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GM-CSF does not increase CD20 antigen expression on chronic lymphocytic leukemia lymphocytes
Leukemia Research 29 (2005) 735–738
GM-CSF does not increase CD20 antigen expression on chronic lymphocytic leukemia lymphocytes M¨unci Ya˘gcı ∗ , ˙Irem Akar, G¨ulsan T¨urk¨oz Sucak, Rauf Haznedar Gazi Medical School, Department of Hematology, Birlik mah, 18 sok 5/5, Ankara, Turkey Received 11 November 2004; accepted 28 November 2004 Available online 7 March 2005
Abstract CD20 antigen expression in B-chronic lymphocytic leukemia (B-CLL) is at significantly lower levels than in non-Hodgkins lymphoma, which may affect the degree of anti-CD20 antibody binding. Low density of CD20 expression on malignant cells may explain the lower response rates to anti-CD20 monoclonal antibody, observed in B-CLL. Upregulating the antigen receptor intensity on tumor cells may enhance the response rates. In this study, we examined the influence of granulocyte macrophage-colony stimulating factor (GM-CSF) on the expression level of CD20 antigen and percent of cells expressing CD20 antigen on B-CLL lymphocytes, in vivo. CD20 antigen expression was studied by flow cytometry at baseline, 12 and 24 h after GM-CSF injection. However neither upregulation of CD20 antigen nor a change of the proportion of CD20 positive cells was observed after a dose of 5 g/kg GM-CSF. Strategies other than GM-CSF priming needs to be evaluated in order to increase the efficacy of anti-CD20 monoclonal antibodies in B-CLL. © 2005 Published by Elsevier Ltd. Keywords: Chronic lymphocytic leukemia; CD20; Rituximab; Cytokine; GM-CSF
1. Introduction Despite advances in the treatment of chronic lymphocytic leukemia (CLL), most patients do not achieve complete response and the disease remains incurable. There is a need for new treatment modalities with different mechanisms of action and toxicity profiles for the management of patients with B-CLL. CD20 is a transmembrane protein that is expressed exclusively on cells of the B-cell lineage. Rituximab is a chimeric monoclonal antibody directed against the CD20 antigen and has potential application in a large number of CD20 positive B-cell malignancies [1]. In contrast to promising effects observed in non-Hodgkin’s lymphoma the response rate in advanced CLL was less than 25% when rituximab was used at the standard doses [2]. It was not clear why CLL
∗
Corresponding author. Tel.: +90 312 4958914; fax: +90 312 2236714. E-mail address: [email protected] (M. Ya˘gcı).
0145-2126/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.leukres.2004.11.021
appeared less responsive to rituximab therapy than folicular lymphoma. Several possible explanations include the low density of CD20 expression on malignant cells in these patients, a shorter half-life in patients with high white blood cell counts, and possibly circulating CD20 [3]. CD20 is expressed at a high density (100,000 molecules per cell) on most lymphoma B cells although the CD20 antigen density is considerably lower (approximately 10,000 molecules per cell) in B-CLL [1]. Enhancing the expression of the antigen on the surface of B-CLL cells and/or inducing its expression on the surface of cells that are negative for the antigen, could enhance the binding of the antibody to the tumor cells, increase the cell kill and thereby enhance the response rates to the antibody. Venugopal et al. have demonstrated an upregulation of CD20 expression by interleukin-4, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-␣ in vitro [4]. In this study, we examined the influence of GM-CSF on the expression level of CD20 antigen and percent of cells expressing CD20 antigen on B-CLL lymphocytes, in vivo.
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M. Ya˘gcı et al. / Leukemia Research 29 (2005) 735–738
2. Materials and methods 2.1. Patients Eighteen patients with previously untreated or treated BCLL as defined by the National Cancer Institute (NCI) Working group [5] were considered for entry into the study. Exclusion criteria were: age > 75 years, pregnancy or lactation, heart failure grade III or IV according to the New York Heart Association Functional classification and institution of chemotherapy for CLL within the last 6 months. 2.2. Study design A dose of 5 g/kg GM-CSF (Leukine, Immunex, Seattle) was administered subcutaneously on outpatient basis. Peripheral blood (PB) samples were obtained for flow cytometric analysis at baseline, 12 and 24 h after GM-CSF injection. Paracetamol 500 mg orally was given as clinically indicated.
