4 groups: (1) untreated; (2) in complete or partial remission; (3) disease in progression; and (4) diagnosed with SLL. Flow cytometry of peripheral blood cells included gating of the CD5þCD19þ tumor population, within which mean fluorescence intensity of fluorescein isothiocyanate (FITC) conjugated with anti-CD20 or anti-CD52 antibody was measured. The resulting expression of the 2 antigens was deduced from the calibration curve using Quantum FITC particles. Results: Expression of CD20 showed no significant differences among the 4 groups of patients. However, significantly greater expression of surface antigen CD52 was recorded in patient group 2 in complete or partial remission (P < .001). Conclusion: The residual population of CLL cells after therapy is characterized by increased surface detection of CD52. Although the exact cause of this phenomenon is unknown, our results provide a basis to consider the potential for CLL consolidation therapy using alemtuzumab.

Clinical Lymphoma, Myeloma & Leukemia, Vol. 14, No. 5, 411-8 ª 2014 The Authors. Published by Elsevier Inc. All rights reserved. Keywords: CD20, Remission, Residual disease, Small lymphocytic lymphoma, Surface CD52

Introduction disease, with the exception of when hematopoietic trans- Despite considerable progress in its therapy in recent years,1 plantation (HSCT) is pursued. CLL is a tumor disease originating chronic lymphocytic leukemia (CLL) remains an incurable from the B-cell lineage. Antigens CD20 and CD52 expressed on the

*This is an open access article under the CC BY-NC-ND license (http:// Submitted: Oct 17, 2013; Revised: May 9, 2014; Accepted: Jun 4, 2014; Epub: creativecommons.org/licenses/by-nc-nd/3.0/). Jun 17, 2014

1International Clinical Research CentereIntegrated Center of Cellular Therapy and Address for correspondence: Martin Klabusay, MD, PhD, St Anne’s University Regenerative Medicine, St Anne’s University Hospital Brno, Brno, Czech Republic Hospital, International Clinical Research Center, Integrated Center of Cellular Therapy 2Department of Internal MedicineeHematooncology, University Hospital Brno, Brno, and Regenerative Medicine, Pekarska 53, 65691 Brno, Czech Republic Czech Republic E-mail contact: [email protected]

2152-2650/$ - see frontmatter ª 2014 The Authors. Published by Elsevier Inc. All rights reserved. - http://dx.doi.org/10.1016/j.clml.2014.06.006 Clinical Lymphoma, Myeloma & Leukemia October 2014 411 CD52 Detection in Chronic Lymphocytic Leukemia

