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Clinical Cancer Research Vol. 12, 1686-1692, March 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Cyclooxygenase 2 Expression in Colorectal Cancer with DNA Mismatch Repair Deficiency

Antoni Castells1, Artemio Payá3, Cristina Alenda3, Francisco Rodríguez-Moranta1, Rubén Agrelo5, Montserrat Andreu2, Virgínia Piñol1, Sergi Castellví-Bel1, Rodrigo Jover4, Xavier Llor6, Elisenda Pons6, J. Ignasi Elizalde1, Xavier Bessa2, Javier Alcedo7, Joan Saló8, Enrique Medina9, Antonio Naranjo10, Manel Esteller5, Josep M. Piqué1 for the Gastrointestinal Oncology Group of the Spanish Gastroenterological Association

Authors' Affiliations: 1 Department of Gastroenterology, Institut de Malalties Digestives, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona; 2 Department of Gastroenterology, Hospital del Mar, Barcelona, Spain; Departments of 3 Pathology and 4 Gastroenterology, Hospital General Universitario de Alicante, Alicante, Spain; 5 Cancer Epigenetic Laboratory, Spanish National Cancer Centre, Madrid, Spain; 6 Department of Gastroenterology, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 7 Department of Gastroenterology, Hospital Clínico, Zaragoza, Spain; 8 Department of Gastroenterology, Hospital General de Vic, Vic, Spain; 9 Department of Gastroenterology, Hospital General Universitario, Valencia, Spain; and 10 Department of Gastroenterology, Hospital Universitario Reina Sofía, Córdoba, Spain

Requests for reprints: Antoni Castells, Department of Gastroenterology, Hospital Clínic, Villarroel 170, 08036 Barcelona, Catalonia, Spain. Phone: 34-93-227-54-18; Fax: 34-93-227-93-87; E-mail: castells{at}clinic.ub.es.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 Appendix A. Investigators from...
 References
 
Background: Cyclooxygenase 2 (COX-2) overexpression is a frequent but not universal event in colorectal cancer. It has been suggested that COX-2 protein expression is reduced in colorectal cancer with a defective mismatch repair (MMR) system, a phenomenon commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC) but also present in up to 15% of sporadic tumors.

Aim: To assess COX-2 expression in a large series of fully characterized colorectal cancer patients with respect to the MMR system and to dissect the mechanisms responsible for altered COX-2 expression in this setting.

Patients and Methods: MMR-deficient colorectal cancer were identified in a nationwide, prospective, multicenter study (EPICOLON project). Control MMR-proficient colorectal cancer patients were randomly selected. COX-2 expression was evaluated by immunohistochemistry. Personal and familial characteristics, as well as MSH2/MLH1 expression and germ line mutations, were evaluated.

Results: One hundred fifty-three patients, 46 with MMR deficiency and 107 with MMR proficiency, were included in the analysis. Overall, tumor COX-2 overexpression was observed in 107 patients (70%). COX-2 overexpression was observed in 85 patients (79%) with a MMR-proficient system, but only in 22 patients (48%) with a MMR-deficient colorectal cancer (P < 0.001). The lack of COX-2 overexpression was independently associated with a MMR-deficient system (odds ratio, 3.89; 95% confidence interval, 1.78-8.51; P = 0.001) and a poor degree of differentiation (OR, 3.83; 95% CI, 1.30-11.31; P = 0.015). In the subset of patients with a MMR-deficient colorectal cancer, lack of COX-2 overexpression correlated with a poor degree of differentiation, no fulfillment of Amsterdam II criteria, absence of MSH2/MLH1 germ line mutations, presence of tumor MSH2 expression, and lack of tumor MLH1 expression. CpG island promoter hypermethylation of COX2 was observed in 6 of 18 (33%) tumors lacking COX-2 expression in comparison with 2 of 28 (7%) tumors expressing this protein (P = 0.04).

Conclusions: Up to half of MMR-deficient colorectal cancer do not show COX-2 overexpression, a fact observed almost exclusively in patients with sporadic forms. COX2 hypermethylation seems to be responsible for gene silencing in one third of them. These results suggest the potential utility of nonsteroidal anti-inflammatory drugs in HNPCC chemoprevention and may explain the lack of response of this approach in some sporadic tumors.

