Cancer Research UK Medical Oncology Unit, University of Oxford, Churchill Hospital, Oxford
    Cancer Research UK Translational Oncology Laboratory, Barts and the London, Queen Mary's School of Medicine and Dentistry, Charterhouse Square, London, United Kingdom

    ABSTRACT

    PURPOSE: Convincing data support the link between inflammation and ovarian cancer. Tumor necrosis factor-alpha (TNF-), a major mediator of inflammation, is chronically produced in the ovarian tumor microenvironment and may enhance tumor growth and invasion by inducing the secretion of cytokines, proangiogenic factors, and metalloproteinases. Etanercept is a recombinant human soluble p75 TNF receptor that binds to TNF- and renders it biologically unavailable. In the current study, we sought to determine the toxicity, biologic activity, and therapeutic efficacy of etanercept in recurrent ovarian cancer.

    PATIENTS AND METHODS: We initiated a phase I-B, nonrandomized, open-label study in patients with recurrent ovarian cancer. Etanercept was administered subcutaneously at a dose of 25 mg twice weekly (cohort one) and 25 mg thrice weekly (cohort two) until disease progression.

    RESULTS: Thirty patients were recruited (cohort one, 17 patients; cohort two, 13 patients). Eighteen of the 30 patients (cohort one, 11 patients; cohort two, seven patients) completed  12 weeks of treatment. Six patients achieved prolonged disease stabilization (cohort one, two patients [40 and 25 weeks]; cohort two, four patients [34, 24, 22, and 24 weeks]). A significant rise in immunoreactive TNF was seen in all patients (pretreatment compared with end of treatment). A phytohemagglutinin-stimulated whole-blood cytokine assay showed a significant fall in interleukin-6 (cohort one [11 of 17]) and CCL2 (cohort one [13 of 17]) levels. Common adverse effects were injection-site reactions and fatigue.

    CONCLUSION: We provide evidence for the biologic activity and safety of etanercept in recurrent ovarian cancer. Our data suggest possible clinical activity that must be confirmed in future studies.

    INTRODUCTION

    Advanced ovarian cancer is the most common cause of death resulting from gynecological cancer and is the fourth leading cause of cancer mortality in women. Despite advances in therapy, the overall survival of patients with recurrent ovarian cancer remains poor. Several chemotherapeutic agents have shown significant antitumor activity in recurrent disease,1 but treatment-related toxicity causes considerable morbidity. Hence, the development of novel therapies to improve patient outcomes remains a high priority.

    Convincing data support the link between inflammation and ovarian cancer.2-5 Tumor necrosis factor-alpha (TNF-) is a major mediator of inflammation.6,7 TNF- is a 17-kd polypeptide that binds as a homotrimer to p55 and p75 TNF- receptors (TNFRs I and II, respectively). These receptors exist both on the cell surface and in soluble form. The soluble receptors are involved in the regulation of bioavailability of TNF-.8,9 TNF- has a bimodal role in cancer.10 Local administration of high-dose TNF- is antiangiogenic and has a powerful antitumor effect.11 On the other hand, endogenous TNF- chronically produced in tumor microenvironment enhances tumor growth and invasion by inducing other cytokines/chemokines involved in cancer progression (such as interleukin-6 [IL-6] and CCL2), proangiogenic factors (such as vascular endothelial growth factors, basic fibroblast growth factor, CXCL8, and thymidine phosphorylase), and matrix metalloproteinases (MMPs).10,12-16 Direct evidence for the role of TNF- in malignancy comes from mice in which TNF- has been deleted. TNF- and TNFR knockout mice are resistant to skin carcinogenesis.17-20

    Several preclinical studies confirm the importance of TNF- in ovarian cancer pathogenesis. TNF- functions as an autocrine and a paracrine growth factor in ovarian cancer.21 TNF- converts ascitic tumor xenografts to solid peritoneal tumors,22 and tumor cells secreting TNF- show enhanced metastasis in nude mice.23 TNF- induces MMP-9, IL-6, and CCL2 production in tumor microenvironment that are important for ovarian cancer progression.24-26 Stimulation of ovarian cancer cell proliferation by IL-1? can be blocked partially by an antibody against TNF-, by soluble (s)TNF- receptor (which reduces the bioavailability of TNF-), and by TNF- antisense mRNA.21,27

