Presence of Villin, a Tissue-Specific Cytoskeletal Protein, in Sera Of

[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

DÃ©partementdeMÃ©decineinterne [L. J., B. C., G. A. C.J, Clinique Chirurgicale [Y. C., P. Bo.J, and Poste de Transfusion sanguine Â¡F.P.], HÃ´pital Cochin, 27, rue du Faubourg Saint Jacques, 75674 Paris Cedex 14; DÃ©partementdeMÃ©decineoncologique [P. Be., P. P.], Laboratoire de radiopathologie Â¡H.M.], and Service de Chirurgie [R. J. S.], Institut Curie, 26 rue d'Ulm, 75005 Paris; Service de Gastroenterologie, Pr. Y. Le Quintrec, HÃ´pital Rothschild, 75012 Paris [J. B.]; Pharmacie HÃ´pital SalpÃ©triÃ©re,83bd de l'HÃ´pital, 75013 Paris [A. P. B.]; Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex [M. T. M.J; and UnitÃ©deBiologie des Membranes, DÃ©partementdeBiologie MolÃ©culaire,Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15 [B. D., S. R., D. L.], France

ABSTRACT This restricted tissue specificity of villin is also conserved during neoplasie processes. In this respect, it is particularly Villin is an actin-binding protein found in a few normal adult epithelia, worth recalling that villin continues to be produced in colorectal namely epithelial cells in the digestive and urogenital tracts. Moreover, tumors at all stages of epithelial cell transformation, but that villin production is maintained in malignant cells. We assumed that cell lysis and necrosis of solid tumors producing villin might result in villin villin is conspicuously absent from tumors arising from cell release into blood. We analyzed the villin content of sera from 788 types in which it is not normally found (5, 6). patients and controls using an enzyme-linked immunosorbent assay. These observations are in contrast with the more generalized Patients and controls were classified into healthy donors, patients with production of CEA,3 a tumor marker that is routinely assayed benign diseases of the gastrointestinal tract, patients with colorectal for screening and follow-up of patients with tumors of the cancers, and patients with malignant nondigestive diseases. In the panel digestive tract. For example, it is now well established that of sera analyzed, the sensitivity of the assay for colorectal cancers was CEA is found in several normal cells of numerous origins (7- 50.5%, and its overall specificity for malignant digestive tumors was 9). 94.5%. Results were statistically analyzed comparing each group of sera The above mentioned properties of villin prompted us to with each other. We conclude that the presence of villin is indicative of a pathological state in the gastrointestinal tract (/' < 0.001). Finally, we determine whether it is released into the blood circulation, due followed villin levels after tumor resections (60 patients). We found that to lysis of malignant cells or by means of another unknown the villin level in sera remains low in remissions but is raised in recur mechanism, which could indicate the existence of a lesion in rences. the digestive tract. We therefore investigated the presence of We suggest that the villin assay may have clinical utility as a diagnostic villin in the sera of 788 patients and controls with an ELISA adjunct for adenocarcinoma of the gastrointestinal tract. It may also have developed for this study in our laboratory. Our initial data some value in monitoring patients with advancing colorectal carcinomas showed that the occurrence of villin in human sera corre after resection of these tumors. 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 INTRODUCTION of the presence of villin in human sera. We discuss the value of Enterocytes, the epithelial cells of the intestinal mucosa, villin as a diagnostic adjunct for the diagnosis and follow-up of exhibit a functional and morphological polarity. Their apical patients with colorectal carcinomas. plasma membrane shows an ordered array of microvilli forming the brush border. Each microvillus is supported by a cytoskel- MATERIALS AND METHODS eton based on actin microfilaments. These actin microfilament bundles are associated with a few actin-binding proteins (1). Clinical Specimens. Age- and sex-matched healthy controls were Among them, villin, a M, 95,000 protein, is able to bind actin provided by a serum bank from the Centre National de Transfusion in a calcium-dependent process. Production of villin and its sanguine (HÃ´pitalCochin, Paris, France). The mean age of these donors regulation were previously studied in our laboratory in vivo and was 37 Â±15 yr. They included a roughly equal proportion of men and in cell cultures (2-4). During ontogenesis of the human diges women. The mean age of donors from whom sera contained detectable amounts of villin was 39 Â±11yr. Coded serum collections from patients tive tract, villin is found in immature intestinal cells as early as bearing gastrointestinal tumors, adenocarcinoma of other organs, or the eight wk of gestation of the human fetus, before the final benign diseases were obtained from three hospitals (Departments of stages of histogenesis. Similarly, villin is found in adult undif- Internal MÃ©decineand Surgery). For this study, the diagnosis and ferentiated crypt cells of the intestinal mucosa. In the adult follow-up of patients bearing digestive tumors were carried out with human, high levels of villin were primarily found in epithelial routine clinical and endoscopie examinations. All specimens (healthy cells with a well-organized brush border, namely the epithelial donors' sera, benign and malignant disease patients' sera) were coded cells in the small and large intestine and in cells lining the and collected during the same period of time. All of them were aliquoted and stored in the same conditions at â€”¿20Â°C.Wehave observed that proximal tubules of the kidney. villin is stable in these storage conditions. Provided that the assay was Received 2/27/89; revised 7/13/89; accepted 9/25/89. performed on frozen and thawed samples, there was no significant The costs of publication of this article were defrayed in part by the payment variation in the measured villin concentration. Medical histories of 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. patients were checked to validate this information and to secure addi 1This work was supported by grants from the Institut National de la SantÃ©et tional data about the serum withdrawal date and about the ultimate de la Recherche MÃ©dicale(No. 86-7008), by the Fondation pour la Recherche MÃ©dicaleFranÃ§aise,by the Association pour la Recherche sur le Cancer (No. 3The abbreviations used are: CEA, carcinocmbryonic antigen; ELISA, enzyme- 6379), thÃ©CNRS(UA-1149), and thÃ©LigueNationale FranÃ§aisecontre le Cancer. linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum 1To whom requests for reprints should be addressed. albumin. 438

