Cancer Letters, 67 (1992)
47
47-54
Elsevier Scientific Publishers Ireland Ltd.
Detection of carcinoembryonic specific anti-CEA monoclonal M. Tobi”, D. O’Kieffeb, ‘Department Confield,
of Medicine,
Detroit,
Pathology, 20037
MI,
The George
L.E. Nochomovitzc
N. Trujillob,
Division
48201
and
Washington
antigen in colonic effluent by antibodies
of Gastroenterology, bDepartment
Wayne
of Medicine,
Uniuersity Medical
Center,
State Division
2150
and W.M. Steinbergb Uniuersity
of Medicine,
Pennsylvania
Auenue,
and N. W.,
540
East
Department
of
Washington
DC
(USA)
(Received
12 July 1992)
(Accepted
1 September
1992)
Summary To characterize the CEA in colonic effluent, anti-CEA monoclonal antibody COL-4 was used in a qualitative radioimmunoassay in both fractionated and unfiactionated colonic effluent. Both effuent samples and tissue extracts, were subjected to Western blotting and
staining. We conclude that in colonic effluent and is ated, but the ouerlapping samples do not make this diagnosis of CRC.
tissue sections to immunohistochemistry. Quantitative leuels of colonic effluent CEA were determined by a kit (Abbott-HA). Higher
Keywords:
mean ualues of CDL-4 binding activity were seen only in patients with a past history of polyps (P < 0.01). Quantitated CEA correlated with the presence of colorectal cancer (CRC) as compared to normal subjects, (1133 f 875 us. 459 ng/ml f 602, P < 0.05) but not when standardized for protein content. COL-4 reacted with an 180 000 M, CEA in the effluent and actiuity was associated with membrane fraction of the effluent, but bore no relation to the immunohistological
lotrodactioo
Correspondence School
School
of Gastroenterology
to:
of Medicine,
Martin Tobi, Dtdision
Wayne
State
University
of Gastroenterology,
Hospital, 3990
John R Street, Detroit, MI 48201,
Abbreviations:
CEA,
Harper
USA.
carcinoembryonic antigen; TAM, tumor
associated marker; MAb, rnonoclonal antibody; CRC, colorectal cancer.
0304-3835/92/$05.00
carcinoembryonic antigen; monoclonal antibody; colonic effluent
We have shown that colonic effluent binding of a MAb which recognizes an early tumor antigen, correlates with the presence of CRC [l] but this antigen has not been characterized, nor is a test for its detection available commercially. CEA has been shown to be present in increased amounts in colorectal carcinomas and adenomas. This increase may be reflected in an increase of CEA in colonic washings or contents [l - 31, with the increment in the fetes preceding the rise in blood levels [4]. Lavage CEA is identical to that of tumor origin and its major source is the colon [5]. The association of CEA with various components of lavage fluid has not yet been fully explored, but this
0 1992 El sevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
CEA is detectable membrane associualues in effluent a useful test in the
48
information is important in developing immunological methods for the early detection of colorectal neoplasia as has been recently demonstrated for mutated K-ras oncogenes in fetes [6]. This study using COL-4, a highly specific MAb which recognizes the 180 000 M, moeity of CEA [7], was undertaken (i) to determine if precolonoscopy effluent contains detectable CEA as measured by a monoclonal antibody assay, (ii) to analyse the source and character of the CEA by comparing it with CEA derived from biopsy samples of specific colonic neoplastic lesions; and (iii) to compare effluent binding by COL-4 with that of a commercially available kit (CEA-EIA) and determine if a correlation exists with the presence of CRC.