Colorectal cancer is the second leading cause of cancer-related death in Western countries in spite of progressive refinements in preventive, diagnostic, and therapeutic approaches (1). In the last two decades, a tremendous advance has been experienced with respect to the knowledge of molecular mechanisms involved in its pathogenesis (2, 3). At present, two main pathways are well established: first, the "suppressor pathway," involving activation of oncogenes and inactivation of tumor suppressor genes and responsible for most sporadic colorectal cancer forms (2), and second, the "mutator pathway," in which an altered DNA mismatch repair (MMR) system leads to replication errors in tumor cells (4, 5). This alteration underlies the majority of hereditary nonpolyposis colorectal cancer (HNPCC) patients (6, 7), but it can also be observed in up to 15% of sporadic colorectal cancer cases (8). Whereas HNPCC is caused by germ line mutations in MMR genes, mainly MSH2, MLH1, and MSH6 (9), defects on this pathway are due to MLH1 promoter methylation in sporadic forms (10).

A large body of evidence indicates that the use of nonsteroidal anti-inflammatory drugs (NSAID) can reduce the risk of colorectal cancer (11). Experimental studies have shown that NSAIDs decrease the incidence of carcinogen-induced colon tumors in rodents (12, 13), and several epidemiologic investigations have also shown a 40% to 50% reduction in the risk of colorectal adenoma and cancer development in patients taking NSAIDs (14–17). Moreover, individuals with familial adenomatous polyposis taking sulindac or celecoxib experience a reduction in adenoma size and number (18–20). These chemopreventive effects of NSAIDs may be largely related to inhibition of cyclooxygenase 2 (COX-2; refs. 21, 22), the inducible isoform of COX that catalyzes the conversion of arachidonic acid to prostaglandins (23).

COX-2 overexpression is a frequent but not universal event in colorectal neoplasms. Indeed, ~50% of adenomas and 80% of colorectal cancer express high levels of COX-2 mRNA and protein in neoplastic tissue (24). Interestingly, a reduced COX-2 protein expression has been shown in colorectal cancer with defective MMR system (25) in a similar manner as in other MMR-deficient neoplasms (26, 27). In a seminal study (28), it was suggested that this reduced expression might affect HNPCC patients, although the mechanism underlying this phenomenon was unclear. Moreover, a parallel investigation from the same group showed promoter hypermethylation of COX2 in colorectal cancer samples, thus suggesting that the reduced expression of COX-2 may also affect sporadic MMR-deficient forms (29). In addition to these apparently opposing results, it is important to note that COX2 hypermethylation did not correlate with the presence of microsatellite instability in this latter investigation (29). Altogether, these results emphasize the fact that the putative low COX-2 expression in MMR-deficient colorectal cancer, a relevant issue with potential implications in prevention and prognosis, is poorly understood. Specific characteristics of previous studies and a possible selection bias in the recruitment of patients might have precluded to elucidate this issue.

In 2001, a prospective, multicenter, nationwide, population-based study was launched to ascertain the incidence of HNPCC and other familial colorectal cancer forms in Spain (30), as well as to establish the most effective and efficient strategy for the detection of MSH2/MLH1 gene carriers in newly diagnosed colorectal cancer patients (31). Besides these primary goals, the EPICOLON study provided a unique opportunity to prospectively assess the expression of COX-2 in a large series of colorectal cancer patients fully characterized with respect to the MMR system and to dissect the mechanisms responsible for the altered COX-2 expression in this setting.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 Appendix A. Investigators from...
 References
 
Patient population. Between November 2000 and October 2001, all newly diagnosed colorectal cancer patients in 25 hospitals were included in the EPICOLON study (30). Eighteen of these 25 centers agreed to participate in a nested case-control study to evaluate COX-2 expression in tumors with MMR deficiency. Exclusion criteria were familial adenomatous polyposis, personal history of inflammatory bowel disease, and patient refusal to participate in the study. The study was approved by the institutional ethics committee of each participating hospital, and written informed consent was obtained from all patients.

Cases were identified among those patients with MMR-deficient tumors, shown by microsatellite instability and/or lack of MSH2/MLH1 protein expression. Two control colorectal cancer patients with MMR proficiency were randomly selected per each case subject, after stratification by center. Patients receiving aspirin or other NSAIDs were excluded.

Demographic, clinical, and tumor-related characteristics of probands, as well as a detailed family history, were obtained using a preestablished questionnaire, as described elsewhere (31).