1 × 106 cells were incubated with the monoclonal antibodies according to the manufacturer’s instructions. After incubation with the antibodies a total of 10,000 events/tube was acquired using a Becton Dickinson FACScalibur device and analyzed with CELLQUEST v.3.3. Flow cytometer was calibrated daily by using CaliBRITE beads (Becton Dickinson). For determination of the expression of the CD20 antigen and percent of cells expressing CD20 antigen, a sequential gating strategy was used (Fig. 1). CD20 expression was evaluated as relative mean fluorescence intensity (MFI) calculated as the ratio of CD20 mean/negative control mean [6]. Analysis of MFI of CD20 antigen and percent of cells expressing CD20 antigen was performed on PB collected prior to GMCSF injection, and at 12 and 24 h after GM-CSF injection. 2.4. Statistical analysis The results were calculated as mean ± S.D. Paired t-test was used to compare the means; p-values lower than 0.05 were considered significant.
2.3. Flow cytometric analysis 2.5. Ethical approval PB specimens were collected in EDTA containing tubes. The monoclonal antibodies used for the diagnosis of CLL were directed against CD5, CD10, CD11c, CD19, CD20, CD23, CD25, CD79b, CD103, FMC7, IgM, IgD, and . In addition, IgG1 fluorescein isothiocyanate (FITC), IgG2a phycoerythrin (PE) and IgG1 peridinin chlorophyll (PerCp) antibodies were used as an isotypic control. All monoclonal antibodies except CD79b (Pharmingen) were purchased from Becton Dickinson. A simultaneous triple labeling with the monoclonal antibodies CD20 FITC, CD5 PE and CD19 PerCp were used for determination of the expression of the CD20 antigen and percent of cells expressing CD20 antigen.
The study protocol was a part of another study, which was approved by the Ethics Committee of Gazi Medical School. Each patient gave written, informed consent before participation.
3. Results MFI and % positivity results are given as mean (median, range). MFI of CD20 antigen before GM-CSF injection, 12 h after GM-CSF injection and 24 h after GM-CSF injection
Fig. 1. Flow cytometric analysis of CD20 antigen expression at baseline (top row) and 24 h after GM-CSF injection (bottom row). Expression of the CD20 antigen was determined by a sequential gating strategy. Left to right: CD5 positive events on side scatter vs. CD5 (R1 and R4); CD19 positive events on side scatter vs. CD19 (R2 and R5); CD5 and CD19 positive events on side scatter vs. forward scatter (R3 = R1 + R2 and R6 = R4 + R5); histogram of isotypic control on R3 and R6; histogram of CD20 on R3 and R6.
M. Ya˘gcı et al. / Leukemia Research 29 (2005) 735–738
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Table 1 Mean fluorescence intensity (MFI) and % positivity of CD20 antigen on B-CLL lymphocytes at baseline, 12 and 24 h after GM-CSF injection
MFI of CD20 % Positivity of CD20
t=0
t = 12
t = 24
p
21.90 ± 13.76 60.48 ± 24.80
24.44 ± 18.66 65.14 ± 24.49
22.84 ± 14.48 61.08 ± 29.94
>0.05 >0.05
ing of CLL cells before treatment with anti-CD20 antibodies. It is possible that different dose and administration schedules of GM-CSF may enhance CD20 expression on CLL lymphocytes. In a recent clinical study rituximab and GM-CSF were added in an attempt to improve the efficacy of the chemotherapy regimen in previously treated B-CLL patients. The addition of rituximab and GM-CSF did not enhance the regimen efficacy, which was consistent with our results [8]. Further studies are needed to evaluate the role of anti-CD20 antibodies in B-CLL. Other strategies need to be evaluated in order to increase the efficacy of anti-CD20 monoclonal antibodies in B-CLL including combining with chemotheraupetic agents [9], priming with cytokines other then GM-CSF [10] or new anti-CD20 antibodies with enhanced cytolitic activities [11]. Fig. 2. Effect of GM-CSF on mean fluorescence intensity (MFI) and % positivity of CD20 antigen on B-CLL lymphocytes.