Patients and Methods
One hundred-thirty two patients undergoing routine total colonoscopy for different indications provided the effluent samples for study. Individuals to be included in the study had to have a detectable effluent protein concentration of >30 pg/ml (minimum for RIA) by the Lowry method as described previously [l]. In addition, the entire colon must have been visualized. The patient could not have been prepared with GolytelyTM which interfers with the ELISA [8]. Patients with inflammatory bowel disease (n = 22)) family history of CRC (n = 12), or those who did not satisfy the above conditions were excluded. The latter exclusions numbered 12 (9 with insufficient protein, 1 with incomplete examination and 2 patients were prepared with GolytelyTM). Eighty six patients qualified for inclusion and more initially evaluated by the qualitative assay (normal = 26; single adenomas = 11; multiple adenomas = 18; history of adenomas 18; history of resection 7; colorectal cancer = 6). The quantitative CEA assay was performed later when the number of cancer patients included had risen to 8. Colonic evaluation was based on macroscopic findings at the time of
colonoscopy and histologic review of available tissue samples. Collection and preparation of effluent Bowel preparation routine was as described previously [l] consisting of a liquid diet and purgative self-administration. A tap water enema was administered by the endoscopy unit nurse on the day of the procedure. The first return of the enema was discarded. A 100ml quantity of the second return was collected and clarified by centrifugation. The supernatant was filtered in a 0.8 pm filtration unit (Nalgene) and a Lowry protein determination was performed on the filtrate. The effluent sample was divided into aliquots and stored at - 2o”c. Radioimmunoassay Effluent samples, at 1.5 pg protein/well diluted in phosphate buffered saline (PBS) at pH 7.2 were dried overnight at 37OC on a 96well microtiter plate (Dynatech Labs Inc., Alexandria, VA). The plates were blocked with 5% bovine serum albumin (BSA) and washed once with 1% BSA in PBS. The wells were then aspirated and dried by inverting and gently tapping onto absorbent paper. All subsequent washes were performed in this manner. A 50-~1 quantity of tissue culture supernatant from the COL-4 hybridoma line in RPM1 medium (Gibco, Grant Island, NY) was added to each well and incubated for 1 h at 37OC. The plates were aspirated and washed twice. Goat anti-mouse IgG labelled with 1251(at an activity of 75 000 counts/min in 25 pi/well), diluted in RPM1 medium and 1% BSA, was added to each well, incubated for 1 h and washed as above. The wells were then cut out and counted in duplicate in a gamma counter (Beckman, USA). The counts in counts/min were subtracted from background (as established by substituting RPM1 medium (Gibco) for the antibody and expressed as counts/min per unit (1.5 pg) of protein. To assess reproducibility, frozen effluent samples from 7 patients were subjected to a COL-4 immunoassay after they had been stored for 3 years.
49
Farther investigations patients
in selected
Effluent fractionation Randomly selected effluent samples taken from 3 individuals with normal colonoscopic findings and low COL-4 effluent binding activity were subjected to a membrane enrichment procedure. This was achieved by ultracentrifugation on a 20 -40% discontinuous sucrose gradient [7,9]. The membrane and soluble factions were confirmed as such by light microscopy, assayeNd for protein and RIA was performed as described above.
Western blotting of effluent compared to tissue extracts Effluent samples with sufficient protein (> 1 mg/ml, i.e. conferring suitability for a Western blot experiment) were obtained from 3 patients and run on electrophoretic gels. The following were the respective colonoscopic diagnoses: 1 sigmoid adenocarcinoma, 1 large (3 cm) sigmoid tubulovillous adenoma, and 1 normalappearing mucosa. ‘For comparison of reactivity, tissue extracts [l] were prepared from corresponding samples taken from a cancer, a pool of fragments from tubulovillous adenomas and from normal colonic mucosa. These extracts were run on the same gels with the effluent samples, at identical protein concentrations. These were then transferred from the gels to nitrocellulose paper and reacted with COL-4 MAb in a sl;andard Western immunoblot technique as described [9]. Effect of polypecl:omy on COL-4 effluent binding In another patient, effluent was collected prior to colonoscopy, during which a polypectomy was performed. This patient later experienced a moderate delayed postpolypectomy bleed and was re-colonoscoped a week later. This provided an opportunity to compare the pre- and post-polypectomy effluent samples.