    In human studies, TNF- mRNA and protein expression is seen predominantly within ovarian tumor epithelial islands.28 TNFRI (p55 receptor) is localized to tumor and stromal cells, and TNFRII (p75 receptor) is localized to the leukocyte infiltrate.28 A positive correlation is seen between tumor grade and the extent of TNF- expression in ovarian cancer.28 Immunohistochemical analysis of tissue specimens suggests that TNF- positivity is confined to malignant tissue and the normal ovarian tissue is negative for TNF- staining.29 Patients with epithelial ovarian cancer have elevated serum levels of TNF- and p55 and p75 receptors.30,31 TNF- is implicated in the induction of CCL2, a predominant chemokine in the ovarian tumor microenvironment that regulates CD8/CD68 infiltrate.32,33 It also causes defective expression of the chemokine receptor CCR2 in the ovarian cancer microenvironment.34 TNF- stimulates IL-6 production, a potent growth factor that directly stimulates angiogenesis.35 Patients with ovarian cancer have elevated levels of IL-6 in serum and ascitic fluid.31,36 Hence, TNF- blockade is a novel approach to ovarian cancer therapy.

    TNF- plays a central role in the pathogenesis of rheumatoid arthritis (RA), which lead to the development of anti-TNF- therapy for RA. Etanercept (Enbrel; Immunex Corp, Seattle, WA) is a recombinant dimer of human soluble p75 TNF- receptor (sTNF- receptor).37,38 It acts as a competitive inhibitor of TNF-, binding to cell surface TNF- receptor, and hence inhibits biologic activity of TNF-. Etanercept binds reversibly to TNF-. Etanercept has an approximately 50-fold greater affinity for TNF- in a binding-inhibition assay and is at least 1,000 times more efficient than the monomeric sTNF- receptor. The half-life of etanercept is five times that of monomeric sTNF- receptor. These characteristics of etanercept result in its greater ability to neutralize the biologic effects of TNF-. The pharmacokinetics of etanercept has been studied in 11 human trials involving 285 subjects. It is absorbed slowly from a subcutaneous (SC) injection site and achieves a maximum serum concentration approximately 48 hours after a single dose. The elimination half-life is 70 hours with a bioavailability of 76%.39

    Based on the above biochemical, preclinical, and clinical data, we initiated a study to evaluate the role of etanercept in patients with recurrent ovarian cancer. The primary objectives of the study were to evaluate toxicity, biologic activity, and clinical activity of etanercept in patients with recurrent ovarian cancer.

    PATIENTS AND METHODS

    Patient Selection

    This study was conducted in compliance with the declaration at Helsinki, Tokyo, Venice, and Hong Kong (1989). Local research ethics committee approval was obtained. All patients provided written informed consent. Patients were eligible to participate in the study if they had advanced, histologically confirmed ovarian cancer with measurable or evaluable lesions and documented progression within 2 months before entry into the study. Patients  18 years of age, nonpregnant, nonlactating, using effective contraception if liable to become pregnant, with a performance status (World Health Organization) of 0, 1, or 2, and expected survival longer than 3 months were considered for the study. An absolute granulocyte count of more than 1.5 x 109/L, platelet count  100 x 109/L, adequate renal function (serum creatinine  0.15 mmol/L or EDTA clearance > 40 mL/min), adequate hepatic function (bilirubin, alkaline phosphatase, or transaminases  2x the upper limit of normal; if there were liver metastases, then ALP or transaminases  5x the upper limit of normal), and a normal coagulation profile were essential. Patients were also required to self-inject etanercept or have a designee who could do so.

    Treatment Plan

    The dose of etanercept in cohort one was 25 mg twice weekly given SC (similar to the dose used in patients with RA). In view of the biologic and clinical effect in cohort one, we extended the study to recruit patients who would receive etanercept 25 mg thrice weekly (cohort two). This was to investigate if it would achieve a better therapeutic response and biologic effect. All patients were expected to receive at least 12 weeks of treatment, and responding patients continued therapy until disease progression.

