Quantification of Expression of Antigens Targeted by Antibody-Based ...

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May 11, 2013 - Key Words: Chronic lymphocytic leukemia; Antibody therapy; Rituximab; .... of CD22 expression (eg, hairy cell leukemia) compared with.
AJCP / Original Article

Quantification of Expression of Antigens Targeted by Antibody-Based Therapy in Chronic Lymphocytic Leukemia Prashant R. Tembhare, MD,1 Gerald Marti, MD, PhD,2 Adrian Wiestner, MD, PhD,3 Heba Degheidy, MD, PhD,2 Mohammed Farooqui, DO,3 Robert J. Kreitman, MD,4 Gregory A. Jasper,1 Constance M. Yuan, MD, PhD,1 David Liewehr, MS,5 David Venzon, PhD,5 and Maryalice Stetler-Stevenson1

Key Words: Chronic lymphocytic leukemia; Antibody therapy; Rituximab; Alemtuzumab; Quantitative flow cytometry DOI: 10.1309/AJCPYFQ4XMGJD6TI

ABSTRACT Objectives: Anti-CD20 (rituximab), anti-CD52 (alemtuzumab), anti-CD22 (BL22, HA22), and anti-CD25 (Oncotac) are therapeutic options that are the mainstay or in preclinical development for the treatment of chronic lymphocytic leukemia (CLL). Studies suggest that levels of surface antigen expression may affect response to monoclonal antibody–based therapy. Methods: Using the flow cytometric Quantibrite method (BD Biosciences, San Jose, CA) to determine antibodies bound per cell, we quantified the levels of surface expression of CD20, CD22, CD25, and CD52 in CLL cells from 28 untreated patients. Results: The CLL cells in all cases expressed CD20, CD22, and CD52 but 4 (14%) cases were negative for CD25. Although the ranking of levels of expression from highest to lowest was CD52, CD20, CD22, and CD25, the level of antigen expression on any specific case could not be accurately predicted. Conclusions: Quantification of antigens might be useful in evaluating new antigens to target for therapy and may provide a systematic approach to selecting individualized therapy in CLL.

© American Society for Clinical Pathology

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CME/SAM

From the 1Flow Cytometry Unit, Laboratory of Pathology, 4Laboratory of Molecular Biology, and 5Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; 2Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD; and 3Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

Upon completion of this activity you will be able to: • discuss the intensity of antigen expression in chronic lymphocytic leukemia cells and potential impact on therapeutic response. • describe the method for determining antigen expression by the leukemic cells, even when nonneoplastic cell populations may be present. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 913. Exam is located at www.ascp.org/ajcpcme.

Chronic lymphocytic leukemia (CLL) is the most common type of chronic lymphoproliferative disease in Western countries, with approximately 14,990 new cases and 4,390 deaths reported in the United States in 2010.1 Although CLL is primarily an indolent disease, most patients eventually require therapeutic intervention. Standard cytotoxic treatments with alkylating agents or purine analogs are effective but result in immune deficiency because of the destruction of normal lymphoid cells; a subset of patients develop myelodysplasia and secondary leukemias with resulting morbidity and mortality.2 For these reasons, specific targeted antibodybased therapies have been explored in CLL, as well as other lymphomas/leukemias. Currently, several monoclonal antibodies are the mainstay among the therapeutic options for CLL, including anti-CD20 (rituximab, ofatumumab) and anti-CD52 (alemtuzumab).3-7 Others are in clinical