Comparison of COL-4 reactiuity in tissues and corresponding effluent sample Immunoperoxidase histochemistry by the ABC technique with the COL-4 MAb was performed [lo] on fixed tissue sections of colonoscopically biopsied lesions from another 3 study patients having colorectal neoplasia with known COL-4 effluent reactivity. Quantitation of CEA in effluent samples Quantitative levels of CEA were determined in the effluent samples of 36 patients with CRC, adenomas or a normal-appearing colonic mucosa. A combined poly- and monoclonal antibody ELISA, CEA-EIA (Abbott Diagnostics, Chicago, II), was used. Statistical methods Statistical analysis was by Student’s twotailed t-test. This study meets the established criteria of the George Washington University Committee on Human Research. Results Figure 1 is a scattergram of the COL-4 effluent binding values of all patients entered into the study according to their diagnosis. In patients with a past history of polyps, COL-4 activity was increased (P < 0.01) and a trend to lower values among those with a previous colonic resection for colorectal carcinoma was observed. No statistically significant difference was observed between the normal, adenoma and cancer groups. COL-4 binding reactivity was reproduced in the samples from 7 patients reassessed after prolonged storage with a correlation factor of R = 0.9 (data not shown). Figure 2 shows a representative COL-4 MAb binding curve of one of the 3 patients, for the membrane versus the soluble antigen fractionated by ultracentrifugation. The curves show the dominance of reactivity in the membrane-enriched over the non-membranebound fractions. On the same graph, the original (unfractionated) effluent sample is shown for comparison, with minimal binding. Binding curves in the other 2 patients were similar (data not shown).
50
COL-4 Mab REACTIVITY IN COLONIC EFFLUENTS AT 1.5 pG PROTEIN IN SOLID-PHASE RIA 14,000 .
12,ooa -
.
10,ooo F z
m ‘3, T F E a 0
.
a.cGG-
. :
l
.
6.000 -
.
:. . 4.ooo-
.
L :
! mo-&
f 1,000
:
-iI H ..
a-
:
.
!
:
E
r n
Multiple Polyps
History of Polyps
T c
-
Normal
Single POlYP
Ry$$m
l
Carcinoma
0
Fig. 1. COL-4 monoclonal antibody reactivity in colonic effluents at 1.5 pg protein in solid phase RIA. Scattergram of study patients grouped according to clinical findings. Horizontal lines denote arithmetic means. Binding in the history of polyps (adenomas) group is significant compared to normal (P < 0.01).
0
1.5
Antigen
0.3
0.15
0.06 0.030.015
Dilution (pG/well)
Fig. 2. The effect of membrane,fractionation on COL4 binding curves. The fractions are derived by ultracentrifugation of effluent from a normal patient. The original level of binding (WJ is shown starting at 1.5 pg protein of antigen with serial dilutions on a log scale (abscissa), contrasted with the membrane-bound (0) and soluble (A) fractions at identical protein concentrations. There is an approximate 5-fold enrichment of the CEA in the membrane-associated fraction.
The Western immunoblot preparations show that, at equal protein concentrations, similar reactivity is seen in the effluents and the corresponding tissue extracts (Fig. 3). Furthermore, in these selected specimens, there is increased activity in the effluent from a cancer-bearing patient and in extracts derived from cancerous tissue. This contrasts with the lesser activity observed in the pooled adenoma extract and the effluent from a patient with a large adenoma. There was no binding activity apparent in the normal preparations. The antigen precipitated in all these preparations is bound by COL-4 and has a M, of approximately 180 000, corresponding to that of the CEA molecule for which COL-4 is specific. There was no decline in reactivity between the preand post-polypectomy effluent samples in the polypectomized patient studied (data not shown). In the immunohistochemically stained tissue
51
IMMUNODETECMlON OF CEA BY WESTERN BLOTTING IN HUMAN COLON EXTRACTS AND EFFLUENTS USING MAB COL-4
- 200
-
-
92 6643-
- 25.71 ,;S3 I
of CEA by Western blotting in human colon extracts and effluents using monoclonal antibody Fig. 3. lmmunodetection COL-4. Autoradiograph of 3 extracts (left box) of colon tissue after immunoblotting with COL-4 MAb detected by 1251IgG goat anti-mouse antibody. Auto-radiographs of effluent samples are depicted in the right box. Position of relative molecular weight markers are shown between the boxes.
specimens, COL-4 reacted positively with lesions removed at colonoscopy, staining the luminal surfaces of neoplastic cells and luminal debris. Tissue reaction from a carcinomatous Table 1. COL-4 MA.b activity in effluent of 3 patients as compared to their tssue sections. Pathology
R1.A effluent reactivity
lmmunoperoxidase tissue staining
(cpm) Tubulovillous Adenoma (1 cm) Tubulovillous Adenoma (3 cm) Polypoid Carcinoma (1 cm)
2865
++
4538
++
2524
+++
RIA effluent activity (COL-4 MAb counts/min/l.5 g protein) compared to corresponding tissue staining intensity by immunoperoxidase technique. No correlation was demonstrated.
polyp was more intense than that from a large (3 cm) tubulovillous adenoma. There was no correlation between the RIA effluent activity and the staining intensity of lesions removed at colonoscopy (Table I). As can be seen in Table II, the CEA-EIA effluent assay shows the positive correlation Table II. samples.