are 21.90 ± 13.76 (21.03, 2.97–58.74), 24.44 ± 18.66 (21.71, 3.43–57.61) and 22.84 ± 14.48 (26.80, 3.00–43.45), respectively. Percent of cells expressing CD20 antigen before GMCSF injection, 12 h after GM-CSF injection and 24 h after GM-CSF injection are 60.48 ± 24.80 (68.21, 15.15–88.08), 65.14 ± 24.49 (74.69, 18.05–96.42) and 61.08 ± 29.94 (74.57, 11.81–87.93), respectively (Fig. 2). When the percent and MFI of the cells expressing CD20 antigen of cells exposed to GM-CSF at 12 and 24 h were compared to that of the cells without exposed to GM-CSF, an increase in MFI and % positivity of cells expressing CD20 was statistically insignificant (p > 0.05) (Table 1).
4. Discussion The response to rituximab would potentially be increased if upregulation of CD20 was possible especially in B-CLL, a lymphoproliferative disease with usually low CD20 surface expression. Venugopal et al. have demonstrated an upregulation of CD20 expression on B-CLL lymphocytes by a variety of cytokines including GM-CSF, in vitro [4]. Data reported by Chow et al. are in contrast with this study demonstrating a significant upregulation of CD20 expression by GM-CSF [7]. Both previous studies are in vitro and results are inconsistent. Taken together, these are the basis for our study. In this study, we, in vivo, examined the effect of a single subcutaneous dose of 5 g/kg GM-CSF on MFI of CD20 antigen and percent of cells expressing CD20 antigen in B-CLL lymphocytes. However, neither upregulation of CD20 nor a change of the proportion of CD20 positive cells was observed. Our results do not establish a theoretical basis for GM-CSF prim-
References ¨ [1] Lundin J, Osterborg A. Advances in the use of monoclonal antibodies in the therapy of chronic lymphocytic leukemia. Semin Hematol 2004;41(3):234–45. [2] Huhn D, von Schilling C, Wilhelm M, Ho AD, Hallek M, Kuse R, et al. Rituximab therapy of patients with B-cell chronic lymphocytic leukemia. Blood 2001;98(5):1326–31. [3] Manshouri T, Do KA, Wang X, Giles FJ, O’Brien SM, Saffer H, et al. Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic significance. Blood 2003;101(7):2507–13. [4] Venugopal P, Sivaraman S, Huang X, Nayini J, Gregory SA, Preisler HD. Effects of cytokines on CD20 antigen expression on tumor cells from patients with chronic lymphocytic leukemia. Leuk Res 2000;24(5):411–5. [5] Cheson Bd, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia, revised guidelines for diagnosis and treatment. Blood 1996;87:4990–7. [6] Del Poeta G, Venditti A, Del Principe MI, Maurillo L, Buccisano F, Tamburini A, et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003;101(6):2125–31. [7] Chow KU, Sommerlad WD, Boehrer S, Schneider B, Seipelt G, Rummel MJ, et al. Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases. Haematologica 2002;87(1):33–43. [8] Tsimberidou AM, Kantarjian HM, Cortes J, Thomas DA, Faderl S, Garcia-Manero G, et al. Fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone plus rituximab and granulocyte-macrophage-colony stimulating factor (GM-CSF) alternating with methotrexate and cytarabine plus rituximab and GMCSF in patients with Richter syndrome or fludarabine-refractory chronic lymphocytic leukemia. Cancer 2003;97(7):1711–20. [9] Byrd JC, Peterson BL, Morrison VA, Park K, Jacobson R, Hoke E, et al. Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results
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from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 2003;101(January (1)):6–14. [10] Kimby E. Beyond immunochemotherapy: combinations of rituximab with cytokines interferon-alpha2a and granulocyte colony stimulating factor. Semin Oncol 2002;2(Suppl. 6):7–10.
[11] Teeling JL, French RR, Cragg MS, van den Brakel J, Pluyter M, Huang H, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 2004;104(6):1793–800.
