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Clinical Cancer Research Vol. 11, 6634-6640, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Targeting Cyclooxygenase-2 in Recurrent Non–Small Cell Lung Cancer: A Phase II Trial of Celecoxib and Docetaxel

Ildiko Csiki1,4, Jason D. Morrow2,4, Alan Sandler2,4, Yu Shyr3,4, John Oates2,4, Myles K. Williams2,4, Thao Dang1,2,4, David P. Carbone1,2,4 and David H. Johnson2,4

Authors' Affiliations: Departments of 1 Cancer Biology, 2 Medicine (Hematology-Oncology and Clinical Pharmacology), and 3 Biostatistics, and 4 the Vanderbilt-Ingram Cancer Center, Nashville, Tennessee

Requests for reprints: David H. Johnson, Vanderbilt-Ingram Cancer Center, 777 Preston Research Building, Nashville, TN 37232-6307. Phone: 615-343-9454; Fax: 615-936-2236; E-mail: david.johnson{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in prostaglandin (PG) synthesis and is overexpressed in 70% to 90% of non–small cell lung cancers (NSCLC). Preclinical studies suggest inhibition of COX-2 can enhance the cytotoxic effect of docetaxel. To test this concept clinically, we administered celecoxib (400 mg p.o. twice daily) plus docetaxel (75 mg/m2 every 3 weeks) to a cohort of patients with recurrent, previously treated NSCLC. Patients first received single agent celecoxib for 5 to 10 days to ascertain the effectiveness of COX-2 inhibition, which was determined by measuring pre- and post-celecoxib levels of urinary 11{alpha}-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major metabolite of prostaglandin E2 (PGE2). We enrolled 56 patients (35 men, 21 women; median age, 61 years). All patients had received at least one prior chemotherapy regimen. The overall response rate was 11% and median survival was 6 months, similar to that observed with docetaxel alone. Pre-celecoxib urinary PGE-M decreased from a mean level of 27.2 to 12.2 ng/mg Cr after 5 to 10 days of celecoxib (P = 0.001). When grouped by quartile, patients with the greatest proportional decline in urinary PGE-M levels experienced a longer survival compared to those with no change or an increase in PGE-M (14.8 versus 6.3 versus 5.0 months). Our data suggest that combining celecoxib with docetaxel using the doses and schedule employed does not improve survival in unselected patients with recurrent, previously treated NSCLC. However, in light of the apparent survival prolongation in the subset with a marked decline in urinary PGE-M levels, further investigation of strategies designed to decrease PGE2 synthesis in NSCLC seems warranted.

Cyclooxygenase-2 (COX-2), a cyclic endoperoxidase that catalyzes the rate-limiting step in prostaglandin (PG) synthesis, is frequently overexpressed in human premalignant pulmonary lesions such as atypical adenomatous hyperplasia and carcinoma in situ, as well as in invasive carcinomas (1–4). Preclinical and clinical data indicate that tumors with up-regulation of COX-2 synthesize high levels of prostaglandin E2 (PGE2; refs. 5–11). In turn, high PGE2 levels are associated with increased production of proangiogenic factors, altered immune responses, and enhanced metastatic potential (12–16). These findings suggest that increased COX-2 expression may play a significant role in the development and growth of malignancies, such as non–small cell lung cancers (NSCLC), and possibly in the acquisition of an invasive and metastatic phenotype (17, 18). Thus, selective inhibition of COX-2 could prove useful both in understanding the role of eicosanoids in lung cancer pathogenesis as well as in the management of established malignancies (19, 20).

