Enhanced efficacy of gemcitabine in combination with anti-CD20 monoclonal antibody against CD20+ non-Hodgkin's lymphoma cell lines in vitro and in scid mice

Background Despite exciting new targeted therapeutics against non-Hodgkin's lymphoma (NHL), chemotherapy remains a cornerstone of therapy. While purine nucleoside analogs have significant activity in low grade NHL, the pyrimidine nucleoside analog gemcitabine has been less extensively studied, but has important activity. Use of the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy for B-NHL is becoming prevalent in clinical practice, but has not been extensively studied in pre-clinical models. Methods We have tested the activity of gemcitabine ± rituximab in vitro and in scid/human NHL xenograft models. We used two t(14;18)+, CD20+ follicular B cell NHL cell lines, DoHH2 a transformed NHL line and WSU-FSCCL isolated from pleural fluid of a patient with indolent NHL. Results Gemcitabine is cytotoxic to DoHH2 and WSU-FSCCL cells in vitro, and the IC50 is 2–3 fold lower in the presence of rituximab. Apoptosis is also enhanced in the presence of rituximab. Clearance of NHL cells from ascites in scid mice is prolonged by the combination, as compared with either agent alone. Most importantly, survival of scid mice bearing human NHL cells is significantly prolonged by the combination of gemcitabine + rituximab. Conclusion Based on our pre-clinical data showing prolonged survival of mice bearing human lymphoma cell line xenografts after treatment with gemcitabine + anti-CD20 antibody, this combination, expected to have non-overlapping toxicity profiles, should be explored in clinical trials.

fludarabine, cladribine and pentostatin have been extensively studied and have significant activity against certain non-Hodgkin's lymphoma subtypes, particularly indolent forms. Though less well studied, an increasing body of data indicates activity of gemcitabine against lymphoma, both Hodgkin's and NHL [2][3][4][5][6][7]. The precise place of gemcitabine in the therapeutic armamentarium for NHL remains to be elucidated.
The chimeric anti-CD20 monoclonal antibody rituximab is active as a single agent in B cell NHL [8]. In addition, it may sensitize cells to the action of chemotherapeutic and other biologic agents pre-clinically [9-12], as well as in patients [13,14]. While mechanisms of rituximab action include direct apoptotic induction, complement activation and antibody dependent cytotoxicity, which of these is important may depend on the experimental conditions, and the relative importance in patients remains to be determined (reviewed in [15]). The same mechanisms, as well as intracellular signaling [16], may account for chemosensitization, but again the exact means by which this occurs in patients remains to be fully elucidated. One previous report has demonstrated in vitro sensitization of aggressive B cell NHL cell lines to gemcitabine by rituximab [10]. Here we extend these in vitro results to additional human CD20+ lymphoma cell lines that carry the t(14:18) translocation, perhaps more analogous to the clinical use of rituximab. More importantly, we demonstrate that gemcitabine + rituximab enhances survival in vivo in a human B-NHL cell line/scid mouse xenograft model.

Apoptosis and cell cycle
Cell cycle was analyzed by DNA content per cell, by propidium iodide (PI) staining of nuclei from hypotonically lysed cells [20]. Apoptosis was determined by dual staining of 1 × 10 5 intact cells in 100 µl calcium binding buffer containing 5 µl of fluoroscein isothiocyanate (FITC)labeled annexin V (Pharmingen) and 5 µg/ml PI for 15 minutes in the dark, followed by analysis by flow cytometry (FACScan).

Scid/human xenograft
Female CB17 scid mice were bred, housed and treated in the Fox Chase Cancer Center Laboratory Animal Facility under an approved protocol. Mice 4-8 weeks old were injected ip with either 1 × 10 7 WSU-FSCCL cells or 5 × 10 6 DoHH2 cells. Mice were observed daily and euthanized when they appeared ill. Lymphoma involving diffuse adenopathy, splenomegaly, infiltration of liver and bone marrow, along with ascites developed in untreated mice with each model, at 8-11 weeks with WSU-FSCCL and 4-6 weeks with DoHH2. Cells were collected sequentially from ascites by peritoneal washings and analyzed, or mice were followed for survival. For ascites clearance, mice bearing DoHH2 received 20 µg rituximab intraperitoneally (ip) on day 3 after lymphoma cell injection and/or gemcitabine 120 µg/gm ip on day 4, while mice bearing WSU-FSCCL received 100 µg rituximab intraperitoneally (ip) on day 7 after lymphoma cell injection, and/or gemcitabine 120 µg/gm ip on day 8. For survival, mice bearing DoHH2 received 5 µg rituximab on days 2, 9 and 16 and/ or gemcitabine 120 µg/gm ip on days 3, 10 and 17.