Quantitative
CEA assay in colonic effluent
Group (n)
Mean CEA ng/ml f SD.
CEA ng/mg protein l S.D.
Cancer (8) Adenoma (14) Normal (14)
1133 zt 875’ 581 f 653 459 * 602
1027 zt 512 1784 zt 2449 1136 zt 1470
CEA assay, CEA-EIA (Abbott Diagnostics, Chicago, II). n, number of patients. ‘Denotes statistical significance between cancer and the normal group at P < 0.05. S.D., standard deviation.
52
between CRC and normal groups for quantitative CEA, expressed in ng/ml (means 1133 f 875 vs. 459 f 602 CEA ng/ml res., P < 0.05). However, when CEA is standardized for protein content of effluent as was done in the COL-4 qualitative assay, the correlation is not maintained. The difference between the protein in effluent samples from patients with cancer or normal colonoscopic examinations was not significant (1.15 f 2.03 vs. 1.28* 1.00 mg/ml respectively, P-0.9). Discnssion This study demonstrates than an MAb can specifically detect CEA in colonic effluent with acceptable reproducibility of binding after storage. Using MAbs to detect CEA in cofonic effluent we have shown that quantitative CEA levels in colonic washings uncorrected for protein correlate with the presence of CRC regardless of location. However, in contrast with the work of Winawer et al. [2] and in agreement with that of Vellacott et al. [3] we find that levels of CEA in lavage fluid standardized for protein does not correlate with malignancy. Furthermore, in this study, qualitative levels of CEA in effluent samples using COL-4 MAb also fails to differentiate between cancer and normal groups. We do however confirm an earlier report showing that CEA is detectable in material from subjects with a macroscopically normal colon [l l] and work showing that CEA positive ceils in the colonic effluent may be likewise found [12]. The CEA-EIA assay, with CEA content expressed in ng/ml gave the only correlation with malignancy. The CEA, as expressed in ng/mg of protein, did not reflect this correlation, despite the fact that the mean protein content of the normal and cancer effluent samples was similar. The numbers of patients are too small to support the use of this assay as a diagnostic aid when examining colonic effluent material, but the potential should be evaluated. We have not performed these quantitative estimations in patients with a past history of adenomas because of the cost of the
quantitative assay (as compared to the COL-4 qualitative assay) and are therefore unable to confirm the increased qualitative CEA levels observed in this particular group of patients. Dominant CEA reactivity resides in the membrane-bound fraction of the effluent, as shown by the ultracentrifugation data. This confirms recent data showing that fetal CEA exists as a membrane-bound form [13]. The major source for the CEA reactivity in colonic effluent may be the region of neoplastic predisposition rather than from the neoplasm alone. This supposition is supported by the failure of lavage CEA activity to decline in a post-polypectomy effluent sample (as seen in the polypectomized patient). This phenomenon has been previously reported [3]. The lack of correlation between the CEA effluent binding and the COL-4 immunohistochemistry in the actual lesion suggests that it is unlikely that the CEA originates from the neoplasm alone. These data are in agreement with the observation that the CEA content of neoplasttc gastrointestinal lesions does not correlate with plasma CEA levels 1141. The similarity in reactivity between effluent and actual tissue extracts as demonstrated in the Western immunoblot experiment is striking. The observation that an effluent sample (representative of a large mucosal area) may be as antigenically reactive as an extract derived from a biopsy sample is encouraging evidence for the further investigation of the use of effluent material for screening or diagnosis of CRC as has been realized recently using genetic markers in fetes [6]. However, in comparing relative reactivity, it should be noted that the protein standard creates a bias that results in increased effluent binding in effluent samples. This is because much of the protein in biopsy material may come from noncancerous, non-epithelial tissue, whilst effluent appears to have a more substantial membranederived protein content where the predominant CEA activity resides (Fig. 2). The two published studies have reported a statistical correlation between the amount of CEA in colonoscopically or sigmoidoscopically
