Download 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
Départementde 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; Départementde 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, Départementde 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 cytoskeleton 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 undifferentiated 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 Santéet
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), thé
CNRS (UA-1149), and thé
Ligue Nationale Françaisecontre le Cancer.
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 (79).
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 ±11 yr. 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, enzymelinked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum
albumin.
438
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VILLIN, A CYTOSKELETAL
PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS
course of the disease. Laboratory assays, such as that for CEA, were
determined in parallel on the same sample and were available for
correlation with the results of the villin assays.
Determination of Villin Levels. Sera were analyzed by an ELISA
using a mouse monoclonal anti-villin (BDID2C3) antibody obtained and
characterized as described in Ref. 4. This monoclonal antibody recog
nizes a well-conserved epitope of villin across species. This epitope is
located in an actin-binding domain at the COOH-terminal end of the
molecule.
Primary sequence analysis of this domain carried out by our group
(10) has revealed that the carbo\>-terminal end of villin is villin specific
and displays no sequence homologies with other actin-binding proteins
nor with any other known proteins currently listed in a protein data
library.
In addition, cell extracts prepared with various cell lines established
from cells not expressing villin in viro, when investigated for villin
content with this ELISA, prove to be negative (2, 11).
Moreover, if a known amount of exogenous villin were added to
these extracts, villin could be quantitatively assayed in these conditions.
Altogether, these data illustrate the specificity of the antibody for villin
and the lack of cross-reactivity for ubiquitous cytoskeletal proteins,
such as gelsolin, tropomyosin, calpactin, actinin, as well as the lack of
interference with the assay of structural and cellular proteins unrelated
to villin.
Polystyrene microtiter plates were coated with 10 Mg/rnl of purified
monoclonal anti-villin IgG in SO mivi potassium phosphate buffer (pH
8), using 50 n\ per well. Coating was carried out at 37°Cfor 2 h and
then at 4°Covernight. After five washes with 0.1 % Tween 20 in PBS,
plates were saturated for 30 min at 4°Cwith 1% (w/w) BSA in PBSTween 20. Serial 2-fold dilutions (from 1/2 to 1/32) of the sera in PBSTween 20-BSA (60 ^I/well) were incubated overnight at 4°C.After five
washes with PBS-Tween 20 buffer, the second antibody, a /J-galactosidase-Iabeled polyclonal rabbit anti-villin IgG diluted in PBS-Tween 20BSA, was added, and the incubation was continued for 3 h at 4°C.After
five more washes with PBS-Tween 20 buffer, hydrolysis of p-nitrophenyl /3-D-galactopyranoside was determined as described (12) and
nitrophenol was measured at 414 nm, using a MK 700 ELISA spectrophotometer (Dynatech, South Windham, ME).
Each plate contained a serial dilution of pure porcine villin (a
generous gift from Dr. V. Gerke and Dr. K. Weber, Max-Planck
Institute, Göttingen,West Germany) diluted in villin-frce human serum
as a standard. False-positive assays due to nonspecific binding to the
plate were detected by the following assay. Sera containing over 20 ng
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 falsepositive 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 coloréela!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
by dividing the number of patients with normal concentrations of the
marker by the total number of healthy donors, patients with benign
colorectal disease, and patients with extradigestive disease.
Statistical Analysis. The classical x2 test was used to analyze the
statistical significance of the presence of villin in sera of patients and
controls.
RESULTS
Sensitivity and Specificity of the Villin ELISA Assay. The
ELISA assay for villin designed in our laboratory uses a highly
specific monoclonal antibody raised against villin and able to
recognize a unique epitope found only in villin (4). The assay
for villin used in this study is a sandwich ELISA (see "Materials
and Methods"). Fig. 1 shows a typical standard curve, made
with villin concentrations ranging from 2 ng/ml to 150 ng/ml.
