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© 2003 American Society for Clinical Oncology Apoptotic-Regulatory and Complement-Protecting Protein Expression in Chronic Lymphocytic Leukemia: Relationship to In Vivo Rituximab Resistance
From the Hematology-Oncology Service, Madigan Army Medical Center, Tacoma, WA; Division of Hematologic Malignancies, Johns Hopkins University, Baltimore, MD; Burnham Institute, Cancer Research Center, La Jolla, CA; and Division of Hematology-Oncology, The Ohio State University, Columbus, OH. Address reprint requests to John C. Byrd, MD, Division of Hematology-Oncology, Starling Loving Hall, The Ohio State University, Columbus, OH 43210; email: byrd-3{at}medctr.osu.edu.
Purpose: Rituximab has clinical activity in patients with chronic lymphocytic leukemia (CLL) and has a variety of proposed mechanisms, including apoptosis, complement-dependent cell lysis (CDC), and antibody-dependent cellular cytotoxicity (ADCC). Here we examine pretreatment biologic features that promote resistance to apoptosis and CDC in CLL patients and correlate it with clinical outcome to rituximab-based therapy. Patients and Methods: Pretreatment samples from 21 CLL patients treated on a prospective, single-agent rituximab trial were examined for quantitative expression of apoptotic and CDC regulatory proteins, and the level of expression of these proteins was correlated with clinical outcome. Results: Of the 21 patents for whom samples were available, 10 attained a partial response and 11 failed to respond to rituximab therapy. The mean pretreatment expression of Bcl-2, Mcl-1, XIAP, and the ratio of Bcl-2/Bax were higher but not statistically increased in nonresponding patients versus those responding to treatment. In contrast, the pretreatment Mcl-1/Bax ratio was significantly elevated (0.82 ± 0.28 v 0.39 ± 0.29, P < .016) in nonresponding patients compared with patients responding to rituximab therapy. Although pretreatment expression of CD55 and CD59 was not associated with response to rituximab therapy, significantly higher levels of CD59 were observed in the CLL cells that were not cleared from the blood at completion of therapy than the level observed at baseline levels (P = .02). Conclusion: These data indicate that baseline expression of the Mcl-1/Bax ratio, but not CD55 and CD59, predict for clinical response to rituximab therapy in CLL patients. Further study of disrupted apoptosis in CLL as a potential mechanism of resistance to rituximab appears warranted.
THE CHIMERIC monoclonal anti-CD20 antibody rituximab has clinical activity in both untreated1,2 and previously treated chronic lymphocytic leukemia (CLL).3–6 In addition, rituximab increases the frequency of complete response to fludarabine phosphate7 or to regimens based on fludarabine phosphate plus cyclophosphamide8 in previously untreated CLL patients. Multiple mechanisms of action have been postulated to explain cell killing by rituximab, including antibody-dependent cellular cytotoxicity (ADCC),9 induction of apoptosis,10,11 and complement-dependent cell lysis (CDC).12,13 In CLL cells, we have recently demonstrated that caspase-dependent apoptosis contributes to in vivo clearance of CLL cells following rituximab treatment.14 To date, little is known about the biologic features that can predict for either response to rituximab or factors that promote resistance to this antibody. In vitro studies have indicated that expression of the cell surface proteins CD55 and CD59 contribute to the sensitivity of CLL cells to complement-mediated cell lysis.10 The classic complement pathway is triggered by the recognition of the antigen-antibody complex by the complement protein C1q, which binds to the Fc domain of antigen-bound IgG or IgM. Mutations in the C1q binding site on the human IgG1 Fc region of rituximab have recently been shown to decrease complement activation in vitro.15 The complement cascade triggered by C1q leads to cleavage of C3 to C3b, a required complement activator, and the eventual assembly of the membrane attack complex (MAC), which is formed by complement proteins C5, C6, C7, C8, and C9. This cascade can be inhibited by CD55 and CD59. In addition, neutralizing CD55 or CD59 with antibodies can enhance lysis of CLL cells10 and other types of tumor cells in vitro.16–20 Previous studies of drug resistance in CLL have focused on pathways that can disrupt apoptosis. Indeed, factors interfering with caspase activation21–26 such as Mcl-1 protein overexpression have been shown to be relevant in predicting patients’ response to fludarabine phosphate therapy.26 One mechanism whereby Mcl-1 increased resistance may occur is through the formation of heterodimers with the proapoptotic protein bax.27,28 Other apoptosis-suppressing proteins such as XIAP also inhibit caspase activation, and these proteins have been correlated with resistance to chemotherapy in other hematologic malignancies (eg, acute myeloid leukemia).29,30 Although caspase-dependent apoptosis appears to contribute to the elimination of tumor cells in CLL patients receiving rituximab, no previous study has examined the quantitative expression of these apoptotic-regulatory proteins relative to response to rituximab therapy. Therefore, we performed an assessment of the potential resistance factors to both complement-mediated cell lysis and apoptosis in patients with CLL who were receiving rituximab therapy.