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. VILLIN, A CYTOSKELETAL PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS course of the disease. Laboratory assays, such as that for CEA, were RESULTS determined in parallel on the same sample and were available for correlation with the results of the villin assays. Sensitivity and Specificity of the Villin ELISA Assay. The Determination of Villin Levels. Sera were analyzed by an ELISA ELISA assay for villin designed in our laboratory uses a highly using a mouse monoclonal anti-villin (BDID2C3) antibody obtained and specific monoclonal antibody raised against villin and able to characterized as described in Ref. 4. This monoclonal antibody recog recognize a unique epitope found only in villin (4). The assay nizes a well-conserved epitope of villin across species. This epitope is for villin used in this study is a sandwich ELISA (see "Materials located in an actin-binding domain at the COOH-terminal end of the and Methods"). Fig. 1 shows a typical standard curve, made molecule. with villin concentrations ranging from 2 ng/ml to 150 ng/ml. Primary sequence analysis of this domain carried out by our group The slope of the curve depends upon the villin-free medium (10) has revealed that the carbo\>-terminal end of villin is villin specific used for the pure villin dilution. The minimal detectable amount and displays no sequence homologies with other actin-binding proteins is as low as 0.5 ng/ml in PBS and up to 4 ng/ml in some villin- nor with any other known proteins currently listed in a protein data free sera obtained from healthy donors. In all cases, a linear library. response up to 75 ng/ml was obtained. Such differences may In addition, cell extracts prepared with various cell lines established be due to interference by serum proteins and were not investi from cells not expressing villin in viro, when investigated for villin gated further. Specificity was checked on every serum sample content with this ELISA, prove to be negative (2, 11). Moreover, if a known amount of exogenous villin were added to with villin levels above 20 ng/ml by preincubation with an excess of monoclonal antibody against villin as described in these extracts, villin could be quantitatively assayed in these conditions. "Materials and Methods." Only samples exhibiting a total Altogether, these data illustrate the specificity of the antibody for villin and the lack of cross-reactivity for ubiquitous cytoskeletal proteins, inhibition after preincubation with the monoclonal antibody such as gelsolin, tropomyosin, calpactin, actinin, as well as the lack of were considered to contain villin. interference with the assay of structural and cellular proteins unrelated Determination of Villin Levels in Sera from Healthy Donors to villin. and from Patients with Benign Digestive Diseases and Malignant Polystyrene microtiter plates were coated with 10 Mg/rnl of purified Digestive and Extradigestive Diseases. The distribution of monoclonal anti-villin IgG in SO mivi potassium phosphate buffer (pH serum villin concentrations in healthy donors and in patients 8), using 50 n\ per well. Coating was carried out at 37Â°Cfor 2 h and with benign diseases is shown in Fig. 2. Among the healthy then at 4Â°Covernight. After five washes with 0.1 % Tween 20 in PBS, donors (421 cases), 408 displayed serum villin levels below 20 plates were saturated for 30 min at 4Â°Cwith 1% (w/w) BSA in PBS- ng/ml, while sera from 13 donors (3%) contained more than Tween 20. Serial 2-fold dilutions (from 1/2 to 1/32) of the sera in PBS- Tween 20-BSA (60 ^I/well) were incubated overnight at 4Â°C.After five 20 ng/ml of villin (Fig. 2A). Among the sera from patients with benign diseases of the washes with PBS-Tween 20 buffer, the second antibody, a /J-galactosid- digestive tract (132 cases), 17 were found to contain significant ase-Iabeled polyclonal rabbit anti-villin IgG diluted in PBS-Tween 20- BSA, was added, and the incubation was continued for 3 h at 4Â°C.After amounts of villin (12.9%) (Fig. 2B). Among 19 samples ob five more washes with PBS-Tween 20 buffer, hydrolysis of p-nitro- tained from patients with duodenal ulcers, only 2 contained phenyl /3-D-galactopyranoside was determined as described (12) and levels of villin slightly above the threshold (22 and 46 ng/ml, nitrophenol was measured at 414 nm, using a MK 700 ELISA spectro- respectively). Villin assays were also carried out on sera of 20 photometer (Dynatech, South Windham, ME). patients with gastric ulcers: 3 patients had serum villin levels Each plate contained a serial dilution of pure porcine villin (a above the threshold. Interestingly enough, one of these patients generous gift from Dr. V. Gerke and Dr. K. Weber, Max-Planck had an ulcer with a severe dysplasia (50 ng of villin/ml of Institute, GÃ¶ttingen,West Germany) diluted in villin-frce human serum serum), and the other two had an ulcer with intestinal meta as a standard. False-positive assays due to nonspecific binding to the plasia (76 and 107 ng of villin/ml of serum). Among 40 sera plate were detected by the following assay. Sera containing over 20 ng from patients with Crohn's disease, 5 patients (12.5%) showed of villin per ml of serum were incubated with an excess of monoclonal anti-villin IgG (35 Mg/ml) for 3 h at room temperature in order to saturate specific villin binding sites prior to incubation with the plate. Control experiments carried out with dilutions of purified villin added to normal human sera showed a 98% inhibition in these conditions. Sera for which this competition was ineffective were considered as false- positive and scored as negative. Determination of CEA Levels. To compare villin and CEA as markers from malignant digestive diseases before and after surgery, CEA levels were assayed in some sera with a solid phase enzyme immunoassay (Abbott diagnostic kit). Levels of CEA over 5 ng/nl of serum were considered to be abnormally high. Determination of Sensitivity and Specificity Scores. The sensitivity of a marker, defined here as the ability to identify true-positive cases in patients with colorÃ©ela!cancer, is calculated by dividing the number of patients with above-normal concentrations of the marker by the total number of patients with colorectal cancer. The specificity of a marker, defined here as the ability to identify true-negative cases in patients without colonie malignancy, is calculated 9 ia7 375 75 150 villin ng/ml by dividing the number of patients with normal concentrations of the Fig. 1. Quantification of villin in sera using an ELISA. A standard curve was marker by the total number of healthy donors, patients with benign constructed using serial dilutions of pure porcine villin in villin-frec human sera colorectal disease, and patients with extradigestive disease. (D). This standard curve is compared with a curve obtained for the same dilutions Statistical Analysis. The classical x2 test was used to analyze the of pure porcine villin in PBS-Tween 20-BSA (â€¢)or of a positive patient serum (A). When an excess of antibody (35 Â»ig/ml)isadded to a serum containing villin, statistical significance of the presence of villin in sera of patients and 98% of inhibition of the specific response is observed (O). No villin is detected in controls. a normal human serum (* ) 439