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development, including anti-CD22 (BL22, HA22),8-10 antiCD25 (Oncotac),11 and recently, anti-CD23 (lumiliximab). Response to therapy with these agents has been highly variable among patients with CLL8,11-13 and side effects vary from primarily infusional toxicity with rituximab to significant immunosuppression with resultant increased risk of viral and other opportunistic infections with alemtuzumab.3,7,14 Furthermore, monoclonal antibody therapy is very costly, potentially resulting in economic strain for the patient and health care system. Several studies have suggested that the level of cell surface antigen expression may affect the response to monoclonal antibody–based therapy. The response to monoclonal antibody therapy in B-cell lymphoproliferative processes with characteristically different levels of target antigen expression differs.8,9,15 The level of surface antigen expression can be accurately quantified using quantitative flow cytometry, allowing one to precisely determine the relationship between levels of antigen expression and response to monoclonal antibody therapy.15-19 A study correlating the level of CD20 expression by the malignant cells with response to rituximab in a series of B-cell non-Hodgkin lymphoma revealed a “cutoff” value for CD20 expression associated with good response.20 Relatively lower expression of CD20 in CLL compared with most B-cell lymphomas has been thought to be a reason for the inferior response rate to single-agent rituximab.13 Indeed, in a recent study, CLL response to rituximab therapy correlated with the level of cell surface CD20 expression and higher CD20 expression correlated with trisomy 12.19 Response to alemtuzumab (Campath-1H, Genzyme, Cambridge, MA) therapy in CLL and T-cell leukemias correlated with the level of cell surface expression of CD52.15 The response to an anti-CD22 immunotoxin was significantly higher in B-cell leukemia/lymphoma known to have relatively high levels of expression of CD22 expression (eg, hairy cell leukemia) compared with those with lower levels (eg, CLL).9 These studies illustrate that the intensity of cell surface expression of target antigen by the leukemic cells may have an effect on the outcome of monoclonal antibody–based treatment regimens. As the concept of “personalized medicine” gains popularity, it is highly desirable to discover a prognostic indicator of response to this type of therapy, and more specifically, to identify the antibody-based regimen that is likely to be most effective in an individual patient. Pretreatment quantification of target antigen expression may aid in guiding patient care and choice of monoclonal antibody therapy, especially if levels of expression of targeted antigens vary significantly. Hence, in the present study, we quantified the levels of cell surface expression of CD20, CD22, CD25, and CD52 in CLL cells from patients and correlated them with each other as well as the absolute B-lymphocyte count at presentation. 814 814

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Materials and Methods Case Selection Peripheral blood samples from 28 patients with untreated CLL undergoing evaluation for research protocol eligibility were submitted to the flow cytometry laboratory for confirmation of diagnosis and quantitative assessment of cell surface expression of CD20, CD22, CD25, and CD52 by malignant B cells. The average age of patients was 60.4 years (range, 31 to 79 years; 9 women and 19 men). All patients signed institutional review board–approved informed consent to be screened for eligibility. Clinical data were obtained through medical record review and by contacting the patients’ National Institutes of Health staff physicians. Immunophenotyping Cell surface expression of CD20, CD22, CD25, and CD52 by CLL cells was evaluated. Specimens were stained within 12 hours of collection with a panel of antibodies as previously described.16 Viability was determined by 7-aminoactinomycin D staining.21 In evaluating levels of CD20, CD22, CD25, and CD52 expression, specimens were stained with separate cocktails containing phycoerythrin (PE)–labeled antibodies with labeled known fluorescence to protein level and other antibodies selected to allow gating on the malignant B cells ❚Table 1❚. Additional antibody combinations were used to confirm diagnosis and immunophenotype of the malignant cells. The antibody concentrations for CD20, CD22, CD25, and CD52 were at the saturation level and the concentrations of other antibodies used were according to the manufacturer’s recommendations. Normal lymphoid cells in specimens served as internal controls. Three- to 8-color flow cytometry was performed using a BD Biosciences FACS CANTOII flow cytometer (BD Biosciences, San Jose, CA). The sensitivity of fluorescent detectors was monitored using CST beads (BD Biosciences) according to the manufacturer’s recommendations, and no significant variation was detected during the period of these studies. The antibody-binding capacity (ABC) per cell of the malignant lymphoid cells was determined for anti-CD20 ❚Table 1❚ Antibody Cocktails for Antibody-Binding Capacity Determinations Quantitated Antigen

FITC

CD20 – CD22 – CD25 – CD52 –

PE

PerCP 5.5

APC

CD20 CD22 CD25 CD52

CD19 CD5 CD19 CD5 CD19 CD5 CD19 CD5

APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP 5.5, peridinin chlorophyll protein 5.5.

