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Keywords:

  • renal carcinoma;
  • COX-2;
  • VEGF;
  • bFGF;
  • interferon alpha

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Cyclooxygenase-2 (COX-2) plays a major role in the development of cancer through numerous mechanisms. COX-2 is expressed in the majority of renal cell carcinoma (RCC) tumors and correlates with stage, grade, and microvessel density. Based on potential additive or synergistic antitumor effects, interferon-alpha (IFNα) and celecoxib, an oral COX-2 inhibitor, were given to metastatic RCC patients in a Phase II trial.

METHODS

Patients with untreated, metastatic RCC received IFNα 3 million units (MU) daily and celecoxib 400 mg orally (p.o.) twice daily continuously until disease progression or unacceptable toxicity. Pretreatment, paraffin-embedded RCC tumor samples were immunohistochemically stained for COX-2 expression and plasma basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) levels were assayed to determine predictive or prognostic potential.

RESULTS

There were three partial responses among 25 patients treated (objective response rate, 12%; 95% confidence interval [CI], 3–31%). The observed median time to disease progression (TTP) for the entire cohort was 3.3 months. A significant association between maximal COX-2 staining and clinical response was observed: all patients who experienced an objective response demonstrated 3+ COX-2 tumor immunostaining (trend test: P = 0.03). Therapy was well tolerated without cardiac or other notable toxicity.

CONCLUSIONS

The addition of celecoxib to IFNα did not increase the objective response rate or TTP of this unselected cohort. Maximal COX-2 tumor immunostaining may identify RCC patents more likely to achieve clinical benefit with COX-2 inhibition in combination with IFNα. Further investigation of this combination in 3+ COX-2-overexpressing RCC tumors is warranted. Cancer 2006. © 2005 American Cancer Society.

Metastatic renal cell carcinoma (RCC) is a largely incurable disease for which few effective treatment options exist. Cytokines such as interleukin-2 (IL-2) and interferon-alpha (IFNα) are standard initial systemic therapy in metastatic RCC, yet have limited effects on RCC tumor progression in the majority of patients.1 The combination of cytokines with novel agents is warranted to investigate the potential enhancement of clinical activity.

Interferon-alpha exerts an antitumor effect in RCC through various mechanisms including immune cell activation and direct antiproliferative effects.2, 3 Additionally, studies in metastatic RCC cell lines support an antiangiogenic effect of IFNα through down-regulation of basic fibroblast growth factor (bFGF) expression.4 Daily administration of low-dose IFNα has produced significant inhibition of tumor growth, vascularization, and angiogenesis as measured by serum bFGF levels and tumor vessel density in a murine bladder cancer model.5, 6

Cyclooxygenase-2 (COX-2) is an enzyme involved in the formation of prostaglandin E2 (PGE2) from arachadonic acid. COX-2-derived prostaglandins may play a major role in the development of cancer through numerous mechanisms, including stimulation of tumor cell growth, inhibition of apoptosis, and neovascularization.7, 8 Inhibition of COX-2 has been shown in both preclinical and clinical studies to inhibit angiogenesis and tumorigenesis in colon, esophageal, and other solid tumors.9, 10

COX-2 expression may be involved in RCC pathogenesis. COX-2 overexpression has been demonstrated in an RCC cell line (OS-RC-2), and suppression of tumor growth and angiogenesis with COX-2 inhibition was observed when studied in an orthotopic murine model.11 Further, COX-2 expression in human RCC and its relation to clinicopathologic features have been described. Investigation of immunohistochemical COX-2 expression in nephrectomy specimens from RCC patients of all disease stages has been reported.12–14 COX-2 expression was observed in 53.4–100% of surgical specimens in these series. Higher COX-2 expression was significantly associated with higher stage and grade.12, 13 COX-2 expression was also reported as significantly associated with increased microvessel density and Ki-67 labeling index, a marker of cell proliferation.12 COX-2 may thus promote RCC angiogenesis and tumor growth and represents a viable therapeutic target.