Cell growth inhibition by gemcitabine and rituximab
Both DoHH2 and WSU-FSCCL cells are growth inhibited by gemcitabine with an IC 50 of 1 nM after 72 hr incubation ( Figure 1). Addition of rituximab alone or in combination with gemcitabine had little effect on WSU-FSCCL cells in vitro. In contrast, DoHH2 cells are growth inhibited about 35% by 20 µg/ml rituximab. The maximal rituximab effect is seen at 1 µg/ml. This is accord with data that CD20 sites are saturated at this level, while effective serum levels in patients are felt to be 25 µg/ml. Thus, there is essentially no dose response at clinically achievable levels. This precludes the calculation of synergy by the standard approach [22], as has been concluded by other investigators as well [23]. Because of our prior experience that oligonucleotides can interfere with drug and antibody uptake, we separated addition of the two agents by 4 hours to preclude direct physical interaction of the antibody and drug, however, no effect of order of addition within 4 hours was observed with rituximab and gemcitabine (data not shown).

Cell cycle alteration by gemcitabine and rituximab
Continuous exposure to 2.5 nM gemcitabine led to accumulation of DoHH2 cells in the S phase, as has been generally reported for gemcitabine [24]. WSU-FSCCL cells, however, accumulated in the G2/M phase of the cell cycle ( Figure 2). Although rituximab alone had no significant cell cycle effects on either cell line, nor on the S phase accumulation of DoHH2 cells, the combination of gemcitabine + rituximab led to S phase accumulation of WSU-FSCCL cells. Statistical analysis of S phase block in WSU-FSCCL cells comparing 2.5 nM gemcitabine alone versus gemcitabine plus rituximab revealed p < 0.001. Thus, the combination had different cell cycle effects on WSU-FSCCL than did either agent alone. The precise basis of this change is unclear, though presumably involves an alteration in the S phase DNA damage sensor, which appears to involve the ATM gene [25].

Apoptosis induced by gemcitabine and rituximab
Apoptosis was assayed by annexin V staining, which detects the altered location of phosphatidyl serine to the outer surface of the cell membrane. Apoptosis was induced at modest levels by gemcitabine or rituximab, but significantly more apoptosis was induced by the combination in both cell lines studied (Figure 3). When apoptosis was separated into early apoptosis, in which cells still exclude propidium iodide (PI), and late apoptosis where cells are PI permeable, early apoptosis is induced by the combination in DoHH2 cells, whereas both early and late apoptosis are demonstrated in WSU-FSCCL cells.
Apoptosis was also assessed by cleavage of PARP, which occurs when the apoptotic pathway is activated, eventually leading to cleavage of caspase 3 and of other downstream proteins including PARP. In each cell line,

Concentration of Gemcitabine
rituximab and gemcitabine result in PARP cleavage, with additional cleavage using the combination (Fig 4).

In vivo efficacy of gemcitabine ± rituximab
We initially screen for efficacy of therapy in our model by assessing the prevention of growth of cells in ascites fluid [11,17]. The lymphomas grow as bulky mesenteric nodes with development of hepatosplenomegaly, as well as diffuse adenopathy elsewhere, but not with measurable disease. While ascites represents only a small part of the animals' disease burden, it can be repeatedly sampled as an indicator of overall tumor in a mouse. Given the differing rates of growth of the cell lines in mice, treatment was started on day 3 after DoHH2 cell injection and day 7 after WSU-FSCCL cell injection. For DoHH2 ( Figure   5A), gemcitabine delayed growth, so that at day 18 there were fewer lymphoma cells in the ascites fluid, however, by day 32 there was no longer a difference. Rituximab is effective in this model, although cells do eventually reaccumulate in ascites. There is a trend to longer time to recurrence of lymphoma cells with combined therapy. In the WSU-FSCCL model ( Figure 5B), while gemcitabine alone had no effect, it did enhance the rituximab-mediated delay in lymphoma cell re-growth.
The most important endpoint for treatment efficacy, since there is not an externally measurable lesion, is survival of mice bearing the human lymphoma cell lines. We have performed duplicate experiments using mice injected with DoHH2 cells (Figure 6). At the gemcitabine dose of 120 Effect of gemcitabine and rituximab on cell cycle Figure 2 Effect of gemcitabine and rituximab on cell cycle. Cells (0.25 × 10 6 /ml) incubated ± 20 µg/ml rituximab for 4 hr, then ± 2.5 nM gemcitabine for 24 hr. Cells stained with propidium iodide, analyzed by flow and cell cycle parameters calculated (ModFit LT program). A typical flow result is also shown (Figure 2A). weekly for 3 doses, there was modest prolongation of median survival, with 0/5 and 2/6 long-term survivors in the two experiments. We used sub-maximally tolerated doses of rituximab, 5 µg per mouse, which had modest therapeutic effect (1/11 long-term survivors combined). Combination therapy with these two agents at the same dose and schedule, however, markedly prolonged survival (p = 0.04, left; p = 0.01, right for rituximab + gemcitabine vs rituximab), and cured 9 of 11 mice. The surviving mice were euthanized at the end of the experiment and found to be histologically negative for lymphoma.