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(clone L27, BD Immunocytometry Systems [BDIS], San Jose, CA), anti-CD22 (clone S-HCL-1, BDIS), anti-CD25 (clone 2A3, BDIS), and anti-CD52 (clone CF1D12, Invitrogen, Life Technologies, Grand Island, NY) using saturating concentrations of antibody and the BD Biosciences Quantibrite system (Quantibrite standard beads and QuantiCALC software) for fluorescence quantification as previously described.16 The ABC value is the measurement of the mean value of the maximum capacity of each cell to bind the monoclonal antibody. Quantibrite PE Beads (BD Biosciences) are precalibrated standard beads containing known levels of PE molecules bound per bead. Quantibrite beads were acquired on a FACS CANTO II flow cytometer on the same day at the same instrument settings as the individual patient specimens. A standard curve comparing the geometric mean of fluorescence to known PE content of the Quantibrite beads was constructed using QuantiCALC software (BD Biosciences). The regression analysis, slope, intercept, and correlation coefficient were determined. Analysis gates were drawn based on CD5 and CD19 co-positivity to include only the malignant cells for determination of the geometric mean fluorescence of CD20, CD22, CD25, and CD52 staining. The ABC values were generated from the measured geometric mean fluorescence of only the malignant cells using the Quantibrite standard curve.

which included expression of CD5, CD19, dim CD22, CD23, atypical CD38 (negative or unusually homogeneous expression at abnormal level), bright CD43, dim or negative CD79b, dim CD81, and dim κ or l light chain restriction. The CLL cells expressed CD20, CD22, and CD52 in all cases and 4 cases were negative for CD25 (14%). Median CD20 expression (9,560.5) was low, but levels were highly variable (minimum, 2,206; maximum, 35,946; standard deviation [SD], 8,801.5). The expression of CD22 was uniformly dim in all cases. The mean CD22 ABC was 2,378.6, with an SD of 1,102.9 and range of 991 to 5,358. CD25 expression was low (mean CD25 ABC, 881.8) in the CD25-positive cases, with a range from 206 to 1,924 (SD, 500.4). Although CD52 was positive in all 22 cases in which it was studied, levels were highly variable, with a range from 1,464 to 34,752 (mean CD52 ABC, 16,253.4; SD, 9,014.2). The levels of antigen expression as measured by ABC values can be ranked as follows (Wilcoxon signed rank test of log-transformed levels): CD52 was greater than CD20 (P = .030), CD20 was greater than CD22 (P < .0001), and CD22 was greater than CD25 (P < .0001). We studied the correlation among CD20, CD22, CD25, and CD52 ABCs ❚Table 3❚. These ABC values in the data set produced 6 pairwise correlation coefficients. Interestingly we found a significant positive correlation between the CD20 ABC and CD52 ABC values (Spearman rank correlation coefficient 0.60; P = .004 for the null hypothesis of zero correlation, not corrected for the number of tests performed). This correlation is based on results of 22 samples positive for both CD20 and CD52. There were, however, isolated cases in which the expression of CD20 and CD52 were disparate. In 3 cases (2, 5, and 22) the CD20 ABC was below the mean and close to the median whereas the CD52 ABC was notably above the mean and median and in the upper quartile for CLL

Results The cell surface expression of CD20, CD22, CD25, and CD52 was determined using flow cytometry ❚Figure 1❚ and ABC values quantified in 28 patients with untreated CLL. CD20, CD22, and CD25 expression was studied in all cases and the expression of CD52 was studied in 22 of 28 cases ❚Table 2❚. All cases showed classic CLL immunophenotype,22

B

C

2

3

4

5

10 10 10 10 CD19 PerCP Cy55

10

5

10

4

10

3

10

2

64

CD52

48 Count

SSC-A

CD5 APC

A

CD25

32

CD20 CD22

16

2

3

4

10 10 10 10 CD19 PerCP Cy55

5

0 10

0

1

10

2

10

10 PE-A

3

4

10

10

5

❚Figure 1❚ Flow cytometric quantification of levels of antigen expression in chronic lymphocytic leukemia (CLL). Flow cytometric analysis of CLL cells is demonstrated in case 2. A, B-cell analysis gate is drawn around CD19–positive B cells. B, Data from B-cell analysis gate in A is shown. Second CLL analysis gate is drawn around CD19-positive and CD5-positive B cells. C, x-Axis phycoerythrin (PE) antibody staining fluorescence, y-axis cell number. Histogram peaks of saturation staining with anti-CD52PE, anti-CD20PE, anti-CD22PE, and anti-CD25PE on cells in CLL analysis gate labeled directly. © American Society for Clinical Pathology

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❚Table 2❚ Antigen Expression Levels by ABC in Patients With CLL

ABC Values

Case No.

