Introduction

Theophylline is a methylxanthine used for the treatment of asthma, and is known to have anti-inflammatory and immunomodulatory properties.^{1, 2} It inhibits T-cell proliferation in vitro³ and decreases lymphocyte infiltration into bronchial epithelium of asthmatics, while increasing counts for CD4^pos and CD8^pos T lymphocytes.^{4, 5} Perhaps more importantly, theophylline inhibits cyclic nucleoside phosphodiesterase isoenzymes, which leads to accumulation of cAMP⁶ that in turn inhibits proliferation of blood lymphocytes⁵ and induces apoptosis in both B and T lymphocytes.^{7, 8}

Mentz et al⁹ found that theophylline dose-dependently induced apoptosis in chronic lymphocytic leukemia (CLL) B cells with sparing of normal B cells. They reported a patient with asthma whose CLL remained stable for 10 years while taking only theophylline.¹⁰ The same group failed to observe any therapeutic effect of theophylline in eight other CLL patients who were treated for 9–49 days.¹¹ Binet et al¹² reported that the addition of theophylline to chlorambucil allowed for disease control in CLL patients at a lower dose of the alkylating agent than previously required. Finally, we reported hematologic improvement in two of three patients with CLL treated with 200 mg theophylline twice daily, a dose that resulted in serum levels in the therapeutic range for asthma.¹³ Based on these observations, we performed a phase II study within the Eastern Cooperative Oncology Group (ECOG) of theophylline in newly diagnosed patients with CLL, Rai stages 0 or 1, and report the results here.

Patients and methods

The objectives of this study were to assess the response rate in patients with B-CLL treated with theophylline, to evaluate the toxicity of the agent and apoptosis-related changes in CLL cells induced in vitro by incubation with theophylline prior to therapy, and to correlate the laboratory data with clinical observations.

Patients

Eligibility required a new diagnosis of B-CLL Rai stage 0 or 1. Pathology was confirmed by central review. Patients with a second malignancy within 5 years were excluded from the study. No prior treatment was allowed, and patients were required to be asymptomatic from CLL, and to have an ECOG performance status of 0–2. Patients had no known CNS disease (including seizure disorder) or cardiac arrhythmia, and had adequate hepatic and renal function within 2 weeks of enrollment in the study.

Treatment

Theophylline was given at a starting dose of 200 mg orally every 12 h as an extended release tablet. Patients were evaluated for response every 4 weeks, and treatment continued as long as response or stable disease (SD) could be documented. Patients who progressed were taken off study, but followed for survival.

Response criteria

Complete (CR) and partial response (PR), or progressive disease (PD) were defined by standard criteria for CLL.¹⁴ Patients who did not achieve CR or PR and did not have findings consistent with PD were considered to have SD.

Statistical methods

The primary clinical end point for this study was the evaluation of the response rate (CR+PR) to theophylline. The two-stage design of Simon¹⁵ allowed for an early termination of the study if no response was observed among the first 12 enrollees; with at least one response, the study was to enroll a total of 36 eligible patients. The method of Kaplan and Meier¹⁶ estimated time to disease progression. Linear mixed effects models¹⁷ were used to describe the relationship between induction of apoptosis in vitro and two covariates, aminophylline concentration and day of culture. Descriptive statistics were used for flow cytometry data, such as medians and ranges. Antigen expression levels, mean fluorescence intensity ratios, and apoptotic variables were considered continuous variables and were compared with the nonparametric Wilcoxon rank sum test. Associations between two continuous variables were assessed using the Spearman correlation coefficient. P-values reported are two-sided.

Laboratory methods

Baseline immunophenotyping

Mononuclear cells (MNC) were isolated from heparinized peripheral blood by density gradient centrifugation through Ficoll/Hypaque (Atlanta Biologicals, Norcross, GA, USA). Surface antigens were detected by 3-color multiparameter flow cytometry with the B-cell marker CD19 selectively tagging the CLL cells. Direct immunofluorescence detected CD45, CD14, CD38, CD5, CD19, CD22, CD23, CD43, CD11c, CD25, CD20, Fmc7, CD40, CD79a (clone ZL7.4), CD79b, CD95 (Fas), and CD95L (Fas-ligand) (Becton-Dickinson Biosciences-Pharmingen, BD; San Diego, CA, USA; Beckman-Coulter, Miami, FL, USA; DakoCytomation, Carpinteria, CA, USA). Indirect method detected a surface P-glycoprotein determinant reactive with antibody MRK-16 (Kamiya Biomedical Co., Seattle, WA, USA). To establish monoclonality, polyclonal fluorescein-isothiocyanate (FITC)-conjugated rabbit anti-human antiserum was combined with R-phycoerythrin-conjugated rabbit anti-human antiserum and CD19-phycoerythrin-cyanin5 in the presence of rabbit immunoglobulin to block Fc receptors.