Evaluation of the MMR system. Microsatellite instability testing and immunostaining for MMR proteins were done in all patients regardless of age, personal or family history, and tumor characteristics. Microsatellite instability testing was done using BAT-26 mononucleotide marker based on its high sensitivity, as described elsewhere (31). Immunostaining was done using mouse monoclonal antibodies against MLH1 protein (clone G168-15, dilution 1:40; PharMingen, San Diego, CA) and MSH2 protein (clone FE11, dilution 1:35; Oncogene Research Products, Boston, MA) as it is described elsewhere (31).

Patients found to have tumors with microsatellite instability and/or lack of protein expression of either MSH2 or MLH1 underwent germ line genetic testing for MSH2 and MLH1 by both multiple ligation probe amplification analysis and sequencing as described elsewhere (31).

Tumor COX-2 protein expression. One block of formalin-fixed, paraffin-embedded tumor tissue was selected per case. Four-micrometer-thick sections were dewaxed and rehydrated using xylene and alcohol. Before immunostaining, antigen retrieval was done by immersing sections in a 10 mmol/L concentration of citrate buffer (pH 6.0) and boiling in a pressure cooker for 3 minutes. Sections were then incubated for 20 minutes at room temperature with mouse monoclonal antibodies against COX-2 (clone PG 27b, dilution 1:400, Oxford Biomedical Research, Oxford, MI). The Ultra-Vision streptavidin-biotin peroxidase detection kit (DAKO, Carpinteria, CA) was used as secondary detection system. The peroxidase reaction was developed using diaminobenzidine tetrachloride as chromogen.

Staining was scored for intensity (0, 1+, 2+, or 3+) and percentage of cytoplasm staining in malignant cells (1, 0-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%; Fig. 1 ). The sum of intensity and percentage counts was used as the final score (32). Cytoplasm staining in normal epithelial cells (discrete positivity near the nucleus on the apical side of epithelial cells both in the normal crypt and the surface epithelium; ref. 33) in each slide served as internal positive controls. Pathologists (C. Alenda and A. Payá) were blinded to the results of microsatellite instability testing and MSH2/MLH1 immunostaining.


Figure 1
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  		Fig. 1. Examples of colorectal adenocarcinoma with 1+ (x100, A), 2+ (x100, B), and 3+ (x100, C; x40, D) of COX-2 immunostaining intensity.

 
COX2 CpG island methylation analysis. COX2 CpG island methylation status was determined by PCR analysis of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil, using methylation-specific PCR with primers specific for either the methylated or modified unmethylated DNA (34). Primer sequences of COX2 for the unmethylated reaction were 5'-GAGAGGGGATTTTTTGTGTTTTT-3' (sense) and 5'-CCCAAACACTTCCAAAAACC-3' (antisense) and for the methylated reaction, 5'-GAGGGGATTTTTTGCGTTTTC-3' (sense) and 5'-CCGAACGCTTCCGAAAAC-3' (antisense). Primers were located encompassing the transcription start site. The annealing temperature for both unmethylated and methylated reactions was 60°C. DNA from normal lymphocytes treated in vitro with SssI methyltransferase was used as a positive control for methylated alleles. DNA samples from normal lymphocytes and adrenal medulla were used as a positive control for unmethylated alleles. PCR products were loaded onto nondenaturing 3% polyacrylamide gels, stained with ethidium bromide, and visualized under a UV transilluminator. Reverse transcription-PCR studies for COX2 expression in colorectal cancer cell lines were developed using the sense primer 5'-CAGCACTTCACGCATCAGTT-3' and the antisense primer 5'-TCAGGGACTTGAGGAGGGTA-3', encompassing an intron.

Statistical analysis. Continuous variables were expressed as mean ± SD. Differences in qualitative variables were evaluated by means of {chi}2 test or the Fisher's exact test when necessary. Continuous variables were compared by means of the Student's t test.

Tumor COX-2 expression was analyzed both qualitatively and quantitatively. For qualitative analyses, patients were classified as overexpressing COX-2 (immunostaining score from 3 to 7) or nonoverexpressing COX-2 (immunostaining score from 0 to 2).

To identify variables associated with COX-2 expression, a multivariate analysis was done. Variables achieving a P value of <0.2 in the univariate analysis were subsequently included in a stepwise backward logistic regression procedure to identify those factors independently associated with COX-2 overexpression.