surface of CLL cells are the targets for the therapeutic activities of Table 1 Characteristics of Patients (n [ 95) monoclonal antibodies. If rituximab (an anti-CD20 antibody) in combination with chemotherapy or alemtuzumab (an anti-CD52 Characteristic Value antibody) in monotherapy is used in first-line CLL treatment, the Median Age (Range), Years 63 (39-87) 2-4 therapeutic responses achieved are in the range of 83% to 95%. Sex, n 5 The antigen CD20 is a classic marker of B-lymphocytes. CD20 Male 58 is a phosphoprotein with 4 transmembrane domains and both of its Female 37 ends extend into the cytoplasm.6 Outside the plasma membrane Untreated Patients there are 2 loops of , 1 large and 1 small. It is believed that Newly diagnosed 20 CD20 acts as a store-operated calcium channel, which is significant Monitored 17 for the cell homeostasis of calcium and for passage through the cell Patients in CR/PR cycle.7,8 In contrast to other B-lymphoproliferative diseases, the expression level of CD20 in CLL is low.9 CR 18 CD52 is expressed by cells within the immune system (lym- PR 3 phocytes, , macrophages, and eosinophils) and by cells of Patients in Disease Progression the epididymal tissue in the male genital tract.10 CD52 is a glyco- 1 Previous therapy 8 whose peptide chain is composed of just 12 amino acids.11 2 Previous therapies 11 The greater part of the molecule consists of the side hydrocarbon Small Lymphocytic Lymphoma 18 complex bound outside the plasma membrane. The C-terminus of the peptide chain is loosely bound to the outer layer of the cell membrane by a glycosylphosphatidylinositol lipid (GPI) anchor. CDC activity than does rituximab, binds to the small loop closer to The GPI anchor is a posttranslational modification common to a the cell membrane.19 Alemtuzumab mediates CDC lysis of CLL group of typically constituting a component of lipid rafts.12 cells much more efficiently than does rituximab, probably because Lipid rafts are sphingolipid- and cholesterol-enriched membrane of the much greater expression of CD52.20 microdomains.13 Proteins with a GPI anchor might spontaneously transfer from one cell and incorporate themselves into the cell 12 membrane of a different cell. The function of CD52 in the im- Figure 1 (A) CD52 Antibody Titering on CLL Cells. 2 3 105 mune system is not known, and its expression by CLL cells is lower Leukocytes in the Total Volume of 100 mL. CLL Cells than that in, for example, either mantle cell lymphoma or normal are Saturated Using 5 mL of FITC-Conjugated Anti- B-lymphocytes.14 CD52 . (B) Determining the Linearity of a Flow Cytometer Amplification System23 We assume that in addition to cell cycle arrest and direct apoptosis induction the effectiveness of the monoclonal antibodies on tumor cells occurs through effector mechanisms of the immune system, the necrosis-causing complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Although we know neither the complex working of these mecha- nisms in vivo nor their role in individual compartments, studies have been able to illustrate them separately in vitro. After binding to rituximab, the CD20 molecule moves to lipid rafts, where it triggers calcium influx and cross-linking of surface-bound rituximab by a secondary antibody, enhances a calcium influx and signal trans- duction, which leads to apoptosis.15 In contrast, the CD52 is preferentially localized in lipid rafts and cross-linking of the bound alemtuzumab on CLL cells leads to the formation of aggregates of lipid rafts and induction of cell death.16 Even if this constitutes merely a redistribution and increased local concentration of antigens within the cell membrane caused by rituximab or artificially by the secondary antibody, we can conclude that the more antigens that are available for the lipid rafts, the more efficient will be the effect indirectly inducing apoptosis. Nevertheless, it has been demonstrated that only in the presence of a complement does in vitro lysis of CLL cells by rituximab correlate with a quantitatively determined level of CD20 expres- sion.17 The effect of the CDC is again related to the translocation of the CD20 and rituximab complex into lipid rafts.18 Rituximab

binds to the large extracellular loop of CD20. The second- Abbreviations: CLL ¼ Chronic Lymphocytic Leukemia; FITC ¼ Fluorescence Intensity of generation anti-CD20 antibody , which has greater Fluorescein Isothiocyanate; MFI ¼ Mean Fluorescence Intensity.

412 - Clinical Lymphoma, Myeloma & Leukemia October 2014 Michaela Pevna et al

white blood cell (WBC) count (x ¼ 2 105/WBC count), Figure 2 Quantitatively Determined CD20 Expression in 4 fi m Groups of Patients Based on MESF-FITC Units. There was lled to the volume of 100 L using phosphate buffered Was No Significant Difference Among the Patients saline. The total volume of 100 mL for the diluted sample was chosen (to include leukopenia patients after therapy) as a constant volume and number of cells required to obtain the same default reaction conditions for all of the samples. A reduction in the number of cells to 2 105 leukocytes was determined on the basis of the titration curve such that the total volume of the 100-mL sample after adding 5 mLoffluorescent-labeled monoclonal anti- bodies against the target antigen would bring us to a saturating concentration for all cells (Figure 1A). The following antibodies were used to label the sample: anti- CD19 antibody conjugated with R-phycoerythrin and anti-CD5 antibody conjugated with phycoerythrin-Cy5 (Invitrogen, Carls- bad, CA) were used to identify the CLL population. Anti-CD20 and anti-CD52 antibodies conjugated with fluorescein isothiocya- nate (FITC; Invitrogen) were used to determine the level of expression of the target antigens CD20 and CD52. The samples were incubated for 20 minutes at room temperature, then lysed with formic acid and osmolarity was then adjusted using a standard protocol and ImmunoPrep Reagent System reagents (Beckman-