    Evaluation Protocol at Baseline and During Therapy

    History, physical examination, and performance status assessments were performed at baseline and days 1, 8, and 28, and every 4 weeks thereafter. Quality-of-life (QOL) assessments using the European Organization for Research and Treatment of Cancer QLQ-C30 (version 3) proforma were performed on day 1 and every 4 weeks from then on. Biochemical evaluation (serum sodium, potassium, glucose, urea, creatinine, alkaline phosphatase, total bilirubin, AST, and gamma glutamyl transferase) and hematologic evaluation (hemoglobin, white cell count, and platelet count) were performed at baseline, days 1, 8, and 28, and every 4 weeks thereafter. For the assessment of biologic responses, blood samples were collected at baseline, 24 hours postetanercept (day 1), days 7 and 28, and every 4 weeks thereafter (in cohort two, additional samples were collected on days 14, 21, and 77). Radiologic evaluation (computed tomography, x-ray) was performed at baseline and at 12 weekly intervals until disease progression. Complete response was defined as disappearance of all clinical evidence of active tumor for a minimum of 4 weeks and normalization of tumor marker (CA-125) and tumor-related biochemical abnormalities. Partial response was defined as  50% decrease in the sum of the diameters of measured lesions. Stable disease (SD) was defined as less than 25% decrease or less than 25% increase in the sum of the longest perpendicular lesion diameter. An increase of more than 25% in the size of any measured lesion or appearance of new lesions was defined as progressive disease. Patients who achieved either SD or tumor regression at 12 weeks were planned to receive additional courses of etanercept with disease-response evaluation performed at 12 weekly intervals. Therapy was terminated at disease progression, with the occurrence of a serious adverse event (including serious infection requiring hospital admission and antibiotics and serious allergic reactions to etanercept), or at the patient's request.

    Toxicity evaluation was performed by using the National Cancer Institute's Common Toxicity Criteria.

    Laboratory Procedures

    TNF- mediates the production of IL-6 and CCL2 in whole blood when stimulated by phytohemagglutinin (PHA) in vitro. The ability of etanercept to alter this response was assessed in sequential samples by using the whole-blood cytokine-release assay as reported previously.40 Briefly, whole blood from patients was stimulated with PHA, and release of IL-6 and CCL2 was measured after 24 hours of incubation at 37°C in 5% CO2. Fifteen-milliliter sterile, pyrogen-free Falcon tubes were spiked with sterilized, pyrogen-free Heparin (30 units/mL whole blood from CP Pharmaceuticals Ltd, Wrexham, United Kingdom). One tube of each pair was spiked further with PHA (2 μg/mL whole blood from Bio-Stat Diagnostic Systems, Stockport, United Kingdom). Twenty milliliters of blood was collected from the patient and split equally into a pair of prepared Falcon tubes labeled either "heparin + PHA" or "heparin" (control). Blood was incubated at 37°C for 24 hours, then the tubes were centrifuged at 200 g for 10 minutes at 4°C, and plasma was aspirated, frozen, and stored at –80°C until assayed. Ten milliliters of blood was also collected and processed immediately for measuring TNF- levels in unstimulated samples. Similarly, CCL2, MMP-3, sTNFRI, and sE-selectin levels in the plasma were measured in unstimulated samples, because significant reductions have been reported in patients with RA who were receiving anti-TNF therapy.41

    All experiments were performed in duplicate, following the protocols provided by the manufacturer (human TNF ELISA kit [DTA50; R & D Systems, Minneapolis, MN], human IL-6 ELISA kit [D6050; R & D Systems], human CCL2 ELISA kit [DCP00; R & D Systems], human MMP-3 ELISA kit [RPN 2613; Amersham, Biosciences Ltd, Chalfont St Giles, United Kingdom], human sTNFRI ELISA kit [DRT100; R & D Systems], and human sE-selectin ELISA kit [BBE 2B; R & D Systems]). All results are expressed in pg/mL (except for MMP-3 and sE-selectin, which are expressed in ng/mL).