All P values are two sided. A P value of <0.05 was considered to indicate a statistically significant difference. All calculations were done by using the 10.0 SPSS software package (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 Appendix A. Investigators from...
 References
 
Characteristics of patients. From among the centers that agreed to participate in this nested, case-control study, 55 patients with a MMR-deficient colorectal cancer were identified. Simultaneously, 110 colorectal cancer patients matched by center were randomly selected among those with a proficient MMR system. COX-2 immunostaining led to ambiguous results in 12 patients and, consequently, they were excluded from the analysis. Thus, 153 patients, 46 of them with a deficient MMR system and 107 with MMR proficiency, constitute the basis of the present investigation. Demographic, clinical, and tumor-related characteristics of these patients are summarized in Table 1 .


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  		Table 1. Characteristics of patients with colorectal cancer included in the study

 
COX-2 expression in patients with colorectal cancer. In the whole series, 107 patients (70%) had colorectal cancer overexpressing COX-2, whereas in the remaining 46 patients (30%) the tumor did not show COX-2 overexpression. In this latter subset of patients, COX-2 expression was absolutely abolished in 33 of them.

With respect to the MMR status, COX-2 overexpression was observed in 85 patients (79%) with a proficient system, but only in 22 patients (48%) with a deficient MMR system (P < 0.001; Table 2 ). Other characteristics associated with tumor COX-2 expression were site of tumor, degree of differentiation, and tumor-node-metastasis stage (Table 2).


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  		Table 2. Variables associated with tumor COX-2 expression in the whole series (n = 153)

 
The multivariate analysis showed that the lack of COX-2 overexpression was independently associated with a MMR-deficient system (odds ratio, 3.89; 95% confidence interval, 1.78-8.51; P = 0.001) and a poor degree of differentiation (odds ratio, 3.83; 95% confidence interval, 1.30-11.31; P = 0.015).

COX-2 expression in patients with MMR-deficient colorectal cancer. In the subset of patients with a MMR-deficient colorectal cancer, 24 tumors (52%) did not show COX-2 overexpression (Table 3 ). The lack of COX-2 overexpression correlated with a poor degree of differentiation, no fulfillment of Amsterdam II criteria, absence of MSH2/MLH1 germ line mutations, presence of tumor MSH2 expression, and lack of tumor MLH1 expression (Table 3).


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  		Table 3. Variables associated with tumor COX-2 expression in patients with MMR deficiency (n = 46)

 
CpG island promoter hypermethylation of COX2. Considering that the lack of tumor COX-2 overexpression correlated with characteristics commonly associated with MMR-deficient sporadic colorectal cancer (i.e., absence of MSH2/MLH1 germ line mutations, no fulfillment of Amsterdam II criteria, and lack of tumor MLH1 expression), promoter hypermethylation of COX2 seemed as the most attractive explanation for gene silencing in patients with MMR-deficient colorectal cancer. In colorectal cancer cell lines, COX2 hypermethylation was associated with loss of COX-2 expression, while treatment with a DNA demethylating agent restored COX-2 expression (Fig. 2 ).


Figure 2
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  		Fig. 2. COX2 CpG island methylation analysis by methylation-specific PCR in colorectal cancer cell lines (A) and primary colorectal cancer (CRC) samples (B). The presence of a PCR band under lane M indicates methylated genes, although the presence of a PCR band under lane U indicates unmethylated genes. NC, normal colon; IVD, in vitro methylated DNA. In (A), the colorectal cancer cell line RKO shows COX2 hypermethylation (top) in association with loss of gene expression determined by reverse transcription-PCR (bottom); treatment with the demethylating agent 5-aza-2'-deoxycytidine restores COX2 expression (lane +).

 
Tumor COX-2 expression decreased in cases with COX2 hypermethylation with respect to unmethylated lesions (immunostaining score: 1.00 ± 1.93 versus 3.29 ± 2.80, respectively; P = 0.01). Indeed, COX2 hypermethylation was observed in 6 of 18 (33%) tumors lacking COX-2 expression in comparison with 2 of 28 (7%) tumors expressing this protein (P = 0.04; Fig. 2).