Celecoxib is a selective COX-2 inhibitor that has been shown in clinical arthritis trials to lead to effective inhibition of the COX-2 enzyme.15 An analysis of several clinical studies employing celecoxib in doses up to 400 mg twice daily involving over 13,000 subjects reported a low incidence of renal side effects, with peripheral edema (2.1%), headache (0.8%), and exacerbation of preexisting hypertension (0.6%) being the most frequently observed, at an incidence rate similar to placebo.16 More recent data has also identified a potential cardiovascular risk to celecoxib with prolonged use.17

Based on these data, a Phase II trial was conducted in untreated, metastatic RCC patients to determine the clinical effect of combination therapy with low dose, daily IFNα and celecoxib. A substantial improvement in objective response rate compared with IFNα monotherapy was felt to be unlikely. Thus, the present trial sought to demonstrate a prolonged time to disease progression (TTP) compared with historical controls as the signal of activity that would prompt further investigation. Immunohistochemical staining of pretreatment tumor samples for COX-2 expression and measurement of plasma angiokine levels (bFGF and vascular endothelial growth factor [VEGF]) were undertaken to determine their predictive and/or prognostic potential.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patient Selection

Eligible patients had histologically or cytologically confirmed metastatic renal cell carcinoma without prior systemic treatment. Patients had measurable disease defined by Response Evaluation Criteria in Solid Tumors (RECIST) criteria.18 Life expectancy of greater than 3 months and Eastern Cooperative Oncology Group (ECOG) performance status 0, 1, or 2 were required. Patients had normal organ and marrow function as defined by the following: leukocytes ≥ 3000/μL, absolute neutrophil count ≥ 1500/μL, platelets ≥ 75,000/μL, total bilirubin within normal institutional limits, alanine aminotransferase (AST) and aspartate aminotransferase (ALT) ≤ 1.5 × institutional upper limit of normal, creatinine within normal institutional limits, or creatinine clearance ≥ 40 mL/min/1.73 m2 (calculated by Cockcroft-Gault formula) for patients with creatinine levels above institutional normal limits.

Patients with significant cardiovascular disease including congestive heart failure (New York Heart Association Class III or IV), angina pectoris requiring nitrate therapy, or myocardial infarction within the last 6 months were excluded. Patients who had received radiotherapy within 4 weeks before entering the study were excluded. Patients with any current or past history of central nervous system (CNS) metastases were excluded. Patients with clinical signs or symptoms of CNS metastases underwent a magnetic resonance imaging (MRI) or infused computerized tomography (CT) scan of the brain before enrollment. Patients may not have taken any nonsteroidal antiinflammatory drugs (NSAIDs, including aspirin or any aspirin-containing products) or corticosteroids within 2 weeks before beginning therapy or at any time during the protocol treatment. Concurrent antineoplastic therapy including investigational agents was prohibited. Patients with uncontrolled intercurrent illness, pregnancy, or known human immunodeficiency virus (HIV) positivity were excluded. All patients signed a written informed consent approved by the UCSF Committee on Human Research.

Treatment

Eligible patients received 3 million units (MU) of IFNα subcutaneously daily and 400 mg twice daily of celecoxib (Celebrex, Pfizer, Groton, CT) orally continuously throughout each consecutive 4-week cycle. CT scans for disease assessment were performed at baseline and after every 2 cycles. Bone scan was performed at baseline for all patients and repeated after every 2 cycles only if indicative of metastases at baseline or if signs or symptoms of bone metastases developed. Disease response and progression was defined using the RECIST criteria.18 Patients continued on therapy until progressive disease or unacceptable toxicity. Time to disease progression (TTP) was defined as the period from the first day of treatment until the RECIST-defined criteria for progressive disease (PD) were met or until the patient was removed from protocol therapy, whichever came first.

Dose modification

Toxicities were graded according to the CTEP Common Toxicity Criteria v. 3.0. Any Grade 3 or 4 toxicity attributable to therapy required IFNα and celecoxib to be withheld. If toxicity resolved to ≤ Grade 1 within 4 weeks, IFNα was reinstituted with one dose level reduction and celecoxib was reinstituted at 400 mg twice daily. IFNα dose level–1 was 2 MU of IFNα daily and IFNα dose level–2 was 1 MU of IFNα daily. If toxicity did not resolve to ≤ Grade 1 within 4 weeks, or if Grade 3 or 4 toxicity occurred after two IFNα dose reductions had been instituted, the patient was removed from the protocol therapy. Once IFNα was reinstituted with a dose reduction, there was no dose reescalation. There were no dose reductions of celecoxib. Missed doses of IFNα and celecoxib were not made up.