For intracytoplasmic bcl-2 and bax proteins, cell membranes were fixed and permeabilized (Fix&Perm kit, Caltag Labs, Burlingame, CA, USA). Unconjugated anti-bax antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was counterstained with FITC-conjugated goat F(ab')2 anti-mouse immunoglobulin (Caltag Labs) followed by CD19-TC. FITC-conjugated mouse anti-human bcl-2 antibody (DakoCytomation, Carpinteria, CA, USA) was added after fixation/permeabilization of CD19-labeled cells.

Antibody binding was evaluated on a FACSCalibur flow cytometer using CellQuest software (BD) and expressed either as percent CD19-stained CLL cells, that is, the fraction of the cells emitting fluorescence beyond the limit of the control for a given fluorochrome, or as fluorescence intensity. Intensity, a measure of antigen density, was expressed by mean fluorescence channel (MFC) of the specific antibody divided by the MFC of the isotype control (mean fluorescence intensity or MFI ratio).

Cell cultures

MNC (2 10⁶/ml) were cultured in RPMI medium (Atlanta Biologicals) with 10% fetal bovine serum and penicillin/streptomycin at 37°C with 5% CO₂ for 1–3 days without aminophylline, the soluble preparation of theophylline (Roxane Labs, Inc., Columbus, OH, USA), or with 75, 150 or 250 g/ml of drug. Cells were harvested from single cultures and tested for viability by trypan blue exclusion and for apoptosis induction.

Apoptosis

Apoptosis was assessed flow cytometrically and based on the forward light scatter (FSC), the MFI of CD45 antibody staining, and Annexin V binding (Caltag Labs) of CLL cells. Propidium iodide (PI) distinguished between viable cells (Annexin V and PI negative), apoptotic cells with intact plasma membrane (Annexin V positive, PI negative), and cells undergoing secondary necrosis (Annexin V and PI positive).

Results

Therapeutic

A total of 25 patients with typical morphology of CLL were accrued from nine ECOG institutions within 4 months. All patients gave written informed consent. Patient characteristics at study entry are shown in Table 1. Although the first stage accrual goal was 14 patients (for 12 eligible), the study accrued rapidly in the 2-week period after the first stage suspension notice was issued. This led to a total accrual of 25 patients.

Table 1 - Patient characteristics.

Full table

During the course of the study, there were no responders. One patient achieved a CR after 22.5 months of therapy and remains in CR at 27+ months; 18 patients (72%) had SD and six patients (24%) progressed. Table 2 gives reasons for treatment termination in 23 patients; a median of 11 cycles of theophylline were given (range 2–33). The median follow-up time from registration to the date last known alive is 39.2 months; the median time to disease progression (progression-free survival, PFS) was 17.8 months (95% confidence interval (CI) 9.6 months; upper limit of CI not yet reached). Patients whose cells underwent apoptosis in response to aminophylline in culture experienced a longer PFS (median not yet reached) than patients whose cells failed to respond in vitro (median 11 months, 95% CI 8.1–21.9) (P=0.025) (Figure 1).

Figure 1.

PFS by apoptosis status. Patients whose leukemic B lymphocytes underwent apoptosis in response to aminophylline in vitro (apoptosis +) experienced significantly longer PFS (P=0.025) than patients whose leukemic cells failed to undergo apoptosis in vitro (apoptosis -).

Full figure and legend (18K)

Table 2 - Reasons for treatment termination.

Full table

Toxicity

Treatment-related toxicity is shown in Table 3. No grade 4 toxicity was observed. Grade 3 toxicity occurred in four (headache, one patient; gastrointestinal, three patients), grade 1 or 2 in 19 patients (primarily gastrointestinal or headache). Five patients removed themselves from study after 7–17 cycles of treatment.

Table 3 - Toxicity of theophylline.