Sex

Age, y

1 M 44 2 F 61 3 M 63 4 M 72 5 M 65 6 F 66 7 F 77 8 M 60 9 M 31 10 M 75 11 M 61 12 M 62 13 M 60 14 M 50 15 F 62 16 F 55 17 M 63 18 M 45 19 F 66 20 F 67 21 M 58 22 M 47 23 M 52 24 F 64 25 F 62 26 M 58 27 M 65 28 M 79 Mean (SD) 60.4 (10.4) Minimum 31 Lower quartile 56.5 Median 62 Upper quartile 65.5 Maximum 79

CD20

CD22

CD25

CD52

3,928 9,625 27,411 3,729 8,974 7,847 9,496 10,203 12,827 4,717 6,010 5,681 15,233 8,778 9,133 11,609 19,313 11,955 8,950 35,946 21,448 11,360 2,206 35,385 5,876 11,630 3,392 18,385 12,180.3 (8801.5) 2,206 5,943 9,560.5 14,030 35,946

1,624 1,448 1,666 991 1,583 1,808 2,229 1,734 1,364 1,287 1,930 3,604 1,579 3,725 2,726 1,752 3,120 4,196 3,509 3,066 5,358 1,311 3,743 2,169 1,026 1,848 3,133 3,073 2,378.6 (1,102.9) 991 1,581 1,889 3,126.5 5,358

206 1,464 461 23,899 1,061 34,752 825 10,689 977 33,994 1,576 14,459 1,122 13,626 661 9,187 Neg 12,225 1,472 9,828 1,308 5,088 1,924 17,568 206 19,329 1,229 9,014 412 10,458 Neg 12,099 555 16,643 1,238 20,380 1,792 11,659 Neg 18,051 742 18,526 Neg 34,636 1,038 ND 341 ND 310 ND 754 ND 535 ND 417 ND 881.8 (500.4) 16,253.4 (9,014.2) 206 1,464 439 10,458 789.5 14,042.5 1,233.5 19,329 1,924 34,752

ABC, antibody-binding capacity; CLL, chronic lymphocytic leukemia; Neg, negative; ND, not done; SD, standard deviation.

❚Table 3❚ Correlation of Antigen Expression Correlation Antigens Coefficient, r P Value

No. of Patients

CD20 vs CD22 CD20 vs CD25 CD20 vs CD52 CD22 vs CD25 CD22 vs CD52 CD25 vs CD52

28 24 22 24 22 18

0.11 –0.25 0.60 0.31 –0.04 –0.03

.57 .24 .004 .14 .87 .90

observed in different cases, indicating that on a case-by-case basis, the level of targeted therapeutic antigen expression cannot be accurately predicted. We further correlated the CD20, CD22, CD25, and CD52 ABC values with the absolute B-cell counts at presentation (data not shown). No statistically significant association was found between the absolute B-cell count and ABC (largest r = 0.28, P = .20).

Discussion CD52 ABC. In cases 20 and 21, the CD20 ABC was in the upper quartile (more than double the median and in case 20 greater than double the mean) whereas the CD52 ABC was below the upper quartile (Table 2). The distribution of differences between CD52 and CD20 was significantly different from zero (P = .030, Wilcoxon signed rank test of log-transformed levels). Neither CD20 nor CD52 expression levels had an apparent correlation with either CD22 or CD25 levels. The highest CD20, CD22, CD25, and CD52 ABC values were 816 816

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Monoclonal antibody therapy has become an important option for patients with CLL. CD20 and CD52 are currently important therapeutic targets for this disease; CD22, CD25, and, more recently, CD23 are being explored as potential targets in research protocols.8,11-13 Preliminary studies indicate that response to antibody-based therapy may correlate with the levels of cell surface expression of the targeted antigen.9,15,19 Tam et al19 demonstrated an association between CD20 ABC values and cytogenetic findings, with higher levels of CD20 © American Society for Clinical Pathology

