[CANCER RESEARCH 50, 438-443. January 15, 1990] Presence of Villin, a Tissue-specific Cytoskeletal Protein, in Sera of Patients and an Initial Clinical Evaluation of Its Value for the Diagnosis and Follow-up of Colorectal Cancers1 B. Dudouet, L. Jacob, P. Beuzeboc, H. Magdelenat, S. Robine, Y. Chapuis, B. Christoforov, G. A. Cremer, P. Pmiillanl, P. Bonnichon, F. Pinon, R. J. Salmon, A. Pointereau-Bellanger, J. Bellanger, M. T. Maunoury, and D. Louvard2 Département de Médecineinterne [L. J., B. C., G.A. C.J, Clinique Chirurgicale [Y. C., P. Bo.J, and Poste de Transfusion sanguine ¡F. P.], Hôpital Cochin, 27, rue du Faubourg Saint Jacques, 75674 Paris Cedex 14; Département de Médecineoncologique [P. Be., P. P.],Laboratoire de radiopathologie ¡H.M.], and Service de Chirurgie [R. J. S.], Institut Curie, 26 rue d'Ulm, 75005 Paris; Service de Gastroenterologie, Pr. Y. Le Quintrec, Hôpital Rothschild, 75012 Paris [J. B.]; Pharmacie Hôpital Salpétriére, 83 bd de l'Hôpital, 75013 Paris [A. P. B.]; Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex [M. T. M.J; and Unité de Biologie des Membranes, Département de Biologie Moléculaire, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15 [B. D., S. R., D. L.], France ABSTRACT Villin is an actin-binding protein found in a few normal adult epithelia, namely epithelial cells in the digestive and urogenital tracts. Moreover, villin production is maintained in malignant cells. We assumed that cell lysis and necrosis of solid tumors producing villin might result in villin release into blood. We analyzed the villin content of sera from 788 patients and controls using an enzyme-linked immunosorbent assay. Patients and controls were classified into healthy donors, patients with benign diseases of the gastrointestinal tract, patients with colorectal cancers, and patients with malignant nondigestive diseases. In the panel of sera analyzed, the sensitivity of the assay for colorectal cancers was 50.5%, and its overall specificity for malignant digestive tumors was 94.5%. Results were statistically analyzed comparing each group of sera with each other. We conclude that the presence of villin is indicative of a pathological state in the gastrointestinal tract (/' < 0.001). Finally, we followed villin levels after tumor resections (60 patients). We found that the villin level in sera remains low in remissions but is raised in recur rences. We suggest that the villin assay may have clinical utility as a diagnostic adjunct for adenocarcinoma of the gastrointestinal tract. It may also have some value in monitoring patients with advancing colorectal carcinomas after resection of these tumors. INTRODUCTION Enterocytes, the epithelial cells of the intestinal mucosa, exhibit a functional and morphological polarity. Their apical plasma membrane shows an ordered array of microvilli forming the brush border. Each microvillus is supported by a cytoskel- eton based on actin microfilaments. These actin microfilament bundles are associated with a few actin-binding proteins (1). Among them, villin, a M, 95,000 protein, is able to bind actin in a calcium-dependent process. Production of villin and its regulation were previously studied in our laboratory in vivo and in cell cultures (2-4). During ontogenesis of the human diges tive tract, villin is found in immature intestinal cells as early as the eight wk of gestation of the human fetus, before the final stages of histogenesis. Similarly, villin is found in adult undif- ferentiated crypt cells of the intestinal mucosa. In the adult human, high levels of villin were primarily found in epithelial cells with a well-organized brush border, namely the epithelial cells in the small and large intestine and in cells lining the proximal tubules of the kidney. Received 2/27/89; revised 7/13/89; accepted 9/25/89. 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. 1This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (No. 86-7008), by the Fondation pour la Recherche MédicaleFrançaise,by the Association pour la Recherche sur le Cancer (No. 6379), théCNRS (UA-1149), and thé Ligue Nationale Françaisecontre leCancer. 1To whom requests for reprints should be addressed. This restricted tissue specificity of villin is also conserved during neoplasie processes. In this respect, it is particularly worth recalling that villin continues to be produced in colorectal tumors at all stages of epithelial cell transformation, but that villin is conspicuously absent from tumors arising from cell types in which it is not normally found (5, 6). These observations are in contrast with the more generalized production of CEA,3 a tumor marker that is routinely assayed for screening and follow-up of patients with tumors of the digestive tract. For example, it is now well established that CEA is found in several normal cells of numerous origins (7- 9). The above mentioned properties of villin prompted us to determine whether it is released into the blood circulation, due to lysis of malignant cells or by means of another unknown mechanism, which could indicate the existence of a lesion in the digestive tract. We therefore investigated the presence of villin in the sera of 788 patients and controls with an ELISA developed for this study in our laboratory. Our initial data showed that the occurrence of villin in human sera corre sponded to the pathological state of the gastrointestinal tract. Moreover, the presence of villin in sera was very often associ ated with a malignant digestive tumor. This is the first report of the presence of villin in human sera. We discuss the value of villin as a diagnostic adjunct for the diagnosis and follow-up of patients with colorectal carcinomas. MATERIALS AND METHODS Clinical Specimens. Age- and sex-matched healthy controls were provided by a serum bank from the Centre National de Transfusion sanguine (HôpitalCochin, Paris, France). The mean age of these donors was 37 ±15 yr. They included a roughly equal proportion of men and women. The mean age of donors from whom sera contained detectable amounts of villin was 39 ±11yr. Coded serum collections from patients bearing gastrointestinal tumors, adenocarcinoma of other organs, or benign diseases were obtained from three hospitals (Departments of Internal Médecineand Surgery). For this study, the diagnosis and follow-up of patients bearing digestive tumors were carried out with routine clinical and endoscopie examinations. All specimens (healthy donors' sera, benign and malignant disease patients' sera) were coded and collected during the same period of time. All of them were aliquoted and stored in the same conditions at —¿20°C. We have observed that villin is stable in these storage conditions. Provided that the assay was performed on frozen and thawed samples, there was no significant variation in the measured villin concentration. Medical histories of patients were checked to validate this information and to secure addi tional data about the serum withdrawal date and about the ultimate 3The abbreviations used are: CEA, carcinocmbryonic antigen; ELISA, enzyme- linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin. 438 Research. on August 26, 2015. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from