Full table

Laboratory correlates

Immunophenotyping in 24 patients (one institution failed to submit a specimen) revealed classical B-cell CLL: CD5^pos, CD23^pos, CD43^pos, CD22^{weakly pos}, CD20^{weakly pos}. The cells lacked surface CD79a when tested with antibody clone ZL7.4. With this antibody, CD79a surface expression is absent on CLL cells, distinguishing between CLL and mantle cell lymphoma.¹⁸ CLL cells expressed CD79b, the second component of the B-cell antigen receptor. Expression of CD38 varied from 0 to 98% (median 2%). Monoclonality for surface light chains was found in 57%, and for light chains in 29% of cases; in 14%, surface light chains were undetectable. P-glycoprotein was demonstrable on >90% of CLL cells in 50% of patients (median 95%; range 4–99%).

Short-term cultures were established from MNC of 22 patients; for two patients, there was insufficient material. In cultures from two patients, viability after 48 h was below 10%. Thus, data on in vitro induction of apoptosis are available on 20 patients. Linear mixed-effects models were used to estimate the relationship between the extent of apoptosis induced by aminophylline and two covariates, the drug concentration (primary covariate) and the duration of culture. This model incorporated fixed effects (drug concentration, duration of culture) and random effects, associated with individual patients. The parameters analyzed were: (1) The ratio of nonapoptotic to apoptotic cells, based on the decreased FSC typical of apoptosis as a result of cell shrinkage.^{19, 20} (2) The MFI ratio for CD45 in nonapoptotic vs apoptotic cells, given that a reduced expression of surface CD45 is peculiar to apoptotic lymphocytes.²¹ (3) The percentage of Annexin V- or PI-positive cells among nonapoptotic (high FSC) or apoptotic cells (low FSC). Fluoresceinated Annexin V binds to phosphatidylserine residues that in apoptotic cells are redistributed from the inner to the outside of the plasma membrane.^{19, 20, 22}

Even in the absence of aminophylline, a median of 20% of cells underwent spontaneous apoptosis after 24 h, 50% after 48 h, and 47% after 72 h in culture. Spontaneous apoptosis was always below the aminophylline effect.

In the linear mixed-effects model, the ratio of nonapoptotic to apoptotic cells (based on FSC) decreased (P=0.03) and the ratio of the CD45 MFI in nonapoptotic to apoptotic cells declined with increasing drug dose (P<0.01) and length of culture (P=0.01), when compared with drug-free cultures. These changes were consistent with a dose- and time-dependent apoptosis-inducing effect of aminophylline. The percentage of Annexin V binding cells was higher in the apoptotic (median 93%, range 54–99%) than in the nonapoptotic fraction (median 15%, range 1–41%) (P<0.01). In nonapoptotic cells, the expression of CD38 decreased with increasing drug dose (by a median of 36%, range 11–94%) (P<0.01), while no such effect was seen in apoptotic cells. CD95 (Fas), a key regulator of apoptosis, was expressed by >95% of CLL cells in all cases, albeit with weak fluorescence intensity. CD95L, the Fas ligand, was detected on <5% of CLL cells. Neither CD95 nor CD95L expression changed during exposure to aminophylline.

The ratio of nonapoptotic to apoptotic cells decreased with increasing drug concentration by a median factor of 1.4. This threshold yielded a clear segregation between two groups of patients, which differed significantly in the extent of apoptotic response to aminophylline in culture: In nine patients, the ratio changed maximally by a median factor of 3.0 ('apoptosis positive' group), while in 11 patients the ratio decreased by a median factor of 0, suggesting the absence of an apoptotic response ('apoptosis negative' group) (P=0.001). To find a predictive parameter for apoptosis induction in vitro, we retrospectively compared in these groups the baseline intensities of bcl-2 and bax expression on CLL cells (Table 4). The median bcl-2 expression was comparable in apoptosis-negative (median MFI ratio 19.8, range 16.9–40.6) and apoptosis-positive patients (median MFI ratio 20.4, range 10.2–21.4). Bax expression was significantly higher in the apoptosis-positive (median MFI ratio 5.7, range 4.3–11.2) than the apoptosis-negative group (median MFI ratio 3.6, range 2.7–10.3) (P=0.025). Thus, the preculture ratio of bcl-2/bax was lower in cells that in vitro underwent apoptosis when exposed to aminophylline (median ratio 2.6, range 1.88–4.83) than in those who did not (median ratio 5.5, range 3.6–8.0) (P=0.006). There was no association between bcl-2/bax ratio and WBC (P=0.615) or lymphocyte count (P=0.915), or response in vivo (P=0.655).