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expression in CLL with trisomy 12. Furthermore, a higher mean CD20 ABC was observed in patients who responded to rituximab single-agent therapy than those who did not.19 The level of CD52 expression, as determined by quantitative flow cytometry, was also higher in patients with CLL who responded to Campath therapy than those who did not in a study by Ginaldi et al.15 Knowledge of the levels of expression of potential target antigens by the malignant cells could therefore provide a possible rationale for the selection-specific monoclonal antibody for therapy. Quantitative flow cytometry can accurately quantify antigen density, determining the number of therapeutic targets on the cell surface of specific populations of interest. The Quantibrite method allows the determination of the mean number of fluorescently labeled ABCs by using precalibrated standard beads containing known numbers of PE molecules. This method provides a precise measurement of surface antigen expression when performed under staining conditions using saturating antibody levels. By including the antibody of interest in a cocktail containing additional antibodies capable of differentiating malignant from normal lymphoid cells, antigen expression can be quantified on just the CLL cells. Using the Quantibrite method we simultaneously determined the mean ABC values for expression of CD20, CD22, CD25, and CD52 by fresh CLL cells in 28 untreated patients. The level of therapeutic antigen expression by malignant cells varied widely among the patients with CLL (Figure 1). CD20, CD22, and CD52 were expressed at varying levels in all cases studied; however, CD25 was negative in 4 cases, indicating that it is advisable to demonstrate cell surface expression of targeted antigen before initiating therapy. The mean CD20 ABC was 12,180, with a range of 2,206 to 35,946 (Table 2). This is consistent with the ABC values previously reported in CLL.19,23-27 All CD20 ABCs were significantly lower than the 80,000 CD20 ABC values reported for normal B cells.19,23,24,27,28 The mean CD22 ABC in normal B cells was previously reported as 21,642 and 26,283.16,25 CD22 expression was uniformly low in all cases with a mean CLL CD22 ABC of 2,378.6, with an SD of 1,102.9 and range of 991 to 5,358. These values are comparable with those reported previously.16,25 To our knowledge, CD52 and CD25 ABC values have not been previously studied in normal B cells or in CLL. In this study, we documented a mean CD52 ABC of 16,253.2, with an SD of 9,014.4 and a mean CD25 ABC of 881.8, with an SD of 500.4 in CLL. When comparing the mean ABC values for antigen expression with response to single-agent therapy, the levels of expression can be ranked, that is, CD52 was greater than CD20 (P = .030), CD20 was greater than CD22 (P < .0001), and CD22 was greater than CD25 (P < .0001) (see Figure 1). The ranking of levels of expression of these antigens from highest to lowest agrees completely with the reported ranking of single-agent response © American Society for Clinical Pathology

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rates. Single-agent alemtuzumab (CD52) overall response rate ranged from 83% to 87%,6,29 whereas single-agent rituximab (CD20) overall response rate has been reported as 40% and 43%.3,30 In a phase I trial of BL22, an anti-CD22 immunotoxin, only a 27% marginal response was observed in CLL, and the higher CD22-expressing hairy cell leukemias had an 81% response rate (61% complete remission).9 In a phase I trial of the anti-CD25 immunotoxin LMB2, 12.5% of patients with CLL had a partial response.11 Thus quantification of mean levels of antigen expression may be useful in evaluating new antigens to target for therapy. Although a correlation was observed between the CD20 ABC and CD52 ABC values (Spearman rank correlation coefficient 0.60, P = .004), in some isolated cases, the expression of CD20 and CD52 was markedly different in its deviation from mean/median (Table 2, cases 2, 5, 20, 21, and 22). The highest CD20, CD22, CD25, and CD52 ABC values were observed in different cases. Furthermore, the lowest levels of CD20 and CD22 were observed in different cases, with the lowest level of CD25 and CD52 being observed in the same case (No. 1; Table 2). This indicates that on a case-by-case basis, the level of targeted therapeutic antigen expression cannot be accurately predicted. The wide variation in the antigen expression levels studied is of potential interest in antibody-based therapy of CLL. For example, cases 5 and 22 had very high levels of CD52 expression (>2 SD above the mean) whereas the level of CD20 expression was at or below the mean for CLL. Based on previous studies indicating a correlation between levels of antigen expression and response to antibody-based therapy, the possibility that these 2 patients would respond better to alemtuzumab than to rituximab therapy should be considered. An alternative approach to maximize anti-CLL activity has been the combination of rituximab with alemtuzumab.31 Although this approach has shown promise and may be particularly beneficial for patients with relatively low expression of both targets, it may also have unnecessary toxic effects in some patients and unjustified additional cost for other patients. Thus, stratification of patients based on the relative expression of the target antigens could further improve the therapeutic use of these monoclonal antibodies in CLL. More studies are necessary to determine if quantification of potential therapeutically targeted antigens can provide a systematic approach to selection of optimal therapy for an individual with CLL. In summary, several monoclonal antibody–based therapeutic options are available for the management of CLL. Response to therapy with these agents and resulting side effects has been highly variable among patients with CLL3,7,8,11-14 and the cost is significant. We have observed that not all CLL cases have cell surface expression of the therapeutically targeted antigens, and furthermore that the levels of expression of the antibodies may vary widely from patient