Docetaxel is modestly beneficial in the treatment of recurrent NSCLC (21). Notably, recent preclinical data indicate that the cytotoxic effect of docetaxel is enhanced in the presence of a selective COX-2 inhibitor (22). Based on these findings, we undertook a phase II study in which docetaxel was combined with the selective COX-2 inhibitor celecoxib. Because measuring intratumoral levels of PGE2 is impractical in metastatic NSCLC, we assessed levels of urinary 11{alpha}-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), the major urinary metabolite of PGE2 (23). We and others have previously shown that quantification of systemic eicosanoid production in humans is best assessed by measurement of stable excreted urinary metabolites (24, 25). Finally, because COX-2 is an inducible enzyme, not highly expressed in normal tissue but frequently overexpressed in NSCLC, we reasoned that urinary PGE-M could potentially serve as a biomarker of changes in tumor-derived COX-2 activity. The results of our trial are reported herein.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Between January 2001 and October 2003, 56 patients with histologically confirmed recurrent NSCLC were enrolled in a phase II trial combining celecoxib and docetaxel. Eligible patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and had received one or more chemotherapy regimens for metastatic disease. Patients could not take any nonsteroidal anti-inflammatory drugs or COX-2 inhibitors for at least 7 days before study entry. Pretreatment evaluation included a complete history and physical examination, routine laboratory evaluation, and a chest computed tomography scan. Patients were ineligible if they had received more than two prior chemotherapy regimens for recurrent or relapsed NSCLC, renal dysfunction (serum Cr >1.8 mg/dl or CrCl <50 cc/min), concomitant use of warfarin, a pure or mixed small-cell carcinoma histology, or a history of allergy to sulfonamides, celecoxib, or any other nonsteroidal anti-inflammatory drugs. All patients initially received celecoxib 400 mg p.o. twice daily for 5 to 10 days before starting chemotherapy to ensure that serum levels of celecoxib had reached steady state. Patients were instructed to take the celecoxib doses 12 hours apart at 8:00 a.m. and 8:00 p.m. Blood and timed 6-hour urine specimens were obtained from all patients before and after celecoxib and before institution of docetaxel or premedications. The collected blood was spun at 5,000 x g for 15 minutes within 30 minutes of collection and the top layer removed. Urine specimens were kept on ice from beginning of collection until the total volume was quantified and subsequently allocated into two 5-cc tubes for each subject. All blood and urine samples were frozen at –80°C until further analysis. After completing the initial run-in phase of celecoxib therapy, docetaxel was initiated at a dose of 75 mg/m2 i.v. over 1 hour every 3 weeks for up to six cycles. Patients received a premedication regimen consisting of dexamethasone 8 mg p.o. twice daily for a total of 3 days beginning 1 day before docetaxel. Celecoxib was continued during and after docetaxel therapy until either disease progression or unacceptable toxicity ensued. Tumor response was determined after the second cycle of docetaxel (i.e., week 6) using traditional tumor response criteria (26). Briefly, a partial response was defined as a ≥50% decrease in the product of tumor diameters for at least 4 weeks without increase in size of any area of known malignant disease of ≥25% or appearance of new areas of malignant disease. Stable disease was defined as no significant change in measurable or evaluable disease for at least 4 weeks (≥12 weeks for bony metastases). Progressive disease was defined as a ≥25% increase in the size of lesions present at the start of therapy or after a response, or appearance of new metastatic lesions known not to be present at the start of therapy, or stable objective disease associated with deterioration in ECOG performance status of ≥1 level related to malignancy. Protocol therapy was discontinued if any of the following occurred: disease progression at any time during therapy or the follow-up period; unacceptable toxicity; patient elected to withdraw from the study treatment; or development of non-cancer-related illness that prevented continuation of therapy. This trial was reviewed and approved by the Vanderbilt-Ingram Cancer Center Scientific Review Committee and the Vanderbilt University Institutional Review Board. Written informed consent was obtained from all patients.

Urinary PGE-M assay. PGE2 production in vivo was quantified by measuring urinary PGE-M by mass spectrometry (MS) using stable isotope dilution methods with chemically synthesized [2H6]PGE-M as an internal standard (27). Briefly, the procedure is as follows: endogenous urinary PGE-M was converted to an unlabeled O-methyloxime derivative and extracted (28). The internal standard was prepared by converting the chemically synthesized PGE-M to an [2H6]O-methyloxime derivative. During MS, the precursor ions of the unlabeled (m/z 385) and [2H6]-labeled (m/z 391) O-methyloxime PGE-M were subjected to collision-induced dissociation, producing ion m/z 336 representing endogenous PGE-M and ion m/z 339 representing the deuterated internal standard. Levels of endogenous PGE-M in samples were calculated from the ratio of the mass chromatogram peak areas of the m/z 336 and m/z 339 ions.