The slope of the curve depends upon the villin-free medium
used for the pure villin dilution. The minimal detectable amount
is as low as 0.5 ng/ml in PBS and up to 4 ng/ml in some villinfree sera obtained from healthy donors. In all cases, a linear
response up to 75 ng/ml was obtained. Such differences may
be due to interference by serum proteins and were not investi
gated further. Specificity was checked on every serum sample
with villin levels above 20 ng/ml by preincubation with an
excess of monoclonal antibody against villin as described in
"Materials and Methods." Only samples exhibiting a total
inhibition after preincubation with the monoclonal antibody
were considered to contain villin.
Determination of Villin Levels in Sera from Healthy Donors
and from Patients with Benign Digestive Diseases and Malignant
Digestive and Extradigestive Diseases. The distribution of
serum villin concentrations in healthy donors and in patients
with benign diseases is shown in Fig. 2. Among the healthy
donors (421 cases), 408 displayed serum villin levels below 20
ng/ml, while sera from 13 donors (3%) contained more than
20 ng/ml of villin (Fig. 2A).
Among the sera from patients with benign diseases of the
digestive tract (132 cases), 17 were found to contain significant
amounts of villin (12.9%) (Fig. 2B). Among 19 samples ob
tained from patients with duodenal ulcers, only 2 contained
levels of villin slightly above the threshold (22 and 46 ng/ml,
respectively). Villin assays were also carried out on sera of 20
patients with gastric ulcers: 3 patients had serum villin levels
above the threshold. Interestingly enough, one of these patients
had an ulcer with a severe dysplasia (50 ng of villin/ml of
serum), and the other two had an ulcer with intestinal meta
plasia (76 and 107 ng of villin/ml of serum). Among 40 sera
from patients with Crohn's disease, 5 patients (12.5%) showed
9
ia7
375
75
150
villin ng/ml
Fig. 1. Quantification of villin in sera using an ELISA. A standard curve was
constructed using serial dilutions of pure porcine villin in villin-frec human sera
(D). This standard curve is compared with a curve obtained for the same dilutions
of pure porcine villin in PBS-Tween 20-BSA (•)or of a positive patient serum
(A). When an excess of antibody (35 »ig/ml)is added to a serum containing villin,
98% of inhibition of the specific response is observed (O). No villin is detected in
a normal human serum ( * )
439
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VILLIN, A CVTOSKELETAL PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS
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Fig. 2. A, distribution of serum villin levels in healthy donors. Among 421
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
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
46 (10.8%) had villin levels above threshold (Lane C). For colorectal polyposis,
one patient of 19 (5%) had levels above 20 ng/ml (Lane D).
•¿â€¢..•".
t»«••••:.•••i.*.â€
Fig. 3. Distribution of serum villin levels in patients with colorectal cancers
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
levels above threshold (Lane B). Six of 13 (Stage C} patients (46%) had villin
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
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
elevated levels of villin (72 to 2000 mg/ml). Four patients in
this group had liver cirrhosis with ascites. We found no signif
icant statistical difference between each group of patients with
benign digestive diseases for the detection of villin in sera (x2
= 3.2; P = not significant).
Sera of patients with colorectal cancers (95 cases) were ana
lyzed before surgery and at Stages A to D according to a
modified Dukes' classification (Stage A, cancer confined to the
mucosa; Stage B, cancer penetrating deeper, even through the
bowel wall; Stage C, cancer penetrating as in Stage B but with
involvement of regional lymph nodes; Stage D, cancer with
distant métastases).The three Stage A patients had undetectable serum villin levels (below 2 ng/ml). Among the 31 patients
at Stage B, 16 (51%) exhibited elevated serum villin levels
(range, 20 to 1250 ng/ml), while 6 of 13 Stage C patients (46%)
were positive (range, 20 to 2000 ng/ml). We also found that
among 48 patients at Stage D, 26 (54%) had values above
threshold (range, from 20 to 3000 ng of villin/ml of serum).
When all stages were taken together, 50.5% of samples were
above the threshold (Figs. 3 and 4).
The slight differences between the groups have no statistical
significance (x2 = 0.52, P = not significant).