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Fig. 2. A, distribution of serum villin levels in healthy donors. Among 421 â€¢¿â€¢..â€¢". tÂ»Â«â€¢â€¢â€¢â€¢:.â€¢â€¢â€¢i.*.â€¢â€¢:ttt:V:::: healthy donors, 13 (3%) exhibited levels above threshold (20 ng/ml). B, distribu tion of serum villin levels in patients with benign gastrointestinal diseases. For duodenal ulcers, 2 patients of 19 (10.5%) were above threshold (Lane A). For Fig. 3. Distribution of serum villin levels in patients with colorectal cancers gastric ulcers, among 20 patients, 3 (15%) showed levels above 20 ng/ml (Lane B). For patients with Crohn's disease and hemorrhagic rectocolitis, 5 patients of before surgery classified into Stages A and D. Patients (Stage A) with undetectable villin levels (Lane A). Sixteen of 31 (Stage B) patients (51%) exhibited villin 46 (10.8%) had villin levels above threshold (Lane C). For colorectal polyposis, levels above threshold (Lane B). Six of 13 (Stage C} patients (46%) had villin one patient of 19 (5%) had levels above 20 ng/ml (Lane D). levels higher than 20 ng/ml (Lane C). Twenty-six of 48 (Stage D) patients (54%) had values above threshold (Lane D). serum villin levels above threshold (30 to 1280 ng/ml), but 300200HM500Â«M300 none of the 6 patients with ulcerative colitis had a detectable level of villin in their sera. In the case of colorectal polyps, only one patient of 19 had serum villin levels above the threshold (200 ng/ml). This patient had a polyadenoma with a severe displasia. The levels of villin were also determined in 28 sera from patients with liver cirrhosis of various etiologies since this pathological state is often associated with elevated levels of tumor markers, such as CEA. Six sera were found to contain -200^ elevated levels of villin (72 to 2000 mg/ml). Four patients in 100Ã• this group had liver cirrhosis with ascites. We found no signif 902 icant statistical difference between each group of patients with Â»BZ benign digestive diseases for the detection of villin in sera (x2 70I = 3.2; P = not significant). Sera of patients with colorectal cancers (95 cases) were ana 50 "UI lyzed before surgery and at Stages A to D according to a modified Dukes' classification (Stage A, cancer confined to the 30v> mucosa; Stage B, cancer penetrating deeper, even through the 20-,*::::::::::::::::::â€¢â€¢itA1â€¢1â€¢f-iâ€¢*â€¢â€¢*â€¢i::::::::':Â»::::. bowel wall; Stage C, cancer penetrating as in Stage B but with involvement of regional lymph nodes; Stage D, cancer with distant mÃ©tastases).The three Stage A patients had undetecta- I II Fig. 4. Distribution of villin concentration in sera from patients with various ble serum villin levels (below 2 ng/ml). Among the 31 patients extradigestive cancers (Lane I) or with colorÃ©ela!cancers(Lane II) before surgery at Stage B, 16 (51%) exhibited elevated serum villin levels (all stages taken together). In Lane I. extradigestive cancers, 4 patients of 80 (5%) (range, 20 to 1250 ng/ml), while 6 of 13 Stage C patients (46%) had villin levels above 20 ng/ml. This group of patients comprises the following cancers: lung (8); ovary (14); breast (25); uterus (5); prostate (4); melanoma (4); were positive (range, 20 to 2000 ng/ml). We also found that osteosarcoma (3); myosarcoma (5); and a panel of other types of cancers occurring among 48 patients at Stage D, 26 (54%) had values above less frequently (12 cases). In Lane II, colorectal cancers, among 95 patients, 48 threshold (range, from 20 to 3000 ng of villin/ml of serum). exhibited villin concentrations above threshold. When all stages were taken together, 50.5% of samples were above the threshold (Figs. 3 and 4). donors; donors with benign digestive diseases; donors with The slight differences between the groups have no statistical malignant digestive diseases; and donors with malignant non- significance (x2 = 0.52, P = not significant). digestive diseases. Each group was compared with each other, and the values obtained were subjected to the x2 test. The Among 80 patients with various extradigestive cancers, only 4 cases (5%) corresponding to advanced cancers had levels of analysis of the results obtained is summarized in Table 1. serum villin above threshold; these cases are: one primary' Our data show that the occurrence of villin in the serum epidermoid tumor of the esophagus; one breast cancer; one correlated strongly with a pathological benign or malignant ovary tumor with peritoneal carcinomatosis; and one larynx state in the digestive tract. It is particularly noteworthy that, cancer (Fig. 4). when healthy donors are compared to donors bearing malignant Statistical Analysisâ€”Sensitivity and Specificity Scores. Con digestive diseases, the statistical parameters x2 and P were trol donors and patients were divided in four groups: healthy found to be particularly significant. Similarly, the comparison 440