Serum vascular endothelial growth factor measurement. Serum vascular endothelial growth factor (VEGF) levels were measured using the Quantikine kit developed by R&D Systems (Abington, United Kingdom). Briefly, blood was obtained in a serum separator tube and allowed to clot for 30 minutes before centrifugation for 10 minutes at ~1,000 x g. The serum was removed and stored at –80°C. The reagents and standards were prepared following the directions of the manufacturer.

Serum endostatin measurement. Serum endostatin levels were determined using the Chemikine kit developed by Chemicon International (Temecula, CA). Serum was obtained as described above and the reagents and standards were prepared following the directions of the manufacturer.

Tumor prostaglandin E2 measurement. Within the context of a separate Vanderbilt-Ingram Cancer Center Institutional Review Board–approved tissue study, we attempted to obtain tumor biopsies before and after a 5- to 7-day course of high dose celecoxib (400 mg p.o. twice daily) from 19 patients with clinical stage I NSCLC scheduled to undergo "curative" resection. Pre-celecoxib tumor specimens were obtained via core needle or mediastinoscopy biopsy whereas the post-celecoxib biopsy was obtained from the resected tumor specimen taken at the time of thoracotomy. All biopsy specimens were immediately flash frozen in liquid nitrogen. We were able to obtain adequate matched tumor samples from just 6 of the 19 consenting patients for a variety of technical reasons (e.g., the pretreatment specimen being too small for adequate quantification of PGE2 or due to patient intolerance of the biopsy procedure and/or inability to reach the primary lesion for a core needle biopsy). Blood and urine specimens were obtained before initiation of celecoxib and again on completion of the initial run-in phase of celecoxib therapy just before thoracotomy. The collected blood and urine specimens were processed as outlined above. Intratumoral PGE2 levels were quantified using well-established techniques (29). Briefly, free PGE2 was assayed in tissue extracts using a stable isotope dilution method with gas chromatography followed by detection with negative ion chemical ionization MS using selective ion monitoring.

Statistical analysis. The primary end point of the phase II trial was overall survival. With a sample size of 56 patients, the study provides at least 85% power to detect an odds ratio of 0.5 (the estimated median survivals are 6 and 12 months for historical control and this study, respectively) with two-sided significant level of 5%. This power analysis was based on the assumptions of the 14 months of accrual and additional 12 months of follow-up. The secondary end point of the study was to determine if the drop of the PGE-M level had the potential to reduce the odds ratio of 40% with a two-sided {alpha} error of 0.05 and if a power of 0.85 required a sample of 56 patients. The power analysis for the secondary end point was based on the assumption of the drop of the PGE-M level occurring in half of the study subjects. Serum and urinary parameters described above were tested as surrogate markers of efficacy and correlations with clinical outcome. In this report, analyses of study results focused on estimating the association between the change of the PGE-M level and survival. The tests of hypotheses on the mean difference of the study outcomes between pre- and post-celecoxib (e.g., serum VEGF, serum endostatin, and serum VEGF/endostatin) were carried out using the paired t test and Wilcoxon signed-rank test. For lifetime data analyses, the COX proportional hazards model was used for adjusted tests of significance and estimates of odds ratios. The adjusted P values and 95% confidence intervals are reported. The quartile of the change of the PGE-M level was compared for survival with Kaplan-Meier estimates and log-rank tests. All tests of significance are two sided, and differences are considered statistically significant when P < 0.05. All data are expressed as means ± SD. SAS 8.02 and S-Plus 6.2 were used for all analyses.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patient characteristics. The clinical characteristics of the 56 patients enrolled in the phase II study are shown in Table 1. The median age was 61 years, 62.5% were male, and the median ECOG performance status was 1. All 56 patients had recurrent NSCLC that was actively progressing at the time of study entry. Thirty-seven patients (66%) had received one prior chemotherapy regimen and 19 (34%) had received two prior chemotherapy regimens for metastatic disease. All but one patient had received prior cisplatin or carboplatin therapy. The best response to the chemotherapy regimen administered immediately preceding study enrollment was as follows: partial response, 16 patients; stable disease, 23 patients; and progressive disease, 17 patients.