    Statistical Analysis

    This was a phase I-B study with a primary end point of toxicity, biologic activity, and efficacy. It was also planned that the study would be discontinued if no responses were observed in the first 14 patients in each cohort. The Wilcoxon matched-pairs signed-ranks test was performed to analyze the serial changes in blood TNF-, IL-6, CCL2, MMP-3, sTNFRI, and sE-selectin levels. Regression analysis was performed to compare biologic responses between cohorts one and two.

    RESULTS

    Patients

    Baseline characteristics of patients are summarized in Table 1. A total of 30 patients were recruited (cohort one, 17 patients; cohort two, 13 patients). Median age at diagnosis in cohort one was 55 years (range, 35 to 76) and in cohort two was 58 years (range, 39 to 66). Patients had been pretreated with surgery and multiple lines of chemotherapy before starting etanercept. Of the 30 patients, 10 received one line, 10 received two lines, and 10 received more than two lines of systemic therapy before etanercept. Four of 10 patients who had only one line of therapy were platinum resistant. Eleven of 17 patients in cohort one and seven of 13 patients in cohort two received  12 weeks of etanercept therapy.

    Toxicity

    All 30 patients were assessable for toxicity. Treatment was well tolerated in all patients. There were no treatment-related deaths. The most common adverse effects reported during etanercept therapy were injection-site reactions (including skin rash, itching, pain, and swelling), fatigue, and anemia. The incidence and severity of skin rash, itching, and fatigue were higher in cohort two compared with cohort one (Table 2). One patient in cohort two developed Sj?gren's syndrome after 8 months of etanercept therapy. She presented with polyarthropathy, polyneuropathy, erythema nodosum-like rash, dry eyes, and dry mouth. It was unclear whether this was related to etanercept. However, therapy was discontinued. She was started on prednisolone and hydroxychloroquine, with significant symptomatic improvement.

    No significant biochemical abnormalities resulting from etanercept were reported in any patients.

    Disease Response

    Eighteen patients (cohort one, 11 patients; cohort two, seven patients) who had at least 12 weeks of therapy were available for disease-response evaluation. No complete or partial responses were seen. Six patients achieved prolonged SD while on etanercept therapy (two patients in cohort one and four patients in cohort two; Table 3). Patient OV2 and OV11 in cohort one achieved SD for 25 and 40 weeks, respectively, and patients OV19, OV25, OV26, and OV28 in cohort two achieved SD for 22, 34, 24, and 24 weeks, respectively. In patient OV25, etanercept was stopped because she developed Sj?gren's syndrome during therapy. Seventeen of 30 patients were alive after entry into this study and five of 17 who had SD were free of progression at 24 weeks.

    CA-125 Levels in Patients Who Achieved SD

    All patients had a documented rise in CA-125 levels before initiation of etanercept therapy (Fig 1). Patient OV2 had a more than 50% reduction in CA-125 levels during etanercept therapy. Patients OV19 and OV25 had a less than 50% reduction in CA-125 levels while on therapy. No significant change in CA-125 level was seen in patients OV11 and OV28 until disease progression. Patient OV26 had doubling of CA-125 level at the time of disease progression. No CA-125 responses were seen in nonresponders.

    QOL Assessments

    Serial QOL assessments using the European Organization for Research and Treatment of Cancer QLQ-C30 (version 3) was available for 29 of 30 patients (Fig 2). Improvements in mean function scales, mean symptom scales, and global functions were seen in patients who had SD. All others reported overall deterioration in QOL, largely because of symptomatic deterioration resulting from disease progression.

    Biologic Response

    Plasma TNF- levels with therapy. Serial blood samples were available for biologic response analysis in patients (Fig 3). In unstimulated blood samples, TNF- was measured as described previously. Serial changes in plasma TNF- levels in cohorts one and two are summarized in Table 4. Minimal immunoreactive TNF- was detected in pretreatment samples. However, within 24 hours of etanercept administration, a significant rise in immunoreactive TNF- was seen in all patients. In addition, the samples were checked for biologic activity in a TNF- bioassay, which was negative. A statistically significant increase in TNF- was demonstrated (pretreatment compared with end-of-treatment values) in both cohorts one (P = .008) and two (P = .02). Regression analysis did not reveal any significant differences between cohorts (P = .63).