Discussion
This report demonstrates the anti-lymphoma activity of the pyrimidine analog gemcitabine in vitro and in a scid mouse human lymphoma xenograft model. Rituximab is an active therapy for CD20+ NHL as a single agent and in combination with some biologic and chemotherapeutic agents [9-12]. The generalizability and mechanism of chemosensitization by rituximab has not been fully explored. Evidence for several mechanisms of rituximab mediated lymphoma cell death has been presented including direct induction of apoptosis, complement mediated killing and antibody-dependent cell mediated cytotoxicity (ADCC). Much of the data addressing these Apoptosis induced by gemcitabine and rituximab C processes comes from in vitro systems, and the relevance to activity in patients remains uncertain. Our in vitro data in this report rely only on direct apoptosis, as we do not add human serum as a complement source nor effector cells. Direct apoptosis can be affected by degree of crosslinking of the antibody, which in turn can be controlled by humoral or cell surface molecules. Complement activity may be altered by inhibitory factors and may differ between human serum in vitro and mouse complement in murine models. We have found that crosslinking does enhance the degree of apoptosis after rituximab treatment in our two cell lines, while addition of complement has little effect (data not shown). ADCC depends on effector cells, and even the precise effector cells remain uncertain. Scid mice have residual granulocytes and NK cells, and depletion of these cells can abolish rituximab efficacy [27]. To understand the mechanisms of resistance to rituximab will require more complete knowledge of which of these mechanisms of action is or are most important in patients [15]. Recent data suggests that rituximab can alter intracellular signaling, even without inducing apoptosis, in ways that can sensitize cells to chemotherapy effects [16].
Our results demonstrate that the combination of gemcitabine and rituximab inhibits NHL cell growth, induces apoptosis in these cells, and, most importantly, is effective in prolonging survival of mice bearing human t(14;18)+ lymphoma cells. Prior reports have shown additive effects of gemcitabine and rituximab in aggressive NHL cell lines in vitro [10]. In that report, aggressive NHL cell lines that were relatively resistant to both gemcitabine and rituximab were pre-treated with rituximab for 24 hours and then treated with gemcitabine for 18 hours and found to have modest increases in hypodiploid cells and PI positive cells after PI staining.
The cell cycle changes seen after gemcitabine treatment alone, and in combination with rituximab, are cell line dependent. WSU-FSCCL cells, in contrast to most cells, are not blocked in S phase by gemcitabine alone, but are after rituximab is added. This may reflect altered PARP cleavage induced by gemcitabine and rituximab Days FSCCL Concurrent treatment of B cell lymphoproliferative disorders (NHL and CLL) with rituximab and chemotherapy is becoming more common, and data suggests benefit in time to disease progression in indolent disease [14] and also in overall survival in aggressive disease [13]. Questions remain regarding the optimal way to combine rituximab and chemotherapy and whether therapeutic efficacy of specific chemotherapeutic agents is enhanced by rituximab. Pre-clinical studies of the interaction of chemotherapy agents and rituximab may provide insight to guide the development of appropriate clinical trials.
Survival of scid/DoHH2 mice after treatment with gemcitabine and rituximab Figure 6 Survival of scid/DoHH2 mice after treatment with gemcitabine and rituximab. Two experiments in which scid mice injected with DoHH2 cells were treated ± 5 µg rituximab on days 2, 9 and 16 and ± gemcitabine (120 µg/gm) on days 3, 10 and 17. Mice were followed for survival and euthanized when ill. For comparison of rituximab + gemcitabine to rituximab alone, p = 0.04 (left) and p = 0.01 (right). Overall, the combination cured 9/11 mice, compared with 2/11 for gemcitabine and 1/11 with rituximab.

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