Patient Samples and Cell Processing Patients were enrolled in an institutional review board–approved multicenter trial at the Walter Reed Army Medical Center and Johns Hopkins Oncology Center.2 Patients with previously diagnosed CLL, as defined by the modified National Cancer Institute (NCI) criteria,31 received rituximab three times a week for 4 weeks, as previously described.2 Written informed consent was obtained from all patients before participation in the trial and before procurement of circulating leukemia cells. In brief, all patients received 100 mg of rituximab on day 1 and either 250 mg/m2 or 375 mg/m2 on day 3 and thereafter, three times a week for 4 weeks. Response to therapy was judged at 2 months posttherapy according to the modified NCI criteria.31 Of the 33 patients enrolled on this trial, 21 had sufficient circulating leukemia cells to assess mechanisms of rituximab resistance. CLL cells were obtained before rituximab treatment in all patients and at 8 weeks after the start of treatment in select patients who continued to have circulating leukemia cells. Mononuclear cells were isolated from peripheral blood using density-gradient centrifugation (Ficoll-Paque Plus, Pharmacia Biotech, Piscataway, NJ). After washing cells with phosphate-buffered saline (PBS), whole-cell lysates were prepared by pelleting mononuclear cells (1.25 x 108) in a microcentrifuge, aspirating the supernatant, and adding 0.5 mL of cold lysis buffer as previously described.32 The cell suspension was then incubated with constant agitation for 40 minutes at 4°C and centrifuged for 15 minutes at 4°C. The supernatant was recovered, aliquotted, and frozen at -80°C.
Immunoblotting To control for differences between immunoblots, a separate lane with lysate derived from the RS11846 lymphoma cell line was included. The relative ratio for the protein (eg, mcl-1, XIAP, bcl-2, and bax) relative to the ratio of actin was examined in the RS11846 lymphoma cell line. The ratio of the patient sample (ie, patient target protein/actin control) was normalized to the ratio of RS11846 (RS11846 target protein/actin control) for each immunoblot. Separate experiments demonstrated that the interblot variability for the Bcl-2/control protein ratio was 1.451 (± 0.714, 95% confidence) in nine separate immunoblot assays
Flow Cytometry Analysis of CD55 and CD59 Expression
Statistics
Pretreatment Expression of Apoptosis-Regulatory Proteins and Response to Rituximab Previous studies have demonstrated that higher levels of Bcl-2, Mcl-1, and XIAP and an increased ratio of Bcl-2 to Bax are associated with poor responses to chemotherapy or shorter remission duration in patients with hematologic malignancies.22,23,26,30 On the basis of these data, we determined whether expression of Bcl-2, Mcl-1, or XIAP before therapy in CLL patients predicted for clinical response. In addition, given the known interaction between the survival factors mcl-1 and bcl-2 and bax, we also explored the ratio of these factors and response to rituximab. Pretreatment circulating leukemia cell samples were available from 21 patients, of which 10 had partial responses to rituximab. Expression of Bcl-2, Bax, Mcl-1, and XIAP relative to a control RS11846 cell line was examined. The Bcl-2 levels were similar between nonresponders and responders (53.5 ± 30 v 58.9 ± 30; P = .67). The relative expression of Mcl-1 in patients who were not responding (65.0 ± 44.4) to rituximab therapy was higher, albeit not statistically significantly (P = .15), than it was in responders (42.1 ± 41.2; Fig 1  ). A similar association was observed with XIAP (Fig 1), with a relative expression of Mcl-1 of 44.5 ± 27.8 and 33.5 ± 13.8 in nonresponding and responding patients, respectively; however, the difference in XIAP expression did not achieve statistical significance (P = .29). The Bcl-2/Bax ratio was also higher in nonresponding patients (0.89 ± 0.91), albeit not statistically significantly (P = .129), than it was in responding patients (0.49 ± 0.25; Fig 2). In contrast, the Mcl-1/Bax ratio was statistically significantly increased in the rituximab nonresponding patients compared with the responding patients (0.82 ± 0.28 v 0.39 ± 0.29, respectively; P = .016; Fig 3). These preliminary studies indicate that the pretreatment ratio of Mcl-1 to Bax may serve as a useful biologic marker to predict response to rituximab therapy.