Immunostaining of tumor for COX-2 expression

Pretreatment paraffin tumor samples from prior nephrectomy or tumor biopsy were retrieved. Tumor sections were cut at 5 μm from the paraffin block, mounted on polylysine-plus slides, heated to 60 °C for 30 minutes, then deparaffinized and rehydrated through graduated ethanol washes. Antigen retrieval was then performed by boiling in a 10-mM citrate buffer (pH 6.0) for 10 minutes using a microwave. Blocking was accomplished using normal horse serum incubation at room temperature (RT) for 30 minutes. Anti-COX-2 mouse monoclonal antibody (Cayman #160112, Ann Arbor, MI) was then applied at 1:200 dilution and incubated at 4 °C overnight. Excess antibody was then washed off and biotinylated antimouse antibody applied for 30 minutes at RT. The excess was removed and antibody detected using an avidin-biotin complex horseradish peroxidase/DAB-hydrogen peroxide method. Anti-COX-2-stained slides were then reviewed and scored for staining intensity (on a scale of 0 for no stain to 3+ for intense complete membrane and cytoplasmic staining). Percent of tumor cells staining at any degree of intensity was also recorded, along with stromal and normal kidney tissue staining. Control tissue used for immunostaining was a breast tumor with known overexpression of COX-2 enzyme by immunohistochemistry, Western blot, and reverse transcriptase polymerase chain reaction (RT-PCR). Internal positive control was adjacent normal kidney (present in 10 cases) and internal negative control was tumor stroma (negative in all cases, but two; both with 1+ tumor staining) and stromal blood vessels (negative in all cases). A single pathologist (J.S.), blinded to clinical outcome, scored the staining intensity for all patients.

Measurement of plasma angiokine levels

Plasma samples were collected from patients at baseline, after every 2 cycles of therapy, and at the time of disease progression or response. Plasma samples were frozen after collection and stored at –70 °C until the time of assay. Samples were thawed and warmed to RT immediately before assaying. For bFGF analysis, triplicate aliquots of 150 μL were then removed from each plasma sample for ELISA (enzyme-linked immunoadsorbancy assay) as previously described.19 bFGF levels were determined using a commercially available, high-sensitivity assay plate (Quantikine HS) from R&D Systems (Minneapolis, MN). Final bFGF concentrations in unknowns were determined colorimetrically from absorbance readings at 490 nm on a Bio-Rad Model 3550 microplate reader (Hercules, CA), utilizing a standard curve generated from serial dilutions of recombinant human bFGF ranging from 1–64 pg/mL, also assayed in triplicate.

For VEGF, a fluorimetric assay was used to measure VEGF in human plasma, as previously described,20 with two modifications: 1) a change in the number of washes from 3 to 6 after blocking, and 2) a change in the sample diluent pH from 6.3 to 8.2. The standard curve range for this modified assay was 2–128 pg/mL. Based on a minimum 1/20 sample dilution, the minimum quantifiable concentration was 40 pg/mL VEGF in neat RCC plasma.

For the purpose of analysis of angiokine characteristics, patients were retrospectively categorized into three groups: 1) patients with progressive disease (PD) at the first disease restaging at the end of the second cycle; 2) patients with a best response of stable disease (SD) on disease on restaging; and 3) patients who achieved a best response of partial response (PR). A change in angiokine level is reported as both the median percent change from baseline to the end of Cycle 2 (the first disease reevaluation point) and median overall percent change. Overall change was calculated as the percentage difference between the baseline value and the value of the final collection point, which was at the time of disease progression for all patients.

Statistical Considerations

The total required sample size for this trial was based on the primary endpoint of determining the median TTP for metastatic RCC patients treated with IFNα and celecoxib. The null hypothesis for this trial was that untreated metastatic RCC patients receiving IFNα-based therapy have a median TTP of 4.7 months.21 We hypothesized that the combination of IFNα and celecoxib would produce a 50% increase in the median TTP (to 7 mos) versus historical controls. Therefore, assuming an exponential failure rate, accrual of 62 patients was required to detect such an improvement. This assumed a level of significance of 0.05 for a directional test and power of 0.80, with total accrual completed in 15–16 months with an additional 3 months of followup after the last patient was enrolled.