53
obtained lavage material and the presence of large colonic polyp:j and CRC [2] and colonic but not rectal cancer in the other [3]. Most of our patients had Dukes A stage disease (50%) as compared to later stages which may explain the discrepancy which exists when comparing our data to that of earlier studies. In those studies, colonic lavage fluid was collected via sigmoidoscopy and saline lavage at a predetermined insertion length in the left colon [2], or by aspirating fluid directed at the lesion of interest during colonoscopy [3]. This type of fluid collection is expensive, invasive, requires specialized medical personnel and causes some patient discomfort. Patients might more readily accept a simpler and cheaper collection of colonic effluent fluid, obtainable without endoscopy and yielding similar information. Furthermore, the acceptable reproducibility after prolonged storage of effluent samples in this study confirms our previous findings in which a different TAM was employed [15]. Effluent samples may be suitable for the detection of dysplasia-associated antigens or other TAM’s [1,6] in patients at high risk for colorectal cancer. Such an approach may be beneficial in the follow-up of individuals with longstanding ulcerative colitis. A diagnostic profile that include,s MAb testing for TAM’s in effluent may enhance the diagnostic yield in the search for early colonic neoplasia [2]. Such markers should not be components of normal gastrointestinal secretions, as is the CA19-9 defined antigen, which is found in normal pancreatic and biliary secretions in high quantity [16] and which we have shown to have no predictive value in identifying patients with using effluent as test colorectal neoplasia, material [ 15,161.
Alexis Shelokov for their assistance in reviewing the manuscript; J. Peacock and J.P. Henry for performing the CEA assays; G. Friedman and C. Halperin for typographical support. This paper is dedicated to the memory of David Shuman. References 1
Tobi, M., Darmon,
E., Phillips, T. et al. (1990)
expression of a putative
adenoma
precolonoscopic effluent of patients with colorectal cancer. Cancer Lett., 50, 212
Winawer,
S.J.,
25.
Fleischer, M, Green,
cinoembryonic
antigen
S. et al. (1977)
in colonic
Car-
lavage.
Gastroenter-
T.W.
et al. (1982)
ology, 73, 719-722. 3
Vellacott, Tumor
K.D.,
Groom,
D, Balfour,
associated products in colonic lavage fluid. Clin.
Oncol., 4
8, 61-67.
Fujimoto, S., Kisukawa,
Y. and Itoh, K. (1979)
Carcino-
embryonic antigen (CEA) in gastric juice or fetes as an aid in the diagnosis of gastrointestinal
cancer.
Ann.
Surg.,
189, 34-38. 5
Egan, M.L.,
Pritchard,
D.G.,
Todd,
Isolation and immunochemical tion
of
carcino-embryonic
C.W.
et al. (1977)
and chemical characterizaantigen-like
colonic lavages of healthy individuals.
substances
Cancer
Res.,
in 37,
2638 - 2643. 6
Sidransky,
D., Tokino,
T., Hamilton,
S.R.
et al. (1992)
Identification of ras oncogene mutations in the stool of patients
with
curable
colorectal
tumors.
Science,
256,
102 - 105. 7
Muraro,
R.,
Generation
Wunderlich,
D.
and
Schlom,
J.
(1985)
of monoclonal antibodies reactive with human
colon carcinomas. In: Genetic and Phenotypic Markers of Tumors, pp. 117 - 127. Editors: S.A. Aaronson, and R. Verner. 8
L. Frati
Plenum Press, New York.
Tobi, M., Darmon,
E., Rozen, P. et al. (1991)
Oral colon
lavage preparations may interefere with ELISA detection of tumor associated antigens in colonic effluent. Sci., 36, 14489
Tobi,
M.,
Maliakkal,
defined
by
Gastro.,
in press.
Hsu, H.S.,
B.J.,
monoclonal
Alousi,
M.A.
et al. Cellular
techniques:
antibody
Adanb-9.