GM-CSF does not increase CD20 antigen expression on chronic lymphocytic leukemia lymphocytes M¨unci Ya˘gcı ∗ , ˙Irem Akar, G¨ulsan T¨urk¨oz Sucak, Rauf Haznedar Gazi Medical School, Department of Hematology, Birlik mah, 18 sok 5/5, Ankara, Turkey Received 11 November 2004; accepted 28 November 2004 Available online 7 March 2005
Abstract CD20 antigen expression in B-chronic lymphocytic leukemia (B-CLL) is at significantly lower levels than in non-Hodgkins lymphoma, which may affect the degree of anti-CD20 antibody binding. Low density of CD20 expression on malignant cells may explain the lower response rates to anti-CD20 monoclonal antibody, observed in B-CLL. Upregulating the antigen receptor intensity on tumor cells may enhance the response rates. In this study, we examined the influence of granulocyte macrophage-colony stimulating factor (GM-CSF) on the expression level of CD20 antigen and percent of cells expressing CD20 antigen on B-CLL lymphocytes, in vivo. CD20 antigen expression was studied by flow cytometry at baseline, 12 and 24 h after GM-CSF injection. However neither upregulation of CD20 antigen nor a change of the proportion of CD20 positive cells was observed after a dose of 5 g/kg GM-CSF. Strategies other than GM-CSF priming needs to be evaluated in order to increase the efficacy of anti-CD20 monoclonal antibodies in B-CLL. © 2005 Published by Elsevier Ltd. Keywords: Chronic lymphocytic leukemia; CD20; Rituximab; Cytokine; GM-CSF
1. Introduction Despite advances in the treatment of chronic lymphocytic leukemia (CLL), most patients do not achieve complete response and the disease remains incurable. There is a need for new treatment modalities with different mechanisms of action and toxicity profiles for the management of patients with B-CLL. CD20 is a transmembrane protein that is expressed exclusively on cells of the B-cell lineage. Rituximab is a chimeric monoclonal antibody directed against the CD20 antigen and has potential application in a large number of CD20 positive B-cell malignancies [1]. In contrast to promising effects observed in non-Hodgkin’s lymphoma the response rate in advanced CLL was less than 25% when rituximab was used at the standard doses [2]. It was not clear why CLL
∗
Corresponding author. Tel.: +90 312 4958914; fax: +90 312 2236714. E-mail address: [email protected] (M. Ya˘gcı).
0145-2126/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.leukres.2004.11.021
appeared less responsive to rituximab therapy than folicular lymphoma. Several possible explanations include the low density of CD20 expression on malignant cells in these patients, a shorter half-life in patients with high white blood cell counts, and possibly circulating CD20 [3]. CD20 is expressed at a high density (100,000 molecules per cell) on most lymphoma B cells although the CD20 antigen density is considerably lower (approximately 10,000 molecules per cell) in B-CLL [1]. Enhancing the expression of the antigen on the surface of B-CLL cells and/or inducing its expression on the surface of cells that are negative for the antigen, could enhance the binding of the antibody to the tumor cells, increase the cell kill and thereby enhance the response rates to the antibody. Venugopal et al. have demonstrated an upregulation of CD20 expression by interleukin-4, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-␣ in vitro [4]. In this study, we examined the influence of GM-CSF on the expression level of CD20 antigen and percent of cells expressing CD20 antigen on B-CLL lymphocytes, in vivo.
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2. Materials and methods 2.1. Patients Eighteen patients with previously untreated or treated BCLL as defined by the National Cancer Institute (NCI) Working group [5] were considered for entry into the study. Exclusion criteria were: age > 75 years, pregnancy or lactation, heart failure grade III or IV according to the New York Heart Association Functional classification and institution of chemotherapy for CLL within the last 6 months. 2.2. Study design A dose of 5 g/kg GM-CSF (Leukine, Immunex, Seattle) was administered subcutaneously on outpatient basis. Peripheral blood (PB) samples were obtained for flow cytometric analysis at baseline, 12 and 24 h after GM-CSF injection. Paracetamol 500 mg orally was given as clinically indicated.