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  		Table 1. Baseline patient characteristics

 
Response and survival. All 56 patients were evaluable for response. There were 6 (11%) partial responses and 13 (23%) patients with stable disease for an overall disease control rate of 34%. The remaining 37 patients all progressed on therapy. Overall median survival was 6 months (95% confidence interval, 4.7-8.2; range, 0.6-38.3 months) and 1-year survival was 23%. Eight patients completed all six planned cycles of docetaxel. The remaining patients failed to receive the intended six cycles of docetaxel due to disease progression or because of treatment-related toxicities or withdrawal of consent. Ten patients discontinued protocol therapy for the following reasons: deep vein thrombosis, 2; skin rash, 3; pneumonia/acute respiratory distress syndrome/respiratory failure, 1; hypoxia and anemia, 1; increased dyspnea, 1; gastrointestinal ulcerative disease, 1; and dyspnea/fatigue/weight loss, 1. The remaining patients discontinued protocol treatment due to intercurrent death (perforated diverticulitis), 1; osteomyelitis, 1; patient request, 1; and physician recommendation, 2.

Treatment-related toxicities. The main toxicities associated with the celecoxib-docetaxel regimen were hematologic. Grade 3 or 4 neutropenia (National Cancer Institute Common Toxicity Criteria version 2) was observed in 15 patients (27%) and 17 patients (30%), respectively. Eight patients (14%) experienced grade 3 or 4 infections and there were five episodes of neutropenic fever. Two patients (4%) required packed RBC transfusions. Gastrointestinal toxicities were relatively uncommon as only one patient (2%) experienced grade 3 nausea and vomiting. Four patients (7%) experienced other grade 3 gastrointestinal toxicities including diarrhea, dehydration, and anorexia. Additional grade 3 treatment-related toxicities included hemorrhage: 1; hepatic toxicity: 1; musculoskeletal toxicity: 2; and dyspnea: 5. As noted above, two patients experienced thrombotic events that may be related to celecoxib (30).

Modulation of urinary PGE-M levels by celecoxib. Previously, we had reported that urinary PGE-M levels in healthy humans are nearly 2-fold greater in men (10.4 ± 1.5 ng/mg Cr) than in women (6.0 ± 0.7 ng/mg Cr; ref. 31). By contrast, in this cohort of patients with recurrent NSCLC, the mean pre-celecoxib urinary PGE-M levels were 22.5 ng/mg Cr (range: 2.2-81.3 ng/mg Cr) and 32.6 ng/mg Cr (range: 1.71-182.2 ng/mg Cr) in men and women, respectively. Overall, the mean urinary PGE-M dropped from a pretreatment value of 27.2 ng/mg Cr (SE: 32.2) to 12.2 ng/mg Cr (SE: 12.9) after celecoxib (P = 0.001; Fig. 1A). Pretreatment urinary PGE-M levels were higher in former and current smokers compared with never smokers (Fig. 1B) although only four subjects fell into the latter category. The marked variability in pretreatment urinary PGE-M levels presumably was related to the variable biology of the individual tumors and also, in part, to the heterogeneity of the study population (i.e., varied tumor bulk, histology, and interval from last chemotherapy to study enrollment). The decline in urine PGE-M levels occurred independent of gender or tumor histology. Notably, the decrease in PGE-M levels among never and former smokers was greater than that observed in current smokers, consistent with the observation that tobacco smoke induces COX-2 in oral mucosa (Fig. 1B; ref. 32).



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  		Fig. 1. PGE-M in NSCLC patients. A, effect of celecoxib on urinary PGE-M in 44 NSCLC urine samples pre- and post-celecoxib. PGE-M levels were quantified by liquid chromatography/electrospray ionization MS using selected reaction monitoring. Precision of the assay is ±5% and accuracy is 92%. Columns, means; bars, SE. B, pre- and post-celecoxib PGE-M levels in never, former, and current smokers.