Table 4 - Baseline bcl-2 and bax expression by flow cytometry in apoptosis-positive compared with apoptosis-negative patients.

Full table

Although culture with aminophylline induced apoptosis in nine of 20 evaluable patients, only one CR was achieved with theophylline in vivo. Remarkably, however, patients whose cells responded to aminophylline in vitro (apoptosis-positive group) experienced significantly longer PFS (P=0.025) (Figure 1).

Discussion

In vitro studies suggested that theophylline induced apoptosis of B-CLL cells in culture by a mechanism that involved cAMP.^{9, 12} Induction of apoptosis by theophylline was synergistic with chlorambucil, and bcl-2 expression was downregulated by theophylline alone or the two agents together.⁹ Kim and Lerner²³ identifed a phosphodiesterase class (PDE4) whose inhibition by theophylline induced apoptosis in CLL cells. More recently, PDE7A, a cAMP-specific phosphodiesterase, was found to be a theophylline target in CLL.²⁴ These data, together with occasional reports of clinical responses to theophylline by CLL patients,^{10, 13, 25} led us to design this phase II study in patients with indolent B-cell CLL.

Apoptosis sensitivity of B-CLL cells is dependent on the relative expression of bcl-2 and bax,^{26, 27, 28} the level of CD38 expression,²⁹ the intracellular cAMP content,³⁰ and on Rai stage, with early stages being more susceptible.³¹ While bcl-2 protects against apoptosis, bax, a homolog of the bcl-2 protein, is a death promoter.³² In agreement with the concept that CLL is a disease of dysregulated apoptosis,³³ increased bcl-2/bax ratios are associated with high WBC counts.³⁴ Given that the bcl-2/bax interaction is only one of many cofactors regulating apoptosis, a correlation of the bcl-2/bax ratio with clinical response in CLL has not been firmly established, having been suggested by some^{35, 36} but not by others.³⁴ In our data set, the baseline bcl-2/bax ratio was higher in CLL cells which failed to undergo apoptosis in response to aminophylline in vitro than in responsive cells (P=0.006). These findings are in agreement with observations with other apoptosis-inducing agents in vitro.²⁹ However, we could not confirm an association between bcl-2/bax ratio and either WBC or lymphocyte count in our patients.

In this study, the level of expression of CD38 was associated neither with sensitivity to induction of apoptosis nor with survival. The pattern of CD38 expression, V_H mutation status, and genetic abnormalities have been reported to affect survival in B-cell CLL.^{37, 38, 39} Genetic analyses and information on immunoglobulin variable region mutations were not available on our patients.

CD95 (Fas), a surface antigen potentially significant for the induction of apoptosis,¹⁹ was invariably expressed by the CLL cells from our patients, albeit at low density. While this observation contrasts to previous data reporting CD95 expression on a variable number of B cells from patients with CLL, it confirms a generally faint expression in this disease.⁴⁰ Reduced surface expression of Fas is one of the mechanisms allegedly responsible for resistance to Fas-mediated apoptosis.⁴¹ In our study, CD95L, the Fas ligand, was detectable on less than 5% of cells. There was no correlation between expression of CD95 and sensitivity to aminophylline-induced cell death.

Despite a prolonged lifespan in vivo, CLL cells undergo spontaneous apoptosis in vitro, possibly as the result of a lack of essential cytokines present in vivo.⁴² An inverse relationship exists between spontaneous apoptosis and the peripheral lymphocyte count, the Rai and the Binet stage.⁴³ We observed spontaneous apoptosis in all patient cultures; however, it was consistently below the specific apoptotic effect induced by aminophylline in apoptosis-susceptible patients.

Our study confirmed that aminophylline induced apoptosis of CLL cells in a subset of patients in vitro. In favor of an association between apoptosis in vitro and clinical response to theophylline, we noticed that 2/9 apoptosis-positive patients have progressed (median PFS not reached) compared with 9/11 apoptosis-negative patients (median PFS 11 months) (P=0.025). This suggests an association between in vitro induction of apoptosis and progression in B-CLL patients treated with theophylline.

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