1 × 106 cells were incubated with the monoclonal antibodies according to the manufacturer’s instructions. After incubation with the antibodies a total of 10,000 events/tube was acquired using a Becton Dickinson FACScalibur device and analyzed with CELLQUEST v.3.3. Flow cytometer was calibrated daily by using CaliBRITE beads (Becton Dickinson). For determination of the expression of the CD20 antigen and percent of cells expressing CD20 antigen, a sequential gating strategy was used (Fig. 1). CD20 expression was evaluated as relative mean fluorescence intensity (MFI) calculated as the ratio of CD20 mean/negative control mean [6]. Analysis of MFI of CD20 antigen and percent of cells expressing CD20 antigen was performed on PB collected prior to GMCSF injection, and at 12 and 24 h after GM-CSF injection. 2.4. Statistical analysis The results were calculated as mean ± S.D. Paired t-test was used to compare the means; p-values lower than 0.05 were considered significant.
2.3. Flow cytometric analysis 2.5. Ethical approval PB specimens were collected in EDTA containing tubes. The monoclonal antibodies used for the diagnosis of CLL were directed against CD5, CD10, CD11c, CD19, CD20, CD23, CD25, CD79b, CD103, FMC7, IgM, IgD, and . In addition, IgG1 fluorescein isothiocyanate (FITC), IgG2a phycoerythrin (PE) and IgG1 peridinin chlorophyll (PerCp) antibodies were used as an isotypic control. All monoclonal antibodies except CD79b (Pharmingen) were purchased from Becton Dickinson. A simultaneous triple labeling with the monoclonal antibodies CD20 FITC, CD5 PE and CD19 PerCp were used for determination of the expression of the CD20 antigen and percent of cells expressing CD20 antigen.
The study protocol was a part of another study, which was approved by the Ethics Committee of Gazi Medical School. Each patient gave written, informed consent before participation.
3. Results MFI and % positivity results are given as mean (median, range). MFI of CD20 antigen before GM-CSF injection, 12 h after GM-CSF injection and 24 h after GM-CSF injection
Fig. 1. Flow cytometric analysis of CD20 antigen expression at baseline (top row) and 24 h after GM-CSF injection (bottom row). Expression of the CD20 antigen was determined by a sequential gating strategy. Left to right: CD5 positive events on side scatter vs. CD5 (R1 and R4); CD19 positive events on side scatter vs. CD19 (R2 and R5); CD5 and CD19 positive events on side scatter vs. forward scatter (R3 = R1 + R2 and R6 = R4 + R5); histogram of isotypic control on R3 and R6; histogram of CD20 on R3 and R6.
M. Ya˘gcı et al. / Leukemia Research 29 (2005) 735–738
737
Table 1 Mean fluorescence intensity (MFI) and % positivity of CD20 antigen on B-CLL lymphocytes at baseline, 12 and 24 h after GM-CSF injection
MFI of CD20 % Positivity of CD20
t=0
t = 12
t = 24
p
21.90 ± 13.76 60.48 ± 24.80
24.44 ± 18.66 65.14 ± 24.49
22.84 ± 14.48 61.08 ± 29.94
>0.05 >0.05
ing of CLL cells before treatment with anti-CD20 antibodies. It is possible that different dose and administration schedules of GM-CSF may enhance CD20 expression on CLL lymphocytes. In a recent clinical study rituximab and GM-CSF were added in an attempt to improve the efficacy of the chemotherapy regimen in previously treated B-CLL patients. The addition of rituximab and GM-CSF did not enhance the regimen efficacy, which was consistent with our results [8]. Further studies are needed to evaluate the role of anti-CD20 antibodies in B-CLL. Other strategies need to be evaluated in order to increase the efficacy of anti-CD20 monoclonal antibodies in B-CLL including combining with chemotheraupetic agents [9], priming with cytokines other then GM-CSF [10] or new anti-CD20 antibodies with enhanced cytolitic activities [11]. Fig. 2. Effect of GM-CSF on mean fluorescence intensity (MFI) and % positivity of CD20 antigen on B-CLL lymphocytes.