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to patient. Knowledge of the level of expression of potential target antigens provides a basic informative platform for the development of new clinical trials and may give a rational paradigm for the selection of therapeutic alternatives in the new age of individualized therapy. Address reprint requests to Dr Stetler-Stevenson: Flow Cytometry Unit, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Dr, Bethesda, MD, 20892; [email protected].

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16. Jasper GA, Arun I, Venzon D, et al. Variables affecting the quantitation of CD22 in neoplastic B cells. Cytom B Clin Cytom. 2011;80B:83-90. 17. Wang L, Abbasi F, Jasper GA, et al. Variables in the quantification of CD4 in normals and hairy cell leukemia patients. Cytometry B Clin Cytom. 2011;80B:51-56. 18. Clinical and Laboratory Standards Institute. Document ILA24-A: Fluorescence Calibration and Quantitative Measurement of Fluorescence Intensity—Approved Guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2004. 19. Tam CS, Otero-Palacios J, Abruzzo LV, et al. Chronic lymphocytic leukaemia CD20 expression is dependent on the genetic subtype: a study of quantitative flow cytometry and fluorescent in-situ hybridization in 510 patients. Br J Haematol. 2008;141:36-40. 20. Horvat M, Prevodnik VK, Lavrencak J, et al. Predictive significance of the cut-off value of CD20 expression in patients with B-cell lymphoma. Oncol Rep. 2010;24:1101-1107. 21. Pallis M, Syan J, Russell NH. Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34. Cytometry. 1999;37:308313. 22. Rawstron AC, Villamor N, Ritgen M, et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 2007;21:956-964. 23. Ginaldi L, De Martinis, M, Matutes E, et al. Levels of expression of CD19 and CD20 in chronic B cell leukaemias. J Clin Pathol. 1998;51:364-369. 24. Huh YO, Keating MJ, Saffer HL, et al. Higher levels of surface CD20 expression on circulating lymphocytes compared with bone marrow and lymph nodes in B-cell chronic lymphocytic leukemia. Am J Clin Pathol. 2001;116:437-443. 25. D’Arena G MP, Cascavilla N, Dell’Olio M, et al. Quantitative flow cytometry for the differential diagnosis of leukemic B-cell chronic lymphoproliferative disorders. Am J Hematol. 2000;64:275-281. 26. Hsi E, Kopecky KJ, Appelbaum FR, et al. Prognostic significance of CD38 and CD20 expression as assessed by quantitative flow cytometry in chronic lymphocytic leukaemia. Br J Haematol. 2003;120:1017-1025. 27. Rossmann E, Lenkei R, Lundin J, et al. Performance of calibration standards for antigen quantitation with flow cytometry chronic lymphocytic leukemia. Cytometry B Clin Cytom. 2007;72B:363-379. 28. Wang L, Abbasi F, Gaigalas AK, et al. Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells. Cytometry B Clin Cytom. 2006;70B:410-415. 29. Lundin J, Kimby E, Bjorkholm M, et al. Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL). Blood. 2002;100:768773. 30. Wierda WG, Padmanabhan S, Chan GW, et al. Ofatumumab is active in patients with fludarabine-refractory CLL irrespective of prior rituximab: results from the phase 2 international study. Blood. 2011;118:5126-5129. 31. Zent CS, Call TG, Shanafelt TD, et al. Early treatment of high-risk chronic lymphocytic leukemia with alemtuzumab and rituximab. Cancer. 2008;113:2110-2118.

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