    Whole-blood cytokine-release assay (IL-6). A whole-blood cytokine-release assay was performed to assess the effect of etanercept on the release of IL-6 in the whole blood when stimulated by PHA (Fig 4). At each time point, IL-6 levels were measured in either the presence or absence of PHA as described previously. Mean levels in PHA-stimulated samples over the whole time course are summarized in Table 5. In cohort one, a consistent fall in PHA-stimulated IL-6 was seen in 11 of 17 patients; in cohort two, 8 of 13 patients showed a fall in PHA-stimulated IL-6 levels (pretreatment values compared to end-of-treatment values). A statistically significant decrease in PHA-stimulated IL-6 was demonstrated (pretreatment compared with end-of-treatment values) in cohort one (P = .03) but not in cohort two (P = .60). Regression analysis did not reveal any significant differences between the two cohorts (P = .78).

    Whole-blood cytokine-release assay (CCL2). A whole-blood cytokine-release assay was performed to assess the effect of etanercept on the release of CCL2 in the whole blood when stimulated by PHA (Fig 5). At each time point, CCL2 levels were measured in either the presence or absence of PHA as described previously. Mean levels in PHA-stimulated samples over the whole time course are summarized in Table 5. A consistent fall in PHA-stimulated CCL2 levels was seen in the patients (cohort one, 13 of 17; cohort two, 7 of 13). A statistically significant decrease in PHA-stimulated CCL2 was demonstrated (pretreatment compared with end-of-treatment values) in cohort one (P = .006) but not in cohort two (P = .40). Regression analysis did not reveal any significant differences between cohorts (P = .22).

    Plasma levels of CCL2, MMP-3, sTNFRI, and sE-selectin in unstimulated samples. Serial blood samples from the first 13 patients in cohort one were available for analysis. Mean plasma levels of CCL2, MMP-3, sTNFRI, and sE-selectin are summarized in Table 6. A significant decrease in sE-selectin was demonstrated (pretreatment compared with day 7, P = .02). For sTNFRI, a significant reduction was also demonstrated (pretreatment compared with day 1, P = .02).

    DISCUSSION

    This is the first trial of a TNF- inhibitor in ovarian cancer. Etanercept was found to be safe and well tolerated in this study. Adverse effects reported were similar to those seen in patients with RA. Most adverse effects required no medical intervention, although etanercept was stopped in one patient because of the development of Sj?gren's syndrome.

    Because etanercept is likely to be cytostatic rather than cytotoxic, we expected to see SD rather than partial or complete responses. In fact, this was demonstrated in six patients. Patients were evaluable for disease response if they had at least 12 weeks of therapy (a total of 18 patients). Overall, 33% of patients achieved SD (57% [4 of 7] of patients in cohort two achieved SD, and 18% [2 of 11] of patients in cohort one achieved SD). Interesting observations were made in patients who achieved SD. First, these patients had stabilization or reduction in CA-125 levels with therapy. Second, they also had overall improvements in QOL compared to nonresponders, suggesting clinical benefit with therapy. Adverse effects caused by etanercept had little impact on QOL, which is in contrast to conventional chemotherapy used in recurrent ovarian cancer that causes significant morbidity in patients. In patients, withdrawal of cytotoxic treatment can lead to a period of stabilization of disease; however, this is unlikely to explain our result, because all patients had documented disease progression (as assessed by computed tomography and rising CA-125 levels) and had a minimum 1-month washout period before starting etanercept.

    A substantial effect of etanercept on the TNF- pathway was demonstrated for the first time in patients with ovarian cancer in our study. A significant increase in immunoreactive TNF- was seen in patients within 24 hours of initiation of etanercept therapy. This rapid elevation of TNF- is due to binding to etanercept and continued at all subsequent time points (days 7, 28, 56, and 84). TNF- levels reached statistical significance in both cohorts one and two, confirming the biologic effect of etanercept. In addition, the elevated TNF- levels in peripheral blood were not biologically active in a bioassay. Therefore, it is unlikely that the levels of biologically active TNF- are elevated in the tumor microenvironment with etanercept treatment, given our results from the peripheral blood assays and those reported in patients with RA. Studies in rheumatoid joints showed reduction in cytokines such as TNF- with etanercept (reviewed in refs 38 and 39). However, biologic marker analysis in serial tumor samples (or ascitic fluid samples) in future studies may be able to address this issue directly.