Abbreviations: CLL ¼ Chronic Lymphocytic Leukemia; FITC ¼ Fluorescence Intensity of Coulter, Fullerton, CA). Fluorescein Isothiocyanate; MESF ¼ Molecules of Equivalent Soluble Fluorochrome; SLL ¼ Small Lymphocytic Lymphoma. Quantitative Fluorescence Cytometry The samples were analyzed using a Cytomics FC500 (Beck- man-Coulter). The system linearity was tested (Figure 1B).23 The The main mechanism mediating ADCC is probably the domi- gating procedure in all 4 groups of patients involved gating of nant cytotoxic effect of natural killer cells. The ADCC effect of lymphocytes on an forward scatter/side scatter (FS/SS) dot plot, rituximab does not differ among cell lines, despite differences in an exclusion of doublets and subsequently gating the þ þ CD20 expression.21 The level of CD52 expression is not proven to CD5 CD19 population. Mean fluorescence intensity (MFI) of be related to cell lysis via the ADCC mechanism after binding of the target antigen CD20 or CD52 was determined from alemtuzumab.20 a histogram. A total of 10,000 cells were measured at the þ þ CD5 CD19 gate. Quantum FITC-calibrated microparticles Patients and Methods with predetermined values of fluorescence in molecules of Patients equivalent soluble fluorochrome (MESF) units (Bangs Labora- The study group consists of 95 patients diagnosed with CLL and/ tories, Fishers, IN) were analyzed using the same protocol and the or small lymphocytic lymphoma (SLL). Patients were divided into same setting as those of the sample. The calibration curve was 4 groups: (1) CLL patients newly diagnosed or so far untreated; made using Quick-Cal software (Bangs Laboratories) and the MFI (2) CLL patients minimal residual disease (MRD)-positive after values of each peak of the calibrated values. Finally, the level of treatment in complete (CR) or partial (PR) remission22; (3) pre- expression of the target antigen in MESF units of fluorochrome treated patients with CLL in progression; and (4) patients diagnosed FITC was deduced from the calibration curve. with SLL. Samples of peripheral blood were collected from patients in each group. An overview of the patients is given in Table 1. Statistical Analyses The nonparametric KruskaleWallis test was used for the Sample Preparation 4 groups of patients. ManneWhitney U tests were subse- The sample of fresh peripheral blood containing 2 105 leu- quently made for every possible pair combination from the 4 kocytes, with volume in microliters that we obtained from the groups.

Table 2 Median Expression Level in MESF-FITC Units for CD20 and CD52 Antigens

Antigen CLL, Untreated CLL, CR/PR CLL, Progression SLL CD20 34641.0 27491.0 22606.0 31393.5 CD52 305961.5 920386.0 296855.0 432269.0 CD5þCD19þ Population, % (Range) 88.46 (54.95-98.4) 27.8 (5.1-58.9) 81.57 (18.8-97.56) 40.7 (15.41-92.1)

Abbreviations: CLL ¼ chronic lymphocytic leukemia; MESF-FITC ¼ molecules of equivalent soluble fluorochromeefluorescence intensity of fluorescein isothiocyanate; SLL ¼ small lymphocytic lymphoma.

Clinical Lymphoma, Myeloma & Leukemia October 2014 - 413 CD52 Detection in Chronic Lymphocytic Leukemia