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
Appendix A. Investigators from...
 References
 
The results of this study indicate that although COX-2 overexpression was observed in most MMR-proficient colorectal cancer, half of those tumors with a deficient repair system did not overexpress this protein. More importantly, lack of COX-2 overexpression was observed almost exclusively in patients with MMR-deficient sporadic forms, thus suggesting an epigenetic mechanism responsible for gene silencing. This was confirmed by the observation that promoter hypermethylation of COX2 was significantly associated with the lack of tumor COX-2 expression, although this mechanism can only explain one third of them. The strength of this study relies on the fact that it was carried out on a general population basis, which ensures an unbiased selection of patients; it involved a large number of subjects, thus guaranteeing that both HNPCC-related and sporadic MMR-deficient tumors are well represented; all patients were fully characterized with respect to the MMR system, including microsatellite instability testing, immunohistochemistry, and germ line mutational analysis; finally, tumor COX-2 immunostaining was done in a blinded fashion and results were analyzed both qualitatively and semiquantitatively.

Results of the present study suggest that HNPCC patients may benefit from chemoprevention using COX-2 inhibitors because five of six MSH2/MLH1 gene carriers exhibited tumor COX-2 overexpression, although the relatively low number of HNPCC patients included precludes to draw definitive conclusions in such subset of patients. This fact was already proposed by Sinicrope et al. (28), although in their study the frequency and intensity of COX-2 expression was significantly reduced in HNPCC with respect to sporadic forms. Conversely, half of those sporadic MMR-deficient tumors did not show COX-2 overexpression. This difference may also have important implications in terms of colorectal cancer chemoprevention because the lack of COX-2 immunostaining would theoretically imply a less relevant pathogenic role of this molecule (11, 35). In that sense, this observation could explain the lack of benefit from COX-2 inhibition in a significant proportion of patients at risk for colorectal cancer (15–17). It is important to mention, however, that the lack of COX-2 expression does not absolutely exclude the response to NSAIDs because they may also act through non-COX-2-dependent mechanisms (11, 35). Moreover, whether a reduced COX-2 expression in MMR-deficient colorectal cancer is reflecting events occurring during premalignant adenoma stages is also unknown and, therefore, may have no bearing on the preventive efficacy of COX-2 inhibitors in this setting (11).

Regulation of COX-2 expression is not completely uncovered. Data obtained in cancer cell lines suggest that COX-2 overexpression is mainly determined by transcriptional activation (36). In animal models, COX-2 expression increases coincidentally with loss of adenomatous polyposis coli, suggesting that TCF/ß-catenin complexes may also regulate COX2 gene transcription (21, 37). In MMR-deficient colorectal cancer, mutations in the transforming growth factor-ß type II receptor gene have been suggested as a potential regulatory mechanism (37), but results obtained thus far seem to discard this possibility (28). On the contrary, according to results of the present study as well as other previously published investigations in patients with colorectal cancer (29) or gastric cancer (38–40), hypermethylation of COX2 seems to be the most attractive explanation for the lack of COX-2 overexpression in this subset of patients. Indeed, aberrant cytosine methylation of promoter region CpG island has clearly been established as an epigenetic mechanism for tumor-suppressor gene inactivation in many gastrointestinal neoplasia, including colorectal cancer (41–43). It is important to point out that, although a significant association between COX2 hypermethylation and lack of COX-2 expression was observed in MMR-deficient tumors, this phenomenon can only explain one third of them, thus suggesting that other regulatory mechanisms may exist. However, considering that there is some controversy with respect to which regions of the COX2 gene are really involved in the regulation of its expression (40), hypermethylation-mediated transcriptional silencing of COX2 cannot definitely be excluded as the mechanism responsible for COX-2 down-regulation in some additional cases.

In conclusion, the results of this study, in agreement with published data using other antibodies or immunoblotting, indicate that patients with MSH2/MLH1–associated HNPCC may probably benefit from chemoprevention with NSAIDs. On the contrary, the fact that almost half of those patients with MMR-deficient sporadic colorectal cancer forms do not exhibit COX-2 overexpression could explain the lack of response to this prophylactic measure. Although lack of COX-2 expression does not absolutely preclude the response to NSAIDs, because they may also act through non-COX-2-dependent mechanisms, our results justify the stratification of patients according to their MMR status in any study aimed at evaluating the potential utility of this chemopreventive strategy in colorectal cancer.