are 21.90 ± 13.76 (21.03, 2.97–58.74), 24.44 ± 18.66 (21.71, 3.43–57.61) and 22.84 ± 14.48 (26.80, 3.00–43.45), respectively. Percent of cells expressing CD20 antigen before GMCSF injection, 12 h after GM-CSF injection and 24 h after GM-CSF injection are 60.48 ± 24.80 (68.21, 15.15–88.08), 65.14 ± 24.49 (74.69, 18.05–96.42) and 61.08 ± 29.94 (74.57, 11.81–87.93), respectively (Fig. 2). When the percent and MFI of the cells expressing CD20 antigen of cells exposed to GM-CSF at 12 and 24 h were compared to that of the cells without exposed to GM-CSF, an increase in MFI and % positivity of cells expressing CD20 was statistically insignificant (p > 0.05) (Table 1).
4. Discussion The response to rituximab would potentially be increased if upregulation of CD20 was possible especially in B-CLL, a lymphoproliferative disease with usually low CD20 surface expression. Venugopal et al. have demonstrated an upregulation of CD20 expression on B-CLL lymphocytes by a variety of cytokines including GM-CSF, in vitro [4]. Data reported by Chow et al. are in contrast with this study demonstrating a significant upregulation of CD20 expression by GM-CSF [7]. Both previous studies are in vitro and results are inconsistent. Taken together, these are the basis for our study. In this study, we, in vivo, examined the effect of a single subcutaneous dose of 5 g/kg GM-CSF on MFI of CD20 antigen and percent of cells expressing CD20 antigen in B-CLL lymphocytes. However, neither upregulation of CD20 nor a change of the proportion of CD20 positive cells was observed. Our results do not establish a theoretical basis for GM-CSF prim-
References ¨ [1] Lundin J, Osterborg A. Advances in the use of monoclonal antibodies in the therapy of chronic lymphocytic leukemia. Semin Hematol 2004;41(3):234–45. [2] Huhn D, von Schilling C, Wilhelm M, Ho AD, Hallek M, Kuse R, et al. Rituximab therapy of patients with B-cell chronic lymphocytic leukemia. Blood 2001;98(5):1326–31. [3] Manshouri T, Do KA, Wang X, Giles FJ, O’Brien SM, Saffer H, et al. Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic significance. Blood 2003;101(7):2507–13. [4] Venugopal P, Sivaraman S, Huang X, Nayini J, Gregory SA, Preisler HD. Effects of cytokines on CD20 antigen expression on tumor cells from patients with chronic lymphocytic leukemia. Leuk Res 2000;24(5):411–5. [5] Cheson Bd, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia, revised guidelines for diagnosis and treatment. Blood 1996;87:4990–7. [6] Del Poeta G, Venditti A, Del Principe MI, Maurillo L, Buccisano F, Tamburini A, et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003;101(6):2125–31. [7] Chow KU, Sommerlad WD, Boehrer S, Schneider B, Seipelt G, Rummel MJ, et al. Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases. Haematologica 2002;87(1):33–43. [8] Tsimberidou AM, Kantarjian HM, Cortes J, Thomas DA, Faderl S, Garcia-Manero G, et al. Fractionated cyclophosphamide, vincristine, liposomal daunorubicin, and dexamethasone plus rituximab and granulocyte-macrophage-colony stimulating factor (GM-CSF) alternating with methotrexate and cytarabine plus rituximab and GMCSF in patients with Richter syndrome or fludarabine-refractory chronic lymphocytic leukemia. Cancer 2003;97(7):1711–20. [9] Byrd JC, Peterson BL, Morrison VA, Park K, Jacobson R, Hoke E, et al. Randomized phase 2 study of fludarabine with concurrent versus sequential treatment with rituximab in symptomatic, untreated patients with B-cell chronic lymphocytic leukemia: results
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from Cancer and Leukemia Group B 9712 (CALGB 9712). Blood 2003;101(January (1)):6–14. [10] Kimby E. Beyond immunochemotherapy: combinations of rituximab with cytokines interferon-alpha2a and granulocyte colony stimulating factor. Semin Oncol 2002;2(Suppl. 6):7–10.
[11] Teeling JL, French RR, Cragg MS, van den Brakel J, Pluyter M, Huang H, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 2004;104(6):1793–800.