Table 1 Statistical analysis of villin detection in healthy donors, patients with benign digestive diseases, or those with malignant extradigestive and malignant digestive diseases Healthy donors (3%) and 12.9% of patients with benign digestive diseases exhibited serum villin levels above 20 ng/ml. These two groups are significantly different (x2 = 16.9; P < 0.001). Patients with colorectal adenocarcinoma (50.5%) showed serum villin levels above threshold. This group is significantly different from healthy donors (x2 = 162.81, P < 0.001) and from those having benign digestive diseases (x2 = 36.49; P < 0.001). The group of patients with digestive cancers (colorectal carcinoma) is significantly different from the group of patients with malignant extradigestive diseases (x! = 40.9; P < 0.001). On the other hand, there is no significant difference between healthy donors and patients with extradigestive cancers (x2 = 0.280; not significant). No. of patients' sera No. of patients' sera with villin levels with villin levels %HealthyComparison of clinical variables above threshold (1)* % above threshold (2)' P16.91 donors (1) and benign digestive (13/421)3% (17/132)48 12.9% <0.001162.81 diseases(2)Healthy

donors (1) and malignant diges (13/421)12.9% (48/95)48 50.5% Â«0.00136.49 (2)Benigntive diseases

digestive diseases (1) and malig (17/132)5% (48/95)48 50.5% <0.00140.94 (2)Malignantnant digestive diseases

extradigestive diseases (1) and (4/80)3% (48/95)4 50.5% <0.0010.280 (2)Healthymalignant digestive diseases

donors (1) and malignant extra- (13/421)17 5% (4/80)x2 NSÂ° digestive diseases (2)1313174133% Â°NS, not significant. *The numbers (1) and (2) correspond to the donor and patient populations listed in column "Comparison of clinical variables".

Table 2 Comparison of villin and CEA detection in patients' sera with colorectal of patients with malignant digestive diseases and patients with cancers before surgery malignant nondigestive diseases is also significant (Table 1). Threshold for villin is 20 ng/ml and threshold for CEA is 5 ng/ml. From the results reported in this study, we have also deter of pa mined figures for the sensitivity and specificity of the villin as Totalno. of pa of pa tients'serawith tumoral marker. We found a sensitivity of 50.5% (48 of 95) ofpatients'seraanalyzedNo.tients'serawith tients'serawith villinand villinlevels CEAlevels CEAlevels and a specificity of 94.5% (598 of 633) for villin using this abovethresholdNo.above% aboveÃ³ particular panel of sera. threshold Â°,No.threshold % A Comparison of Villin and CEA as Markers of Malignant Stage B 27 12 44.44 10 37.03 17 63 Digestive Diseases before Surgery. Sera obtained from the ma jority of patients with malignant digestive diseases could be Stage C compared before surgery for villin and CEA levels (75 cases). Stage D133561746.15 48.578 3161.53 88.5793269.23 91.42 All of these patients had colorectal tumors and were classified as being at one of the three Dukes' stages (B, C, or D), as defined above. Among the 27 patients of Stage B, 12 (44.5%) POST SUR6ERT FOLLÃ“NUP exhibited serum villin levels significantly above threshold, and REMISSIONS RECURRENCESVillin 10 (37%) had elevated levels of CEA. When both villin and CEA200010001002OIBiiVillin CEA.. CEA were assayed, the sensitivity of the analysis was improved ....*2Oâ€¢â€¢â€¢â€¢200010001001 since 17 patients of 27 (63%) were positive for at least one marker. Similarly, among 13 patients at Stage C, 9 (69.3%) were positive for at least one marker as compared with 6 for the villin test above and 8 if only CEA were assayed. At Stage D, 17 of the 35 patients (48.6%) exhibited serum villin levels above 20 ng/ml, and 31 (88.6%) had CEA levels above 5 ng/ ml. When both villin and CEA were assayed, the existence of colorectal cancers was correctly predicted in 32 cases (91.3%). Fig. 5. Villin and CEA levels in patients with colorectal carcinoma in post- Taken altogether, these observations suggest that patients in surgery follow-up. Normal levels for villin were fixed at 20 ng/ml, and at 5 ng/ whom colorectal cancer has escaped diagnosis by CEA analysis ml for CEA. Remissions and recurrences were determined after clinical exami nation. Among 60 cases, 49 patients were in remission: 46 of these displayed might be positive for villin. These preliminary data suggest that undetectable villin levels (below 2 ng/ml) and, in 47 cases, the CEA level was also a simultaneous search for villin and CEA in sera may be useful, under threshold. Among the 11 recurrences. 9 were positive in the serum villin particularly in the case of patients at Stage B, when tumor test, and 7 had an elevated level for CEA. exÃ©resisgives a better prognosis. It should be mentioned that the comparison of the figures reported here for villin and CEA before surgery. This included 5 patients positive for both villin measured separately and those obtained when both markers and for CEA. After surgery, 10 patients had remission and were were assayed simultaneously is promising but not statistically negative for both markers. The other two patients were positive significant. Therefore, further analysis including a larger num for both villin and CEA. ber of patients should be performed in the future. These data Among the patients for whom the information for villin and are summarized in Table 2. CEA was not available before surgery but available for a post- Villin and CEA Levels during Postsurgery Follow-up. For over surgical follow-up, we found that, among 49 patients clinically 3 to 60 mo, 60 patients bearing colorectal cancers were moni in complete remission, 46 had normal villin and 47 had normal tored after surgery for villin and CEA serum levels. Data for CEA levels (Fig. 5). Villin level remained above threshold in villin and CEA before surgery were only available for 12 pa three cases. Among those, two were positive for both villin and tients. Among those, 7 were positive for at least one marker CEA. The third patient, who had an ascitic cirrhosis, was the 441