Results Table 3 Characteristics of Patients in CR or PR (n [ 21) Antigen CD20 The nonparametric KruskaleWallis test did not demonstrate Characteristic Value inhomogeneity in distribution of the values of the quantitatively Median WBC Count (Range), 3 109/L 6.19 (2.93-10.6) established CD20 among the 4 groups of patients. Also, the Median CD5þCD19þ Population in Gate of 27.8 (5.1-58.9) ManneWhitney U test showed no difference in the distribution Lymphocytes (Range), % of quantitatively determined CD20 values between any possible Median Time Interval From Last Therapy 8 (1-59) pair from the 4 groups of patients. Most different from each other (Range), Months were the groups 1 (patients with newly diagnosed or untreated CLL) Last Therapy and 3 (pretreated patients with CLL in progression), but this dif- FC 8 ference was not statistically significant (P < .076) (Figure 2, FCR 6 Table 2). In the group 2 of patients in CR/PR at least 1 month FC-O 1 elapsed since the end of therapy that included anti-CD20 mono- Alemtuzumab 4 clonal antibody (in 7 of them). NA 2 IgVH Mutation Status CD52 Antigen Mutated 1 For the values of quantitatively determined CD52, the Krus- Unmutated 15 e kal Wallis test did demonstrate inhomogeneity in distribution of NA 5 the 4 monitored groups (P < .001). Subsequent analysis of the individual groups using the ManneWhitney U test showed a Abbreviations: FC ¼ fludarabine with cyclophosphamide; FCR ¼ fludarabine with cyclophos- fi phamide and rituximab; FC-O ¼ fludarabine with cyclophosphamide and ofatumumab; WBC ¼ signi cantly greater value of quantitatively determined CD52 an- white blood cell. tigen (compared with each of the remaining groups) in group 2, which were CLL patients MRD-positive after treatment in CR or PR (P < .001) (Figure 3, Table 2). An overview of this group of MESF-FITC for the CD52 antigen. The remaining 2 patients, patients is shown in Table 3, and flow cytometric analyses of pe- 17 and 33 months after the last treatment, had greater quantitatively ripheral blood from 1 of the patients are shown in Figure 4. There determined values of CD52 antigen expression: 904,861 and was no significant correlation between percentage of residual CLL 1,569,584 MESF-FITC. All of the patients in this group exhibited þ þ population and MESF values (Figure 5). Four of 21 patients in this an increasing proportion of the CD5 CD19 cell population in the group received alemtuzumab during the last therapy, from which at gate of lymphocytes and showed progression of CLL in 3 years after least 6 months passed. In 2 of these patients, 6 and 12 months after collection of the peripheral blood sample for the quantitative fluo- the end of therapy, the measured values were 451,758 and 460,946 rescence cytometry analysis (data not shown). The ManneWhitney U test showed a difference between groups 1 (patients with newly diagnosed or untreated CLL) and 4 (SLL) Figure 3 Quantitatively Determined CD52 Expression Among (P < .03). 4 Groups of Patients in MESF-FITC Units. The Group fi of Patients in Category CR/PR Differs Signi cantly Discussion From the Others (P < .001) Toourknowledge,thisisthefirstreportdrawingattentionto increased surface detection of CD52 on CLL cells in patients after treatment determined using quantitative fluorescence cytometry and, according to our data, the level of expression of CD20 did not change in relation to treatment. Although quantitative fluores- cence cytometry is not yet a standardized method at the inter- laboratory level, the MESF unit is now defined by the National Institute of Standards and Technology,24 and it is sufficient to analyze the variability of the surface expression of CD20 or CD52 within one lab. A potential problem of our work lies in the fact that the results from our group of patients with CLL after therapy might interfere þ þ with the polyclonal physiological CD5 CD19 B cell, because our þ þ gating strategy of residual population is based on the CD5 CD19 population within the gate of lymphocytes. B1 cells are distin- guished from conventional circulating B cells (B2) by their devel- opmental origin, their surface marker expression, and their functions.25,26 B1a cells are CD5-positive, and B1b cells are CD5- þ þ negative. Physiological CD5 CD19 cells dominate in the popu- Abbreviations: CLL ¼ Chronic Lymphocytic Leukemia; FITC ¼ Fluorescence Intensity of Fluorescein Isothiocyanate; MESF ¼ Molecules of Equivalent Soluble Fluorochrome; SLL ¼ lation of B-lymphocytes during fetal life. Their ratio declines Small Lymphocytic Lymphoma. with age, and in healthy adults the cells represent 10% to 25% of

414 - Clinical Lymphoma, Myeloma & Leukemia October 2014 Michaela Pevna et al

Figure 4 Quantitative Fluorescence Cytometry in a Patient With MRD-Positive Complete Remission. (A) Initial FS/SS Dot Plot Used to Gate Lymphocytes; (B) CD5DCD19D Population Gated on Lymphocytes, 2-Color Dot Plot; (C) MFI of the Target Antigen CD52 in Histogram Based on Channel FL1-FITC, Gated on CD5D19D Lymphocytes; (D) Standard Calibration Particles on FS/SS Dot Plot; (E) 5 Peaks of Standard Particles in Histogram Based on Channel FL1-FITC; and (F) Calibration Curve Constructed From MFI Values of Each Peak of Standard Particles. The Level of CD52 Expression in the Patient is 1,199,186 MESF-FITC