Baseline CD55 and CD59 Expression and Response to Rituximab Therapy Pretreatment expression of the complement-inhibiting molecules CD55 and CD59 on patient-derived CLL cells (n = 20) was assessed by flow cytometry. CD55 expression ranged from 3,478 to 13,233 anti-CD55 antibodies bound per cell, whereas CD59 expression ranged from 1,851 to 9,707 anti-CD59 antibodies bound per cell. No statistically significant association was observed between baseline CD55 or CD59 expression and patient sensitivity to rituximab treatment (Fig 4 ), with nonresponding patients having lower mean expression of CD55 and CD59 than responding patients.
CD55 and CD59 Expression Increases from Pre- to Posttherapy in Rituximab-Treated CLL Patients Who Fail to Clear Blood Leukemia Cells Serial assessment of CD55 and CD59 expression from pretreatment to posttreatment (week 8) was only possible in seven patients, all of whom had failed to clear their blood of CLL cells. The remaining 13 patients had none or very few CLL cells in their blood, making serial assessment not possible. A significant increase in CD59 expression among the seven patients who failed to clear CLL cells from their blood after initiation of rituximab treatment was noted (Fig 5 ). Average CD59 expression levels increased from 3,587 antibodies bound per cell (before treatment) to 5,724 antibodies bound per cell (after treatment) (P = .02). Changes in the CD55 expression levels from before treatment to after treatment were not significant (5,992 v 7,503 antibodies bound per cell, respectively; P = .3), although there was a tendency toward higher CD55 expression levels after therapy.
The data in this article represent, to our knowledge, the first study in CLL that examines the biologic features that predict for response to rituximab-based therapy. We have demonstrated that patients who are not responding to rituximab treatment have higher baseline Mcl-1/Bax, Bcl-2/Bax ratios, and XIAP levels than do patients who are responding to treatment, although the differences between the two groups are not statistically significant. In contrast, the ratio of Mcl-1/Bax was statistically significantly higher in patients not responding to rituximab treatment than it was in patients responding to treatment (P < .016). Several studies27,28 have demonstrated that Mcl-1 forms dimers with the proapoptotic protein bax, thereby inhibiting apoptosis via the intrinsic (ie, mitochondrial) apoptotic pathway. This study demonstrates, for the first time, the clinical association of the Mcl-1/bax ratio (or any other antiapoptotic protein) with response to monoclonal antibody therapy (ie, rituximab) in CLL patients. Given our observation14 that rituximab induces apoptosis through the intrinsic pathway in CLL in vivo, it is not surprising that overexpression of biologic proteins preventing this process would predict response to this agent. The lack of statistical significance among other proteins inhibiting this process, including Bcl-2, Mcl-1, and XIAP may be reflective of both the small sample size and heterogeneity of patients studied, which included untreated to heavily pretreated CLL patients. The major factor predicting resistance to rituximab at diagnosis relates to disrupted apoptosis. This scenario is similar to chemotherapy agents and likely represents a nonspecific finding of "resistance to apoptosis." Nevertheless, our data provide support for confirming the value of the Mcl-1/bax ratio as a prognostic predictor of response to rituximab treatment in CLL patients and for conducting further studies of Bcl-2, Bax, Mcl-1, and XIAP (or other apoptosis biomarkers) in more homogeneous CLL patient populations who are receiving rituximab treatment. With respect to complement-mediated cell lysis, we observed pretreatment variability in CLL cell expression of the complement-resistance proteins CD55 and CD59. However, expression of these two proteins before rituximab treatment was not associated with clinical response to rituximab. Similar results were recently reported in a 29-patient series of follicular lymphoma patients treated with rituximab, for whom pretreatment expression levels of CD55 and CD59 were studied.37 Distinguishing our data from the follicular lymphoma study, we were able to perform a serial analysis of CD55 and CD59 expression for a small group