    We also evaluated whether the depletion of biologically active TNF- could have a functional effect on target cells. There was a significant decline in the ability of PHA to stimulate IL-6 and CCL2 in peripheral blood that correlated with duration of treatment, which reached statistical significance in cohort one but not in cohort two. This could be because of variability between patients in the amount of chemotherapy that patients received before etanercept, which could affect lymphocyte function. In patients who had SD, there was a consistent time-dependent decrease in IL-6 and CCL2, suggesting a correlation with response (Fig 6). The effect of etanercept is also suggested by stable CA-125 levels over time in these patients when they were doubling before treatment. Moreover, this reduction is consistent with those seen in patients with RA who show reduction in serum levels of IL-6, chemokines, and acute phase proteins.37 Given the substantial biologic evidence for the success of anti-TNF therapy in RA joints (including diminished cytokine/chemokine [TNF, IL-6, CCL2] production, reduced levels of vascular endothelial growth factor and angiogenesis, and restoration of depressed T-cell immune responses), a similar cytokine response within the tumor microenvironment is likely to have contributed to the prolonged SD seen in six patients in our study. Similarly, in unstimulated plasma samples, there was a significant decrease in plasma sE-selectin and sTNFRI levels.

    We recently reported a phase II study of etanercept in patients with metastatic breast cancer.42 Sixteen patients received etanercept 25 mg SC twice weekly. The most common adverse effects were injection-site reactions, fatigue, loss of appetite, nausea, headache, and dizziness. A brief period of SD was seen in one patient, which lasted for 16.4 weeks. Immunoreactive TNF- was elevated within 24 hours of therapy and persisted until the end of treatment (days 7, 28, 56, and 84). PHA-stimulated cytokine-release assay showed a consistent decrease in IL-6 and CCL2 levels compared with pretreatment values in serial blood samples (days 1, 7, 28, 56, and 84).42 The role of etanercept has also been investigated in hematologic disorders. Although etanercept has shown some clinical activity in myelodysplasia, myelofibrosis, and chronic lymphocytic leukemia,43,44 it did not produce any disease responses in myeloma.45

    A key issue for future analysis is whether higher or more frequent doses of etanercept could produce more tissue depletion in cancer patients and whether blockade with other anti-TNF approaches (some with longer half-lives) could enhance biologic effectiveness. D2E7 is a fully human anti-TNF monoclonal antibody and an irreversible inhibitor of TNF-.46 Trial of D2E7 in ovarian cancer is likely to produce interesting results. Other agents in various stages of development include infliximab (a chimeric immunoglobulin G1 monoclonal antibody targeted against TNF), pegylated recombinant human sTNFR type 1, pegylated humanized anti-TNF fragment (CDP870), and TNF synthesis inhibitors (p38 kinase inhibitors).46 Other potential roles of etanercept include its use in antiangiogenic combinations to block multiple pathways. Etanercept in combination with low-dose methotrexate chemotherapy has been evaluated in patients with RA.47 Combination therapy was found to be safe and efficacious. Whether a combination of etanercept with "metronomic" scheduling of chemotherapy (eg, low-dose cyclophosphamide and methotrexate)48 is likely to produce an enhanced antiangiogenic effect in patients with cancer remains to be established.

    We provide evidence for the biologic activity and safety of etanercept in recurrent ovarian cancer. Our data suggest possible clinical activity that must be confirmed in future studies.

    Authors' Disclosures of Potential Conflicts of Interest

    The authors indicated no potential conflicts of interest.

    Acknowledgment

    We acknowledge the support of Wyeth for the provision of etanercept. We are grateful to patients for consenting to participate in the study.

    NOTES

    Supported by Cancer Research UK.

    Authors' disclosures of potential conflicts of interest are found at the end of this article.

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《临床肿瘤学医学期刊》2005年9月第23卷第9期