Abbreviations: FITC ¼ Fluorescence Intensity of Fluorescein Isothiocyanate; FS/SS ¼ Forward scatter/Side scatter; lin ¼ linear scale/channel; MESF ¼ Molecules of Equivalent Soluble Fluorochrome; MFI ¼ Mean Fluorescence Intensity; MRD ¼ Minimal Residual Disease; PE ¼ phycoerythrin.

þ þ B-lymphocytes. In old age, their number increases again.27 Unlike trend in the ratio of the CD5 CD19 population to the gating of the protocol used by Rawstron et al to detect MRD in CLL,28 lymphocytes. The patients in this group later showed a progression which is highly sensitive, the samples from our patients in CR/PR of disease. þ þ contained more than 5% of CD5 CD19 cells in the gating of One explanation of our data is that only the part of CLL lymphocytes. We believe there is significant indirect evidence as to cells with increased expression of CD52 antigen might survive þ þ the pertinence of the gated CD5 CD19 cells to the malignant the therapy or the therapy might lead to the selection of cells CLL population based on the fact that all patients in the CR/PR with an increased expression of CD52. It is of interest that group during a 3-year monitoring process showed an increasing even after alemtuzumab therapy, the expression level of CD52

Clinical Lymphoma, Myeloma & Leukemia October 2014 - 415 CD52 Detection in Chronic Lymphocytic Leukemia