   Appendix A. Investigators from the Gastrointestinal Oncology Group of the Spanish Gastroenterological Association who participate in the study
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 Appendix A. Investigators from...
References
 
All participants listed below were fully involved in the study:

Hospital 12 de Octubre, Madrid: Juan Diego Morillas (local coordinator), Raquel Muñoz, Marisa Manzano, Francisco Colina, Jose Díaz, Carolina Ibarrola, Guadalupe López, and Alberto Ibáñez. Hospital Clínic, Barcelona: Antoni Castells (local coordinator), Virgínia Piñol, Sergi Castellví-Bel, Francisco Rodríguez-Moranta, Francesc Balaguer, Antonio Soriano, Rosa Cuadrado, Maria Pellisé, Rosa Miquel, J. Ignasi Elizalde, and Josep M. Piqué. Hospital Clínico Universitario, Zaragoza: Ángel Lanas (local coordinator), Javier Alcedo, and Javier Ortego. Hospital Cristal-Piñor, Complexo Hospitalario de Ourense: Joaquin Cubiella (local coordinator), Ma. Soledad Díez, Mercedes Salgado, Eloy Sánchez, and Mariano Vega. Hospital del Mar, Barcelona: Montserrat Andreu (local coordinator), Xavier Bessa, Agustín Panadés, Asumpta Munné, Felipe Bory, Miguel Nieto, and Agustín Seoane. Hospital Donosti, San Sebastián: Luis Bujanda (local coordinator), Juan Ignacio Arenas, Isabel Montalvo, Julio Torrado, and Ángel Cosme. Hospital General Universitario de Alicante: Artemio Payá (local coordinator), Rodrigo Jover, Juan Carlos Penalva, and Cristina Alenda. Hospital General de Granollers: Hospital General de Vic: Joan Saló (local coordinator), Eduard Batiste-Alentorn, Josefina Autonell, and Ramon Barniol. Hospital General Universitario de Guadalajara: Ana María García (local coordinator), Fernando Carballo, Antonio Bienvenido, Eduardo Sanz, Fernando González, and Jaime Sánchez. Hospital General Universitario de Valencia: Enrique Medina (local coordinator), Jaime Cuquerella, Pilar Canelles, Miguel Martorell, José Ángel García, Francisco Quiles, and Elisa Orti. Hospital do Meixoeiro, Vigo: Juan Clofent (local coordinator), Jaime Seoane, Antoni Tardío, and Eugenia Sanchez. Hospital San Eloy, Baracaldo: Luis Bujanda (local coordinator), Carmen Muñoz, María del Mar Ramírez, and Araceli Sánchez. Hospital Universitari Germans Trias i Pujol, Badalona: Xavier Llor (local coordinator), Elisenda Pons, Rosa M. Xicola, Marta Piñol, Mercè Rosinach, Anna Roca, José M. Hernández, and Miquel A. Gassull. Hospital Universitari Mútua de Terrassa: Fernando Fernández-Bañares (local coordinator), Josep M. Viver, Antonio Salas, Jorge Espinós, Montserrat Forné, and Maria Esteve. Hospital Universitari Arnau de Vilanova, Lleida: Josep M. Reñé (local coordinator), Carmen Piñol, Juan Buenestado, and Joan Viñas. Hospital Universitario de Canarias: Enrique Quintero (local coordinator), David Nicolás, Adolfo Parra, and Antonio Martín. Hospital Universitario La Fe, Valencia: Lidia Argüello (local coordinator), Vicente Pons, Virginia Pertejo, and Teresa Sala. Hospital Universitario Reina Sofía, Córdoba: Antonio Naranjo (local coordinator), María del Valle García, Patricia López, Fernando López, Rosa Ortega, Javier Briceño, and Javier Padillo.


   Footnotes
 
Grant support: Fondo de Investigación Sanitaria grants FIS 01/0104-01, FIS 01/0104-02, and FIS 01/0104-03; Instituto de Salud Carlos III grants RC03/02 and RC03/10; Ministerio de Educación y Ciencia grant SAF 04-07190; Merck, Co.; Francisco Rodríguez-Moranta received a research grant from the Instituto de Salud Carlos III, Virginia Piñol a research grant from the Institut d'Investigacions Biomèdiques August Pi i Sunyer, and Xavier Llor has a contract from Programa Ramon y Cajal, Ministerio de Educación y Ciencia.

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 7/22/05; revised 11/11/05; accepted 1/ 6/06.


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 Abstract
 Patients and Methods
 Results
 Discussion
 Appendix A. Investigators from...
 References
 

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