Raine, L., and Fanger, H. (1981)
biotin-peroxidase
Acknowledgments
Dig. Dis.
1452.
distribution of a colonic adenoma-associated
10
complex
a comparison
antibody (PA) procedures.
We thank A.P. ljpence for expert technical assistance, M.L. Aure (B.S.M.T.) and Dr. D. Wilkinson for their patience and permission to University use The George Washington Medical Center Clinical Laboratory facility; Prof. Paul Rozen, Dr. R. Dubrow and Dr.
Increased
associated antigen in
(ABC) between
antigen
as
Scan.
J.
Use of avidin
in immunoperoxidase ABC
J. Histochem.
and unlabeled Cytochem.,
29,
577 - 580. 11
Martin, F., Martin, M.S. and Bourgeaux,
C. (1976)
antigens in human digestive tumors. Eur. J. Cancer,
Fetal 12,
165 - 75. 12
Rozen, P., Tobi, M., Darmon, Colonic Cytology:
E. and Kaufman,
a simplified method
initial results. Acta Cytol.,
34, 627 - 31.
L. (1990)
of collection and
54
13
14
15
Sugano, K., Ohkura, H., Hirohashi, S. et al. (1989) Detection of increased fetal carcinoembryonic antigen and its characterization as a membrane-bound form in colorectal carcinoma and other gastrointestinal disorders. Jpn. J. Cancer Res., 80, 1156- 1160. Wagener, C., Muller-Wallraf, R., Nisson, S. et al. (1981) Localization and concentration of carcinoembryonic antigen in plasma. J. Natl. Cancer Inst., 67(3), 539-47. Tobi, M., Steinberg, W., Henry, J. and Nochomovitz, L.
16
(1991) Cancer associated antigen CA19-9 in colonic effluent of patients with colorectal neoplasia and inflammatory bowel disease. Cancer Lett., 60, 9- 13. Darmon, E., Tobi, M. and Rozen, P. (1991) Newer tests on the colonic effluent, for identifying persons with colorectal neoplasia. In: Advances in Large Bowel Cancer: Policy, Prevention and Treatment, Frontiers of Gastrointestinal Research 18, pp. 236- 247. Editors: P. Rozen, C.B. Reich and S.J. Winawer. Karger, Basel.
47
47-54
Elsevier Scientific Publishers Ireland Ltd.
Detection of carcinoembryonic specific anti-CEA monoclonal M. Tobi”, D. O’Kieffeb, ‘Department Confield,
of Medicine,
Detroit,
Pathology, 20037
MI,
The George
L.E. Nochomovitzc
N. Trujillob,
Division
48201
and
Washington
antigen in colonic effluent by antibodies
of Gastroenterology, bDepartment
Wayne
of Medicine,
Uniuersity Medical
Center,
State Division
2150
and W.M. Steinbergb Uniuersity
of Medicine,
Pennsylvania
Auenue,
and N. W.,
540
East
Department
of
Washington
DC
(USA)
(Received
12 July 1992)
(Accepted
1 September
1992)
Summary To characterize the CEA in colonic effluent, anti-CEA monoclonal antibody COL-4 was used in a qualitative radioimmunoassay in both fractionated and unfiactionated colonic effluent. Both effuent samples and tissue extracts, were subjected to Western blotting and
staining. We conclude that in colonic effluent and is ated, but the ouerlapping samples do not make this diagnosis of CRC.
tissue sections to immunohistochemistry. Quantitative leuels of colonic effluent CEA were determined by a kit (Abbott-HA). Higher
Keywords:
mean ualues of CDL-4 binding activity were seen only in patients with a past history of polyps (P < 0.01). Quantitated CEA correlated with the presence of colorectal cancer (CRC) as compared to normal subjects, (1133 f 875 us. 459 ng/ml f 602, P < 0.05) but not when standardized for protein content. COL-4 reacted with an 180 000 M, CEA in the effluent and actiuity was associated with membrane fraction of the effluent, but bore no relation to the immunohistological
lotrodactioo
Correspondence School
School
of Gastroenterology
to:
of Medicine,
Martin Tobi, Dtdision
Wayne
State
University
of Gastroenterology,
Hospital, 3990
John R Street, Detroit, MI 48201,
Abbreviations:
CEA,
Harper
USA.
carcinoembryonic antigen; TAM, tumor
associated marker; MAb, rnonoclonal antibody; CRC, colorectal cancer.