Among 80 patients with various extradigestive cancers, only
4 cases (5%) corresponding to advanced cancers had levels of
serum villin above threshold; these cases are: one primary'
epidermoid tumor of the esophagus; one breast cancer; one
ovary tumor with peritoneal carcinomatosis; and one larynx
cancer (Fig. 4).
Statistical Analysis—Sensitivity and Specificity Scores. Con
trol donors and patients were divided in four groups: healthy
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II
Fig. 4. Distribution of villin concentration in sera from patients with various
extradigestive cancers (Lane I) or with coloréela!cancers (Lane II) before surgery
(all stages taken together). In Lane I. extradigestive cancers, 4 patients of 80 (5%)
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);
osteosarcoma (3); myosarcoma (5); and a panel of other types of cancers occurring
less frequently (12 cases). In Lane II, colorectal cancers, among 95 patients, 48
exhibited villin concentrations above threshold.
donors; donors with benign digestive diseases; donors with
malignant digestive diseases; and donors with malignant nondigestive diseases. Each group was compared with each other,
and the values obtained were subjected to the x2 test. The
analysis of the results obtained is summarized in Table 1.
Our data show that the occurrence of villin in the serum
correlated strongly with a pathological benign or malignant
state in the digestive tract. It is particularly noteworthy that,
when healthy donors are compared to donors bearing malignant
digestive diseases, the statistical parameters x2 and P were
found to be particularly significant. Similarly, the comparison
440
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VILLIN, A CYTOSKELETAL PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS
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).
%Healthy
Comparison of clinical variables
No. of patients' sera
with villin levels
above threshold (1)*
No. of patients' sera
with villin levels
above threshold (2)'
%
P16.91
donors (1) and benign digestive
(2)Healthy
diseases
(13/421)3%
(17/132)48
12.9%
<0.001162.81
donors (1) and malignant diges
(2)Benign
tive diseases
(13/421)12.9%
(48/95)48
50.5%
«0.00136.49
digestive diseases (1) and malig
(2)Malignant
nant digestive diseases
(17/132)5%
(48/95)48
50.5%
<0.00140.94
extradigestive diseases (1) and
(2)Healthy
malignant digestive diseases
(4/80)3%
(48/95)4
50.5%
<0.0010.280
NS°
donors (1) and malignant extra(13/421)17
5% (4/80)x2
digestive diseases (2)1313174133%
°NS, not significant.
*The numbers (1) and (2) correspond to the donor and patient populations listed in column "Comparison of clinical variables".
of patients with malignant digestive diseases and patients with
malignant nondigestive diseases is also significant (Table 1).
From the results reported in this study, we have also deter
mined figures for the sensitivity and specificity of the villin as
tumoral marker. We found a sensitivity of 50.5% (48 of 95)
and a specificity of 94.5% (598 of 633) for villin using this
particular panel of sera.
A Comparison of Villin and CEA as Markers of Malignant
Digestive Diseases before Surgery. Sera obtained from the ma
jority of patients with malignant digestive diseases could be
compared before surgery for villin and CEA levels (75 cases).
All of these patients had colorectal tumors and were classified
as being at one of the three Dukes' stages (B, C, or D), as
Table 2 Comparison of villin and CEA detection in patients' sera with colorectal
cancers before surgery
Threshold for villin is 20 ng/ml and threshold for CEA is 5 ng/ml.
Totalno.
of pa
of pa
tients'
serawith
tients'
serawith
ofpatients'seraanalyzedNo.
villinlevels
CEAlevels
abovethresholdNo.
above%
threshold
Stage B
27
12
of pa
tients'
serawith
villinand
CEAlevels
aboveó
°,No.threshold
37.03
44.44
10
48.578
3161.53 88.5793269.23
17
%
63
Stage C
Stage D133561746.15
91.42
POST SUR6ERT FOLLÓN
UP
defined above. Among the 27 patients of Stage B, 12 (44.5%)
exhibited serum villin levels significantly above threshold, and
REMISSIONS
RECURRENCESVillin
10 (37%) had elevated levels of CEA. When both villin and
CEA..