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. VILLIN, A CYTOSKELETAL PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS only one to exhibit normal CEA levels with very high villin be carried out to evaluate the usefulness of the villin assay for values (3300 ng/ml). Tumor recurrence was studied in 11 these cancers. The sensitivity score of the villin test is 50%, patients, 9 of whom displayed high villin values (range, from regardless of the stage of invasion of the tumor. This indicates 20 to 2000 ng/ml). Seven of these patients also scored as that the villin assay has a sensitivity equivalent to existing positive for CEA. Very high villin concentrations (2000 ng/ml) biological assays for colorectal cancers. Thus, taking into ac were observed in sera from some cases of cancer recurrence count the specificity score mentioned above, it would appear with liver mÃ©tastases(Fig. 5). The preoperative and follow-up that the main advantage of the villin assay in diagnosis as well investigations of 7 representative patients are summarized in as for follow-up patients after surgery lies in its high specificity Fig. 6. for digestive cancers. The villin test would indeed be primarily Our data demonstrate that, after surgery, villin levels drop advantageous for patients bearing resectable colorectal tumors and remain below the threshold when a complete remission is (such as Stage B tumors). Moreover, significant improvement clinically confirmed. Moreover in cases of tumor recurrence, for all stages of colorectal tumors may be obtained when both villin was observed in the serum in the majority of cases studied. CEA and villin are assayed, but the latter proposal remains to be rigorously tested on a larger number of patients. The value in clinical practice of the CEA or of the CA 19.9 DISCUSSION tests for the diagnosis or prognosis of digestive cancers is still under debate (13, 14). It is widely accepted, however, that none In this study, we have investigated the occurrence of villin in of the available assays is satisfactory. This is especially true for 788 sera. We show that the presence of villin in human serum the early diagnosis of colorectal cancer when tumors are still can often be correlated with the existence of a malignant tumor resectable. The low specificity of these markers can be partly in the gastrointestinal tract. In contrast to existing biological attributed to their wide distribution in malignant and normal assays for colorectal carcinoma, villin concentrations above cell types (15). On the contrary, the high specificity of the threshold indicate a pathological state in the gastrointestinal serological assay observed for villin can be easily explained by tract in almost 95% of cases. We have focused this first study taking into account the tissue specificity of villin expression in of villin in human sera on colorectal cancers, since they are normal or malignant epithelial cells in the gastrointestinal tract. more frequent in Western countries than other cancers which Villin is a nonglycosylated intracellular protein which is pro might release villin into sera, such as pancreatic cancers, duced early during embryogenesis in endodermic cells of the cholangioma, or renal cancers. Indeed, further studies should primitive gut (2-16). During adult life, villin continues to be produced at high levels in normal intestinal cells. This specific POST SURGERY FOLLOW UP pattern of expression for villin is also maintained during neo Â«ILLINng/ml plasie processes (2, 5). Thus, if villin is detected in sera of 2000 patients, one must suppose that it originates from one of a limited number of tissues. Moreover, our study strongly indi cates that the release of villin into serum is very often associated with the existence of a neoplasie tissue in the gastrointestinal 1000 tract. Its entry into the general circulation presumably requires cell lysis. Our results suggest that, even if cell lysis occurs in ulcerative or inflammatory diseases of the large bowel, villin only rarely appears in the blood. This may be due to the fact III that, under these circumstances, the blood supply and the cellular environment do not allow the entry of villin into the general circulation. In cases of highly vascularized colorectal adenocarcinomas, rapid necrosis and death of malignant cells are known to occur. Under these conditions one may assume that efficient release of cell content and discharge into the blood are facilitated. If such mechanism explains the release of villin