of patients (n = 7) with resistant CLL. Posttreatment increases in CD55 and CD59 expression levels were observed in those patients who failed to clear CLL cells from their peripheral blood after rituximab therapy. These data indicate that pretreatment expression levels of CD55 and CD59 do not predict response to rituximab therapy in CLL patients. However, in CLL patients who fail to achieve clinically significant responses to therapy, a subclone of leukemic cells may be selected by means of increased expression of CD55 and CD59. Although there are several interesting results in this study, there are several confounding variables that must be considered when interpreting the results. First, the pretreatment samples resulted from protein lysates, which were extracted from a set amount (1 x 108) of CLL cells and, second, an identical amount of protein was analyzed for each patient for both the target protein (Bcl-2, Bax, Mcl-1, and XIAP) and the control protein (actin). Thus, the size of the CLL cells could confound analysis, as larger cells may contain more protein. This method was chosen for our analysis because most other published reports11,13,21,26 have used similar methodology, and it controls for other confounding variables such as equal lane loading. Other controls for variability among different immunoblots were included, including use a cell-line lysate as a control in all of the gels assayed. Because these results are derived from a small phase II study, confirmation of our results, as part of other studies, should be pursued. Further study of the mechanism(s) of rituximab resistance in CLL patients is required, including ADCC assessment, based upon previously published studies.36 However, the observations presented in this study suggest the importance of apoptosis and provide support to several different strategies for optimizing rituximab therapy in CLL. A variety of antisense molecules directed at proteins such as Mcl-138 are currently in preclinical development and may greatly augment the efficacy of rituximab therapy in CLL. In addition, in vitro synergy between the bcl-2 antisense molecule G3139 and rituximab has been observed.39 With respect to complement-mediated resistance, in vitro studies indicate that neutralizing antibodies directed at these antigens (ie, CD55 and CD59) may be an effective way to increase leukemia cell lysis.16–20 Because CD55 and CD59 are ubiquitously expressed and are likely to be important to normal cell protection from complement-mediated lysis, the use of bi-specific antibodies directed against both the complement inhibitor and the tumor antigen would most likely be required.17 In addition, alternative rapid-dosing regimens for rituximab therapy over a relatively short treatment interval may also reduce emergence of CD55 or CD59 overexpressing complement-resistant CLL cell clones. Other mechanisms of resistance specific to ADCC that were not examined in this trial should be pursued in future investigations. Finally, improved understanding of the mechanisms of response and resistance against rituximab treatment may provide more effective ways to use this therapeutic antibody in the treatment of B-cell malignancies.
We thank Dr. David Lucas for reviewing the manuscript and the patients and medical staff who were instrumental in performing this clinical trial.
This work was supported in part by the National Cancer Institute (P3016058, P01 CA81534-02 and CA98099), National Institutes of Health, Department of Health and Human Services, Bethesda, MD; the Sidney Kimmel Cancer Research Foundation; the Leukemia and Lymphoma Society of America; and the D. Warren Brown Foundation. R.B. and S.K. contributed equally to the production of this work. J.C.B. is a Clinical Scholar of the Leukemia and Lymphoma Society of America.
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38. Moulding DA, Giles RV, Spiller DG, et al: Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells. Blood 96:1756–1763, 2000 39. Auer RL, Corbo M, Fegan CD, et al: Bcl-2 antisense induces apoptosis and potentiates activity of both cytotoxic chemotherapy and rituximab in primary CLL cells. Blood 98:808, 2001 (abstr) Submitted June 4, 2002; accepted January 7, 2003. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
receptor ligation. Blood 94:313, 1999. (abstr)