CEA200010001002OIBii
Villin
CEA were assayed, the sensitivity of the analysis was improved
....*2O••••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 postTaken altogether, these observations suggest that patients in surgery follow-up. Normal levels for villin were fixed at 20 ng/ml, and at 5 ng/
ml for CEA. Remissions and recurrences were determined after clinical exami
whom colorectal cancer has escaped diagnosis by CEA analysis
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
test, and 7 had an elevated level for CEA.
particularly in the case of patients at Stage B, when tumor
exéresisgives 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
CEA was not available before surgery but available for a postare summarized in Table 2.
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
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VILLIN, A CYTOSKELETAL
PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS
only one to exhibit normal CEA levels with very high villin
values (3300 ng/ml). Tumor recurrence was studied in 11
patients, 9 of whom displayed high villin values (range, from
20 to 2000 ng/ml). Seven of these patients also scored as
positive for CEA. Very high villin concentrations (2000 ng/ml)
were observed in sera from some cases of cancer recurrence
with liver métastases(Fig. 5). The preoperative and follow-up
investigations of 7 representative patients are summarized in
Fig. 6.
Our data demonstrate that, after surgery, villin levels drop
and remain below the threshold when a complete remission is
clinically confirmed. Moreover in cases of tumor recurrence,
villin was observed in the serum in the majority of cases studied.
DISCUSSION
In this study, we have investigated the occurrence of villin in
788 sera. We show that the presence of villin in human serum
can often be correlated with the existence of a malignant tumor
in the gastrointestinal tract. In contrast to existing biological
assays for colorectal carcinoma, villin concentrations above
threshold indicate a pathological state in the gastrointestinal
tract in almost 95% of cases. We have focused this first study
of villin in human sera on colorectal cancers, since they are
more frequent in Western countries than other cancers which
might release villin into sera, such as pancreatic cancers,
cholangioma, or renal cancers. Indeed, further studies should
POST SURGERY FOLLOW UP
«ILLIN
ng/ml
2000
1000
III
months
Fig. 6. Serum villin levels in 7 patients with colorectal carcinoma during
postsurgery follow-up. Patient I. a case displaying a postoperative residual tumor
and métastases.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
surgery to 1000 ng/ml, whereas the clinical diagnosis of recurrences and métas
tases was still negative. At 12 mo, the serum villin level was even higher (2000
ng/ml), and liver métastaseswere clinically diagnosed at this stage. Patient 2, a
case similar to the previous one but with no preoperative villin assay available.
This patient was bearing a recurrent colorectal tumor with liver métastases.The
two patients (Nos. 1 and 2) were not treated by chemotherapy and/or radiother
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
tumor; thereafter they exhibited a rapid decrease in villin serum levels. Patient 5,
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/
ml). This recurrence, which was detected by colonoscopy, required readmission
for surgery, after which the serum villin concentration decreased and remained
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
of invasive liver métastases.In contrast to Patients 1 and 2, this patient received
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
levels remaining below threshold.
be carried out to evaluate the usefulness of the villin assay for
these cancers. The sensitivity score of the villin test is 50%,
regardless of the stage of invasion of the tumor. This indicates
that the villin assay has a sensitivity equivalent to existing
biological assays for colorectal cancers. Thus, taking into ac
count the specificity score mentioned above, it would appear
that the main advantage of the villin assay in diagnosis as well
as for follow-up patients after surgery lies in its high specificity
for digestive cancers. The villin test would indeed be primarily
advantageous for patients bearing resectable colorectal tumors
(such as Stage B tumors). Moreover, significant improvement
for all stages of colorectal tumors may be obtained when both
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
tests for the diagnosis or prognosis of digestive cancers is still
under debate (13, 14). It is widely accepted, however, that none
of the available assays is satisfactory. This is especially true for
the early diagnosis of colorectal cancer when tumors are still
resectable. The low specificity of these markers can be partly
attributed to their wide distribution in malignant and normal
cell types (15). On the contrary, the high specificity of the
serological assay observed for villin can be easily explained by
taking into account the tissue specificity of villin expression in
normal or malignant epithelial cells in the gastrointestinal tract.