An early stopping rule for efficacy was also employed. If, after accrual of 25 patients, the observed median TTP was less than 4.8 months, the lower 95% confidence bound under the alternative hypothesis of a 7-month TTP, accrual would be halted and the regimen declared unlikely to prolong TTP by 50% compared with historical controls.

Descriptive statistics were calculated to characterize patients, clinical outcome, and COX-2 staining of archival tumor tissue. The Kaplan–Meier product limit method was used to estimate the probability of disease progression measured from the start of protocol therapy with subsets compared using the log-rank test.

Pretreatment COX-2 immunostaining from archival specimens was correlated with objective response and time to progression. A nonparametric trend test that adjusts for tied observations developed by Cuzick22 was used to analyze the association between COX-2 staining, tumor grade, and response. An exploratory analysis comparing pre- and posttreatment VEGF and bFGF levels in responders versus nonresponders was also undertaken. The nonparametric Wilcoxon matched pair test was used to compare the change in angiokine levels from baseline until Cycle 2 or last measurement and the Kruskal–Wallis test was used to compare the distributions of change by best response achieved. The Spearman correlation coefficient was calculated to assess the relation between angiokine features of change (e.g., baseline and percent change at Cycle 2).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patient Characteristics

Between July 2002 and March 2004, 25 patients were enrolled on this Phase II study. This patient cohort was typical of a metastatic RCC population with a preponderance of lung and lymph node metastases and the majority of patients with three or more metastatic sites (Table 1). Patients were also prospectively characterized by the number of previously described adverse risk factors for untreated metastatic RCC patients entering IFNα-based therapy including: Karnofsky performance status < 80%, hemoglobin ≤ the lower limit of normal, lactate dehydrogenase ≥ 1.5 × upper limit of normal (ULN), corrected serum calcium ≥ 10 mg/dL, and time from diagnosis of RCC to metastases < 1 year.21 By these criteria, 7 (28%) patients had no risk factors (favorable risk), 16 (64%) patients had 1–2 risk factors (intermediate risk), and 2 (8%) patients had 3+ risk factors (poor risk).

Table 1. Patient Characteristics
CharacteristicNo. of patients, n = 25
Age, yrs 
Median (range)60 (29–87)
Gender 
 Male18 (72%)
 Female7 (28%)
ECOG performance status 
 014 (56%)
 110 (40%)
 21 (4%)
Site of metastases 
 Lung19 (76%)
 Lymph node11 (44%)
 Liver7 (28%)
 Adrenal gland7 (28%)
 Bone6 (24%)
 Other15 (60%)
No. of metastatic sites 
 15 (20%)
 28 (32%)
 ≥ 312 (48%)
Prior therapy 
 Nephrectomy23 (92%)
 Radiotherapy, any5 (20%)
Histologic subtype 
 Clear cell22 (88%)
 Papillary2 (8%)
 Chromophobe1 (4%)
No. of risk factors21 
 0, favorable risk7 (28%)
 1, intermediate risk16 (64%)
 2–3, poor risk2 (8%)

Clinical Efficacy

Three partial responses were observed (objective response rate, 12%; 95% confidence interval [CI], 3–31%). Two of these patients came off the study at Day 440 and Day 413 for disease progression; the third patient came off the study at Day 167 due to Grade 4 neutropenia with an ongoing partial response. This patient continues on IFNα in combination with celecoxib 200 mg QD off-study and is currently in PR 670 days from the start of protocol therapy.

The observed median TTP for the entire cohort was 3.3 months (range, 0.2–14.3 mos). The trial was suspended after accrual of 25 patients to evaluate efficacy criteria for continued enrollment. The observed distribution of risk factors indicated an expected weighted median TTP of 5.8 months. The 95% CI for the alternative hypothesis of a 50% increase over 5.8 months excluded the observed median TTP of 3.3 months, and thus a 50% improvement in the TTP for the entire study cohort compared with historical controls was unlikely. As such, the trial was closed to further accrual.