 
Absorption of celecoxib from the gastrointestinal tract is variable and patient compliance also may vary, further contributing to the potential for the differences observed in posttreatment PGE-M levels. To assess absorption and compliance, steady-state serum celecoxib levels were determined in 48 of the 56 subjects. Celecoxib was undetectable in the serum of two patients, one of whom acknowledged not taking the celecoxib. In the remaining patients, steady-state celecoxib levels ranged from 105 to 3,814 ng/mL (median: 865.5 ng/mL), which is consistent with levels observed in individuals with nonmalignant diseases (33). The relationship between the concentration of celecoxib in plasma and effect of the drug on PGE-M excretion was examined to determine whether interindividual differences in pharmacokinetics could account for the marked variability in drug effect. There was no relation between the concentration of celecoxib in plasma and its effect on the biosynthesis of PGE2 (data not shown). This indicates that the variability in response to the drug resides with as yet unexplained heterogeneity of the tumor cells themselves. Possible explanations include differences in disposition of the drug at the cellular level or factors that regulate the action of celecoxib on COX-2.

In a multivariate model accounting for gender, sex, smoking history, and histology, we correlated changes in PGE-M with clinical parameters and observed a strong association with overall survival (Table 2). Patients (n = 11) experiencing the greatest proportional decline in urinary PGE-M levels following celecoxib had an approximate 50% decrease in the relative risk of death, a median survival of 14.8 months, and a 1-year survival of 36% (Table 3). In comparison, patients (n = 11) whose PGE-M levels rose 50% after therapy had a median survival of just 5 months and there were no 1-year survivors.


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  		Table 2. Analysis of maximum likelihood estimates

 

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  		Table 3. Survival relative to change in PGE-M levels

 
Modulation of serum angiogenic factors by celecoxib. Because COX-2 is reported to act as a tumor promoter via stimulation of angiogenesis, we also assessed the effect of celecoxib on the serum levels of VEGF (a potent proangiogenic molecule) and endostatin (a potent antiangiogenic molecule). In keeping with the putative antiangiogenic effect of COX-2 inhibition, celecoxib decreased mean serum VEGF concentration by an average of 30% (P = 0.02) whereas the mean serum endostatin concentration increased by an average of 76% (P = 0.2; Fig. 2A and B). The shift in the ratio of proangiogenic to antiangiogenic molecules (48% reduction; P = 0.02; Fig. 2C) indicates a possible shift toward a less angiogenic phenotype.



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  		Fig. 2. VEGF and endostatin. A, effect of celecoxib on serum VEGF; B, serum endostatin; C, the ratio of serum VEGF to endostatin in serum samples pre- and post-celecoxib.

 
Modulation of intratumoral prostaglandin E2levels by celecoxib. As noted above, we obtained matched pre- and post-celecoxib biopsy specimens sufficient for intratumoral PGE2 determinations in 6 of 19 patients with clinical stage I NSCLC as part of a separate trial. In two of the matched specimens, no change in intratumoral PGE2 levels was detected (data not shown). However, one of the latter specimens was derived from the patient who failed to take celecoxib. The second specimen demonstrating no change in intratumoral PGE2 level had a very low pretreatment level. In the remaining four matched specimens, there was a marked reduction in intratumoral PGE2 levels accompanied by a corresponding reduction in urinary PGE-M levels (Fig. 3). Overall, the mean pre- and post-celecoxib intratumoral PGE2 levels declined from 237.9 to 47.5 pg PGE2/mg tumor, respectively, a 5-fold decline. Paired pre- and post-celecoxib urine samples for PGE-M levels were available in 10 clinical stage I patients (including five of the six patients with paired tumor specimens). Urinary PGE-M levels declined from a mean value of 26.3 to 16.7 ng/mg Cr in the pre- and post-celecoxib samples, respectively. However, neither of the observed changes noted in pre- and post-celecoxib intratumoral PGE2 or urine PGE-M levels reached statistical significance due to the small sample size.