Villin is a nonglycosylated intracellular protein which is pro
duced early during embryogenesis in endodermic cells of the
primitive gut (2-16). During adult life, villin continues to be
produced at high levels in normal intestinal cells. This specific
pattern of expression for villin is also maintained during neo
plasie processes (2, 5). Thus, if villin is detected in sera of
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
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
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
into the blood, one would also expect a direct correlation
between the size and stage of the tumor and the presence of
villin in the serum. Indeed, our results indicate that there are
no significant differences if one compares the percentage of
patients positive for villin at Stages B and D. This implies that
other factors must be taken into account to interpret our data,
such as variability of villin stability in serum and large differ
ences in the extent of tumor necrosis among patients. Alterna
tively, changes in villin organization in tumor cells may account
for the release of villin into the blood. In support of this
proposal one should recall the recent observation (6) reporting
the localization of villin along the basement membrane in some
colorectal adenocarcinoma. The physiopathological conditions
leading to the appearance of villin in the blood are indeed
important to analyze and remain to be studied in detail.
Our current study suggests that villin assays could be espe
cially useful to follow the clinical course and diagnosis of
recurrence of colorectal cancers in addition to CEA assays that
442
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VILLIN. A CYTOSKELETAL
PROTEIN IN SERA OF PATIENTS WITH COLORECTAL CANCERS
display low specificity. Indeed, serum villin levels drop after
surgery and remain below threshold upon complete remission.
In contrast, recurrence is accompanied by a significant increase
in villin level, even before clinical evidence of the recurrence,
especially in cases of liver métastases.Moreover, our prelimi
nary observations strongly suggest that, in case of recurrence,
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
disease. However, these important findings require further in
vestigation.
Since 15% of colorectal carcinomas do not produce CEA
(15), this marker cannot be used during follow-up studies of
some patients. Our work (5) (confirmed in Ref. 6) has estab
lished that villin production is maintained in all colorectal
tumors so far studied (44 cases). Therefore, one would expect
that the detection of tumor recurrence in patients with a nega
tive CEA assay might be established using a villin assay. Our
preliminary comparative study on CEA and villin before and
after surgery supports this proposal.
In spite of the conspicuous production of villin in colorectal
tumors, only 50% of patients bearing colorectal tumors at all
stages had villin in their sera. We have observed by immunoprecipitation analysis that villin circulating in serum is proteolytically digested (data not shown). Moreover, the pattern of
villin digestion by proteolytic enzymes may vary between dif
ferent individuals. Thus, the epitope recognized by our mono
clonal antibodies may be destroyed; consequently, even if villin
is present in the serum, a negative result would be reported.
This implies that further developments of our assay and/or new
monoclonal antibodies for villin may improve the sensitivity of
the test. Other parameters such as protein interference in sera
should also be further investigated. Work in progress in our
laboratory is aimed at answering these important questions.
Most studies on cancer markers are based upon the assump
tion that the transformed cell is capable of presenting at its
surface new antigens, a dogma which is still widely disputed.
Our rationale was the use of a differentiation marker such as
villin, a well-characterized constituent of intestinal brush bor
ders, that is produced very early during embryogenesis in prim
itive endoderm cells. Moreover, the profile of villin gene expres
sion appears to be precisely modulated during the onset of the
intestinal differentiation program (3, 16). The above findings
are consistent with the proposal that molecules displaying these
properties may in the future provide a new set of markers in
cancer research. Such an approach should be assisted by our
increasing knowledge of proteins playing a key role in cellular
organization and differentiation.
ACKNOWLEDGMENTS
We are indebted to J. F. Colombel, M.D., and M. Thioux for
providing coded sera, E. Coudrier, Ph.D., and F. Traincard for advi-.e
and expert technical assistance, T. Pugsley, Ph.D., for his helpful
comments during the preparation of the manuscript, and R. Carréfor
typing this manuscript.
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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 Cancers
B. Dudouet, L. Jacob, P. Beuzeboc, et al.
Cancer Res 1990;50:438-443.
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