COX-2 Expression and Clinical Outcome

Twenty paraffin-embedded RCC tumor samples were available for analysis of COX-2 expression. The tissue from five patients was unavailable due to local pathology department restrictions or disposal of previously obtained tissue. In general, glomeruli and proximal renal tubules exhibited 3+ staining. Figure 1 depicts representative staining of 1+, 2+, and 3+ intensity in RCC tumors. Nineteen of 20 (95%) tumors evaluated demonstrated some degree of COX-2 expression (Table 2). No significant COX-2 staining was observed in the stroma or stromal blood vessels in this series.

thumbnail image

Figure 1. Representative COX-2 tumor immunostaining in RCC tumors. (A) 1+ COX-2 immunostaining, (B) 2+ COX-2 immunostaining, and (C) 3+ COX-2 immunostaining. All photos taken at original magnification ×200.

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Table 2. COX-2 Expression and Clinical Outcome in Metastatic RCC
Histologic subtypeGradeMaximum COX-2 tumor staining (% of tumor with maximum staining)Overall % of tumor with COX-2 staining of any degree (weak to 3+)Best clinical response (duration)
  1. COX-2: cyclooxygenase-2; PR: partial response; SD: stable disease; PD: progressive disease; RCC: renal cell carcinoma.

  2. Patients are ordered from highest to lowest degree of maximal COX-2 tumor staining.

Clear cell43+ (50)100PR (670+ days)
Clear cell33+ (20)60PR (440 days)
Clear cell43+ (10)80SD (112 days)
Clear cell33+ (10)60PR (413 days)
Chromophobe22+ (40)90SD (341 days)
Clear cell32+ (10)80SD (140 days)
Clear cell32+ (10)75SD (238 days)
Clear cell32+ (10)50PD (73 days)
Papillary31+ (40)70SD (168 days)
Clear cell21+ (20)60PD (34 days)
Clear cell11+ (20)20SD (186 days)
Clear cell21+ (10)20SD (284 days)
Clear cell21+ (5)70SD (279 days)
Clear cell21+ (5)30PD (55 days)
Clear cell21+ (5)20SD (36 days)
Clear cell31+ (5)10SD (56 days)
Clear cell2weak (70)70SD (82 days)
Clear cell2weak (60)60SD (234 days)
Clear cell2weak (30)30SD (56 days)
Clear cell2None0PD (91 days)

A significant association between maximal COX-2 staining and objective response was observed. All three patients who experienced an objective response demonstrated 3+ maximal COX-2 staining (trend test: P = 0.03). Time to disease progression was also examined in relation to COX-2 expression. Patients with 2+ or 3+ COX-2 maximal immunostaining had a median TTP of 167 days with therapy versus 82 days for patients with < 2+ maximal COX-2 immunostaining. In addition, patients with ≥ 50% overall COX-2 immunostaining had a median TTP of 168 days with therapy versus 75 days in patients with < 50% overall COX-2 immunostaining.

There was an increasing trend in maximal COX-2 intensity with an increase in the number of risk factors for disease progression21 (0 vs. 1–2 vs., 3 or more) (P = 0.06) and with increasing tumor grade (P = 0.01). Thus, patients with higher COX-2 immunostaining intensity would be expected to have a shorter TTP, not longer as was observed in this study.

Plasma Angiokine Levels

Plasma bFGF was measured in all 25 patients at baseline. Twenty-four patients had at least two bFGF measurements after the start of protocol therapy and were thus included in the calculations of bFGF changes. The excluded patient had a best response of PD and only a single bFGF sample collection. Plasma VEGF was measured in 24 patients at baseline. One patient had VEGF values below quantifiable concentration (40 pg/mL) on all three samples obtained, and thus this patient was excluded from all analyses of plasma VEGF. Twenty-one patients had at least two VEGF measurements after the start of protocol therapy and were included in the calculations of VEGF changes. In addition to the one patient with undetectable values described above, three patients were removed from study for PD (two patients) or toxicity (one patient) before any posttreatment sample collections and were excluded from analysis of VEGF changes.