Patients achieving an MRD-negative CR survive longer without Figure 5 MESF Values Plotted Against Percentage of Residual CD5D19D Population in Group 2 progression (PFS) and, according to some analyses, have overall survival as good as or better than patients who achieve MRD- positive CR.4,40-42 In cases of CLL after allogeneic HSCT, which now constitutes the only potentially curative CLL treatment, the eradication of MRD is linked with a decreased risk of relapse.43 Although this treatment is based on different principles than is the conventional therapy, and it is loaded with toxicity, it underlines the importance of eliminating MRD in CLL for the long-term success of the treatment. In particular, patients with unfavorable prognostic markers might benefit from the eradication of MRD. A low level of circulating CD52 after CLL treatment using FCR (fludarabine, cyclophosphamide, and rituximab)44 and detection of high expression of CD52 in the residual CLL population in our patients after treatment appear to represent conditions suitable for consolidation therapy using alemtuzumab, which already has been Abbreviation: MESF ¼ Molecules of Equivalent Soluble Fluorochrome. examined in clinical studies.45-49 There is no known optimal treatment scheme for consolidation therapy in the case of CLL on the residual CLL population is high in 2 of 4 patients in using alemtuzumab, and differences include the method of our study. administering alemtuzumab, the interval between the beginning of Although the CD52-I isoform is sensitive to the lysing of the consolidation therapy and previous therapy, the maximum phosphatidylinositol-specific phospholipase C (PI-PLC), the single dose, and the duration of therapy in weeks. Improvement in CD52-II isoform is resistant.29 Reduced degradation of the GPI therapeutic response after consolidation using alemtuzumab varies anchor in the case of isoform CD52-I using PI-PLC can also be considerably among published studies and in a range of 15% to theoretically responsible for the upregulation of CD52 in the CLL 66% of patients. Alemtuzumab is effective during consolidation in residual population. Proteins with a GPI anchor are subject to eliminating CLL cells from the peripheral blood and bone marrow, internalization to a cell by a mechanism different than clathrin- including MRD, and its effect on lymphadenopathy is less. In this dependent endocytosis.30 The change of speed in the recycling of respect, we can state that circulating CD52, which is low after the proteins with a GPI anchor31 back to the membrane might be previous therapy,44 will not probably be the general cause of revealed in the final surface expression of these proteins. alemtuzumab failure during lymphadenopathy. The infectious Although the median levels of circulating CD20 and CD52 were complications might be fatal, and, in particular, reactivation of comparable,32,33 our results in groups 1 and 3 show the level of cytomegalovirus infection and opportunistic infections45-48 are surface antigen expression of CD52 on CLL cells in MESF-FITC limiting factors. However, our results indicate that high levels of units to be 10 times higher than the level of CD20 expression. If the surface CD52 on the residual population in CR patients lasts we take into account that the sources of the circulating antigens for several months. Postponement of the consolidation using consist only from cell apoptosis and CLL cell turnover, then the alemtuzumab for a longer interval, which is necessary for the im- relatively lower level of circulating CD52 with a view to its higher mune reconstitution after therapy, might be important for at least surface expression might suggest a different cycle for the 2 antigens 2 reasons. These are to reduce the risk of opportunistic infections in vivo. It appears that circulating CD52 antigen in the case that its and for functioning of lymphokine-activated killer cells, which structure is intact has an increased affinity for the membranes of play a key role in ADCC, becoming normal in 12 months after an living cells and is retained by them (cell surface painting).12 FCR regimen.50 Transfer of CD52 antigen is known within the male genital tract, where it is released by the epithelial cells of distal epididymis Conclusion and ductus deferens into the seminal plasma and then acquired by The increased surface expression of CD52 antigen on the residual sperm cells, which do not express it.34 Many cells are broken down CLL population after therapy, as detected in our study, apparently during CLL therapy and acquisition of free CD52 antigen by the indicates that the CD52 antigen can be surface-expressed in CLL in residual population also can explain our data. This could explain varying degrees. Because of its unknown function, we cannot say better progression-free survival (PFS) after fludarabine and alem- how the therapy should contribute to the induction of CD52 an- tuzumab combination treatment than after fludarabine mono- tigen increased surface detection, or whether it is a characteristic of therapy.35 We can conclude that high expression of CD52 on the the residual CLL population. An analysis of a larger study group residual CLL population, which persists according to our data for will be needed to clarify these issues. Quantitative fluorescence several months, might be a consequence of increased expression, cytometry analysis draws attention51 in connection with clinical reduced release or cleavage, and increased surface membrane decisions for treatment of CLL, although more data need to be retention of this antigen. analyzed. One clinically important result of our work is that the Chronic lymphocytic leukemia is a disease with a variable clinical residual CLL population shows an increased number of target an- course. New biological prognostic markers allow us to stratify pa- tigens for therapeutic intervention using alemtuzumab. The elimi- tients into different risk groups at the time of diagnosis.36-39 nation of these cells through alemtuzumab consolidation might be

416 - Clinical Lymphoma, Myeloma & Leukemia October 2014 Michaela Pevna et al associated with improved PFS, as in MRD-negative CR. Although 10. Hale G. CD52 (Campath-1). J Biol Regul Homeost Agents 2001; 15:386-91. 11. Hale G. The CD52 antigen and development of the CAMPATH antibodies. alemtuzumab consolidation has already been tested in clinical trials, Cytotherapy 2001; 3:137-43. which revealed its significant side effects, this treatment is not 12. Paulick MG, Bertozzi CR. The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 2008; 47:6991-7000. optimized in the indication of residual CLL population eradication. 13. Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res 2003; 44:655-67. According to our results, the advantageous increased expression of 14. Klabusay M, Sukova V, Coupek P, et al. Different levels of CD52 antigen expression evaluated by quantitative fluorescence cytometry are detected on CD52 antigen is detected on the residual population even after B-lymphocytes, CD34þ cells and tumor cell of patients with chronic B-cell several months; the implementation of the “watch and wait” strat- lymphoproliferative diseases. Cytometry B Clin Cytom 2007; 72B:363-70. 15. Janas E, Priest R, Wilde JI, et al. Rituxan (anti-CD20 antibody)-induced trans- egy and the initiation of alemtuzumab consolidation much later location of CD20 into lipid rafts is crucial for calcium influx and apoptosis. Clin after the last therapy could have a better effect as a result of the Exp Immunol 2005; 139:439-46. 16. Mone AP, Cheney C, Banks AL, et al. 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