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  		Fig. 3. Intratumoral PGE2 levels (dashed line) and corresponding urinary PGE-M levels (solid line) in patients with clinical stage I NSCLC.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There is little doubt that improved insight into the biology of cancer has greatly expanded the spectrum of potential therapeutic targets (34). Drugs that target the epidermal growth factor receptor family, for example, have yielded symptomatic and survival benefits in breast, lung, and colon cancers, often with less toxicity than that occasioned by standard chemotherapy (35–39). Another potentially attractive molecular target for these common malignancies is COX-2 (40). Preclinical data indicate that selective COX-2 inhibitors can prevent carcinogenesis and reduce the growth rate of various tumors in vitro and in vivo (7). As COX-2 and its product PGE2 seem to play a role in the pathogenesis of NSCLC, we hypothesized that inhibition of this pathway might impart therapeutic benefit in patients with advanced disease. To test this hypothesis, we combined the selective COX-2 inhibitor celecoxib with docetaxel in patients with recurrent NSCLC. At the time the study was developed, docetaxel was the only Food and Drug Administration–approved drug for recurrent NSCLC. In addition, the combination of docetaxel and a selective COX-2 inhibitor had shown enhanced cytotoxicity in preclinical models of lung cancer (22). We further hypothesized that celecoxib might inhibit increased COX-2 activity and the resultant increased PGE2 production observed in tumors exposed to taxanes and which theoretically might reduce the therapeutic efficacy of these agents (41). Lastly, we also anticipated celecoxib might alleviate some of the more troubling toxicities of taxanes (i.e., myalgia and arthralgia) that often accompany the use of these agents. Despite the compelling rationale for combining docetaxel and celecoxib, our results were disappointing. The overall response rate of 11% and the median survival of 6 months are not substantially different from what has been reported in similar patient populations with docetaxel alone (21, 42). Therefore, the combination of celecoxib and docetaxel in unselected patients as administered in this study does not seem particularly promising in recurrent NSCLC.

A major problem accompanying human studies of so-called targeted therapies is the frequent lack of confirmation that the desired target is activated in a particular patient's tumor or, if so, whether it is inhibited or affected in a favorable manner. Therefore, an important secondary goal of our study was to assess the activity of the COX enzymes in individual patients and the ability of celecoxib to inhibit this intratumoral COX-2 activity at the dose and schedule employed. We attempted to do this indirectly by assessing pre- and post-celecoxib urine levels of PGE-M, the major metabolite of PGE2. We employed a robust technique, recently developed by our group (31), for assessing PGE-M. We did not assess serum PGE2 because it is a highly labile eicosanoid, rendering its measurement in serum both problematic and, worse, potentially misleading (25, 43). Moreover, previous studies indicate that quantification of systemic eicosanoid production in humans is best assessed by measurement of excreted urinary metabolites (24, 25). Because COX-2 is an inducible enzyme not highly expressed in normal tissue, we reasoned that a majority of PGE2 in lung cancer patients would derive from the tumor rather than potential nonmalignant sources (23). We further reasoned that changes in urinary PGE-M might reflect changes in tumor-derived PGE2 that are dependent on intratumoral COX-2 activity.

We found that urinary PGE-M is elevated in a high proportion of patients with recurrent NSCLC, confirming an observation we first made 30 years ago (23). Moreover, urinary PGE-M levels commonly fell after celecoxib irrespective of the initial value, often to levels observed in normal men or women without cancer. The considerable difference between individuals in the extent to which celecoxib reduced PGE-M, however, is notable. In a few cases, PGE-M levels actually increased slightly. Potential reasons for this variability in the suppression of PGE2 biosynthesis include a possible contribution of COX-1 to the elevated levels of PGE-M or a substantial variability in the concentration of celecoxib at these dose levels within the relevant intracellular compartments within the tumor. In addition, PGE2 levels may be increased for reasons other than increased COX-2 activity. For example, recent reports indicate that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme responsible for PGE2 metabolism and elimination, is frequently down-regulated in colon and lung cancers (44–46). Down-regulation of 15-PGDH may result in increased levels of PGE2 even in the absence of increased COX-2 activity. This recent observation suggests that combinatorial therapy with an inhibitor of COX-2, coupled with an agent that up-regulates 15-PGDH, might be an effective therapeutic strategy for tumors in which PGE2 plays a prominent role. Nonetheless, patients who experienced the greatest proportional drop in urinary PGE-M levels following celecoxib in our study were at substantially reduced risk of death relative to their counterparts with no change or an increase in PGE-M level. This apparent survival advantage was especially impressive among those patients in the upper quartile of PGE-M decline (i.e., ≥72% decrease) and suggests that further studies targeting COX-2 inhibition in this subset of patients are warranted.