Baseline angiokine levels are reported, as well as change in angiokine levels with therapy (Tables 3, 4). There was no difference in the distribution of baseline bFGF or VEGF by best objective response achieved. There was a greater overall percent change from baseline in bFGF with improved objective response that was of borderline statistical significance (trend test: P = 0.06) (Tables 3, 4). No significant association between change in VEGF measurements and objective response was recognized. In general, however, patients achieving a PR had declining levels of both bFGF and VEGF with therapy, patients achieving SD had stable levels of these angiokines, and patients with PD had increasing levels of these angiokines, but small subset sample sizes limited the ability for statistical association with response.

Table 3. Plasma Basic FGF Characteristics
Angiokine characteristicEvaluable cohort (n = 24)Patients with best response of PD (n = 5)Patients with best response of SD (n = 16)Patients with best response of PR (n = 3)
  1. bFGF, basic fibroblast growth factor. PR, partial response; SD, stable disease; PD, progressive disease;

Median baseline bFGF (pg/mL)6.06.75.014.4
Median % change in bFGF from baseline to end of cycle 2−3.9%+19.4%−1.0%+4.5%
Median overall % change in bFGF−3.9%+12.3%−1.3%−66.6%
Table 4. Plasma Basic VEGF Characteristics
Angiokine characteristicEvaluable cohort (n = 21)Patients with best response of PD (n = 4)Patients with best response of SD (n = 14)Patients with best response of PR (n = 3)
  1. VEGF: vascular endothelial growth factor; PR: partial response; SD: stable disease; PD: progressive disease.

Median baseline VEGF (pg/mL)98.0102.093.548.0
Median % change in VEGF from baseline to end of cycle 2−4.3%+3.7%−2.9%−24.0%
Median overall % change in VEGF−12.3%+3.7%−2.2%−40.8%

Toxicity

Therapy was generally well tolerated by patients (Table 5). Cytopenias were as frequent as expected for IFNα monotherapy and managed with dose modification as described above. Constitutional symptoms were minimal. Cardiac toxicity consisted of a single episode of atrial fibrillation observed in a 72-year-old patient with a history of paroxysmal atrial fibrillation, hypertension, hypercholesterolemia, and coronary artery disease status post-coronary artery bypass grafting. This patient had no active cardiac symptoms and was in normal sinus rhythm at baseline without medication. The patient experienced dyspnea 8 days into protocol therapy and was found to be in atrial fibrillation. The patient was placed on amiodarone and returned to normal sinus rhythm without further dysrhythmic or other cardiovascular episodes during an additional 5 months of protocol therapy. No additional cardiovascular toxicity including hypertension or thromboembolic events was observed in other patients. No edema, elevations of creatinine, or other renal toxicity was noted.

Table 5. Treatment-Related Toxicity
 Grade 2 no. (%)Grade 3 no. (%)Grade 4 no. (%)
Hematologic   
 Neutropenia6 (24)01 (4;)
 Leukopenia8 (32)1 (4)0
 Thrombocytopenia4 (16)1 (4;)0
Nonhematologic   
 Fatigue8 (32)5 (20)0
 Elevated AST/ALT6 (24)1 (4)0
 Dizziness03 (12)0
 Nausea2 (8)1 (4)0
 Rash2 (8)00
 Myalgia2 (8;)00
 Hypophosphatemia1 (4)1 (4)0
 Hyperkalemia1 (4)2 (8)0
 Atrial fibrillation01 (4)0
 Hypothyroidism01 (4)0

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Given the established modest clinical effect of low-dose IFNα in RCC and the potential pathogenic role of COX-2, we attempted to augment the IFNα antitumor effect in RCC through combination with COX-2 inhibition. In this unselected cohort, the objective response observed was within the range expected for IFNα monotherapy and the observed median TTP indicated that this combination is unlikely to produce a TTP in RCC patients 50% greater than historical controls.

Staining of archived RCC tumors for COX-2 expression, however, revealed the potential biologic and therapeutic relevance of COX-2 expression in RCC. An association was observed between maximal COX-2 staining and objective response. All patients who experienced an objective response demonstrated 3+ maximal COX-2 tumor staining. Additionally, time to disease progression appeared longer in patients with 2+ or 3+ COX-2 staining and in patients with > 50% overall COX-2 staining despite worse prognostic characteristics. Consistent with prior reports, our series demonstrated overall COX-2 expression in the vast majority (95%) of primary tumors from patients with metastatic disease and maximal COX-2 immunostaining (3+) in a substantial percentage (20%) of patients.