months into the blood, one would also expect a direct correlation Fig. 6. Serum villin levels in 7 patients with colorectal carcinoma during between the size and stage of the tumor and the presence of postsurgery follow-up. Patient I. a case displaying a postoperative residual tumor villin in the serum. Indeed, our results indicate that there are and mÃ©tastases.The serum villin level is low but remained above the threshold level. A sharp rise in serum villin levels was detected between 3 and 10 mo after no significant differences if one compares the percentage of surgery to 1000 ng/ml, whereas the clinical diagnosis of recurrences and mÃ©tas patients positive for villin at Stages B and D. This implies that tases was still negative. At 12 mo, the serum villin level was even higher (2000 other factors must be taken into account to interpret our data, ng/ml), and liver mÃ©tastaseswere clinically diagnosed at this stage. Patient 2, a case similar to the previous one but with no preoperative villin assay available. such as variability of villin stability in serum and large differ This patient was bearing a recurrent colorectal tumor with liver mÃ©tastases.The ences in the extent of tumor necrosis among patients. Alterna two patients (Nos. 1 and 2) were not treated by chemotherapy and/or radiother tively, changes in villin organization in tumor cells may account apy. High villin levels (higher than 1000 ng/ml) were observed in both cases. Patients 3 and 4, two patients positive for villin before surgical removal of their for the release of villin into the blood. In support of this tumor; thereafter they exhibited a rapid decrease in villin serum levels. Patient 5, proposal one should recall the recent observation (6) reporting 10 mo after resection of the colorectal tumor, this patient showed a local recurrence of the tumor with a serum villin level slightly above threshold (35 ng/ the localization of villin along the basement membrane in some ml). This recurrence, which was detected by colonoscopy, required readmission colorectal adenocarcinoma. The physiopathological conditions for surgery, after which the serum villin concentration decreased and remained leading to the appearance of villin in the blood are indeed below threshold. Patient 6, a case having a history similar to the previous ones but exhibiting a lower increase in serum villin levels in relation to the existence important to analyze and remain to be studied in detail. of invasive liver mÃ©tastases.In contrast to Patients 1 and 2, this patient received Our current study suggests that villin assays could be espe a complete course of postoperative 5-fluorouracil and radiotherapy. Patient 7, a case showing complete remission 5 mo and 16 mo after surgery, serum villin cially useful to follow the clinical course and diagnosis of levels remaining below threshold. recurrence of colorectal cancers in addition to CEA assays that 442

display low specificity. Indeed, serum villin levels drop after ACKNOWLEDGMENTS surgery and remain below threshold upon complete remission. We are indebted to J. F. Colombel, M.D., and M. Thioux for In contrast, recurrence is accompanied by a significant increase providing coded sera, E. Coudrier, Ph.D., and F. Traincard for advi-.e in villin level, even before clinical evidence of the recurrence, and expert technical assistance, T. Pugsley, Ph.D., for his helpful especially in cases of liver mÃ©tastases.Moreover, our prelimi comments during the preparation of the manuscript, and R. CarrÃ©for nary observations strongly suggest that, in case of recurrence, typing this manuscript. a high level of villin in serum correlates with a rapid progression of the disease. It would therefore appear that villin may have a predictive value concerning the extent and the prognosis of the REFERENCES disease. However, these important findings require further in 1. Mooseker, M. S. 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B. Dudouet, L. Jacob, P. Beuzeboc, et al.

Cancer Res 1990;50:438-443.

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