Because PGE2 can induce angiogenesis, at least in part, by stimulating production of proangiogenic factors such as VEGF, we also assessed serum VEGF levels pre- and post-celecoxib as a possible surrogate of COX-2 inhibition. Elevated levels of VEGF are common in NSCLC and portend a poor prognosis (47). Moreover, recent reports indicate that antiangiogenesis therapy is beneficial in advanced NSCLC (48). Although we observed a 30% decrease in mean serum VEGF levels after celecoxib administration, the patterns of change in individual patients were not predictive of clinical outcome in a multivariate analysis. Similarly, changes in serum endostatin were highly variable although there was a general trend for levels to increase post-celecoxib. The ratio of VEGF to endostatin levels declined significantly in the post-celecoxib samples, representing a shift that would presumably favor decreased tumor angiogenesis. Nonetheless, the changes in cytokines did not correlate with clinical outcome and therefore do not seem to be a useful surrogate marker of COX-2 inhibition.

Finally, within the context of a separate tissue study conducted in patients with operable clinical stage I NSCLC, we also found that celecoxib 400 mg p.o. twice daily markedly decreased intratumoral PGE2 levels in all four of the five total paired pre- and posttreatment biopsy specimens with elevated PGE2. These findings are consistent with an observation made by Altorki et al. (49) and strongly suggest that the dose and schedule of celecoxib used in this trial was sufficient to inhibit intratumoral COX-2, the intended molecular target. Although the number of patients is small, the results also suggest that changes in urinary PGE-M levels reflect intratumoral PGE2 levels. If confirmed in a larger cohort of patients, these findings indicate that urinary PGE-M levels may be useful for individualizing therapy aimed at inhibiting intratumoral COX-2 activity.

In summary, combining celecoxib with docetaxel did not seem to improve therapeutic outcome in patients with recurrent NSCLC although inhibition of intratumoral COX-2 was achieved. Importantly, however, urinary PGE-M seems to be a useful biomarker for assessing intratumoral COX-2 activity in patients with NSCLC. Indeed, monitoring urine PGE-M levels might be used to titrate optimal COX-2 inhibition in future clinical trials. Further studies are clearly needed to confirm these preliminary observations and more fully define the operating characteristics of the marker. Given the results of the subset analysis indicating that a large proportional decline in urine PGE-M levels is associated with improved survival, it is possible that changes in urinary PGE-M may allow the identification of a subset of NSCLC patients particularly likely to benefit from COX-2 inhibition.


   Acknowledgments
 
We thank research nurses Norma Campbell and Ben Garcia and data managers Debbie Murrey and Darienne Adkins for their invaluable help. Celecoxib was generously provided by Pharmacia.


   Footnotes
 
Grant support: NIH grants CA68485, P50 CA90949 (Specialized Program of Research Excellence in Lung Cancer), CA076321, K24CA080908, CA77839, GM15431, DK48831, and RR00095; Aventis Pharmaceuticals; Burroughs Wellcome Fund Clinical Scientist Award in Translational Research (J.D. Morrow); and Unrestricted Institutional Grant for Cancer Research from the Bristol Myers Squibb Foundation (D.H. Johnson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/28/05; revised 6/10/05; accepted 6/28/05.


   References
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 Results
 Discussion
 References
 

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