These intriguing findings must be cautiously interpreted given several limitations of this study. The objective responses and a longer time to progression in higher COX-2 staining tumors is drawn from a very limited sample size. These observations in 3+ COX-2 overexpressors are a signal, however, that generates the hypothesis that COX-2 inhibition in combination with IFNα, may have therapeutic benefit in this subset of patients. This hypothesis requires prospective validation in a larger number of patients with 3+ COX-2 expression. It is also possible that maximal COX-2 expression is simply a marker for IFNα responsiveness (with or without direct relevance to the biology of IFNα effect). We retrospectively characterized the maximal COX-2 staining of primary tumors from nine patients not on this clinical trial with metastatic RCC who previously achieved an objective PR to IFNα monotherapy. These patients demonstrated a range of COX-2 maximal staining as follows: weak (2 patients), 1+ (6 patients), and 3+ (1 patient). Thus, it is not apparent from this small sample set that 3+ COX-2 staining predicts response to IFN monotherapy. Further, it is unknown whether similar clinical benefit would have been observed in 3+ COX-2 overexpressing tumors treated with celecoxib alone. Importantly, primary tumor expression of a given protein does not necessarily correlate with protein expression in metastatic tissue. We retrospectively characterized COX-2 expression in matched RCC primary and metastatic tissue from eight patients. Six (75%) patients had identical maximal COX-2 staining intensity. The two mismatched patients had 2+ COX-2 staining in the primary tumor and 3+ COX-2 staining in the metastatic tissue. Lastly, the reproducibility of COX-2 staining in RCC and validation of the discriminatory ability of COX-2 intensity in relation to clinical outcome awaits further investigation.

In addition, we sought an improvement in TTP compared with historical controls as the signal of activity for further development of this regimen. Clearly, comparison with historical controls is fraught with methodologic problems, in part due to the difficulty in establishing a null hypothesis before the risk factor distribution of the study cohort is known. Nonetheless, unfavorable comparison with historical controls is a signal that a significant disease stabilizing effect of this combination therapy is unlikely and precludes further development in an unselected RCC population. Last, PGE2 and its inhibition by celecoxib may also have effects on dendritic cell and T-cell function that could impact an antitumor immune response induced by IFNα.23, 24 No studies were undertaken to investigate this or other potential alternative mechanisms of antitumor effect. Lastly, limited sample size precluded statistical significance for some of the differences observed and highlights the need for prospective validation of these observations in a larger cohort of patients selected for maximal COX-2 expression.

To characterize the potential antiangiogenic effect of this therapy, measurement of plasma angiokines was undertaken. Although interesting trends in differential angiokine change with therapy was observed, no statistically significant association was made. In addition, baseline angiokine levels did not correlate with clinical outcome. The lack of effect for this biomarker is perhaps due to limited sample size, lack of effect of the intervention on these biomarkers, or limitations of the biomarkers to predict clinical response. Although elevated serum bFGF and VEGF levels have been demonstrated in RCC patients compared with controls,25–29 no correlation of baseline levels of either of these angiokines with clinical outcome has been shown in multivariate analysis.29, 30 Further, no predictive or prognostic value of serum VEGF levels (baseline or change with therapy) has been demonstrated in a clinical trial with single-agent anti-VEGF therapy in RCC.31, 32 One report of 12 metastatic RCC patients treated with IFNα describes higher median baseline bFGF levels and a greater percent decrease of bFGF at 8 weeks in patients with an objective response.33 We also observed that patients with a best response of PR demonstrated higher baseline bFGF levels and a greater overall decrease with therapy. It is apparent from the present and prior studies that macroscopic measurement of circulating VEGF and bFGF may not provide clinically relevant information in RCC patients treated with IFNα-based therapy. Examination of larger datasets and further investigation into alternative biomarkers in RCC is needed.

In summary, the observation of objective responses and longer TTP in patients with maximal COX-2 staining tumors generates the hypothesis that enrichment of a subpopulation of RCC patients more likely to respond to COX-2 inhibition is possible. Further, prospective investigation is warranted.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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