0304-3835/92/$05.00
carcinoembryonic antigen; monoclonal antibody; colonic effluent
We have shown that colonic effluent binding of a MAb which recognizes an early tumor antigen, correlates with the presence of CRC [l] but this antigen has not been characterized, nor is a test for its detection available commercially. CEA has been shown to be present in increased amounts in colorectal carcinomas and adenomas. This increase may be reflected in an increase of CEA in colonic washings or contents [l - 31, with the increment in the fetes preceding the rise in blood levels [4]. Lavage CEA is identical to that of tumor origin and its major source is the colon [5]. The association of CEA with various components of lavage fluid has not yet been fully explored, but this
0 1992 El sevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
CEA is detectable membrane associualues in effluent a useful test in the
48
information is important in developing immunological methods for the early detection of colorectal neoplasia as has been recently demonstrated for mutated K-ras oncogenes in fetes [6]. This study using COL-4, a highly specific MAb which recognizes the 180 000 M, moeity of CEA [7], was undertaken (i) to determine if precolonoscopy effluent contains detectable CEA as measured by a monoclonal antibody assay, (ii) to analyse the source and character of the CEA by comparing it with CEA derived from biopsy samples of specific colonic neoplastic lesions; and (iii) to compare effluent binding by COL-4 with that of a commercially available kit (CEA-EIA) and determine if a correlation exists with the presence of CRC.
Patients and Methods
One hundred-thirty two patients undergoing routine total colonoscopy for different indications provided the effluent samples for study. Individuals to be included in the study had to have a detectable effluent protein concentration of >30 pg/ml (minimum for RIA) by the Lowry method as described previously [l]. In addition, the entire colon must have been visualized. The patient could not have been prepared with GolytelyTM which interfers with the ELISA [8]. Patients with inflammatory bowel disease (n = 22)) family history of CRC (n = 12), or those who did not satisfy the above conditions were excluded. The latter exclusions numbered 12 (9 with insufficient protein, 1 with incomplete examination and 2 patients were prepared with GolytelyTM). Eighty six patients qualified for inclusion and more initially evaluated by the qualitative assay (normal = 26; single adenomas = 11; multiple adenomas = 18; history of adenomas 18; history of resection 7; colorectal cancer = 6). The quantitative CEA assay was performed later when the number of cancer patients included had risen to 8. Colonic evaluation was based on macroscopic findings at the time of
colonoscopy and histologic review of available tissue samples. Collection and preparation of effluent Bowel preparation routine was as described previously [l] consisting of a liquid diet and purgative self-administration. A tap water enema was administered by the endoscopy unit nurse on the day of the procedure. The first return of the enema was discarded. A 100ml quantity of the second return was collected and clarified by centrifugation. The supernatant was filtered in a 0.8 pm filtration unit (Nalgene) and a Lowry protein determination was performed on the filtrate. The effluent sample was divided into aliquots and stored at - 2o”c. Radioimmunoassay Effluent samples, at 1.5 pg protein/well diluted in phosphate buffered saline (PBS) at pH 7.2 were dried overnight at 37OC on a 96well microtiter plate (Dynatech Labs Inc., Alexandria, VA). The plates were blocked with 5% bovine serum albumin (BSA) and washed once with 1% BSA in PBS. The wells were then aspirated and dried by inverting and gently tapping onto absorbent paper. All subsequent washes were performed in this manner. A 50-~1 quantity of tissue culture supernatant from the COL-4 hybridoma line in RPM1 medium (Gibco, Grant Island, NY) was added to each well and incubated for 1 h at 37OC. The plates were aspirated and washed twice. Goat anti-mouse IgG labelled with 1251(at an activity of 75 000 counts/min in 25 pi/well), diluted in RPM1 medium and 1% BSA, was added to each well, incubated for 1 h and washed as above. The wells were then cut out and counted in duplicate in a gamma counter (Beckman, USA). The counts in counts/min were subtracted from background (as established by substituting RPM1 medium (Gibco) for the antibody and expressed as counts/min per unit (1.5 pg) of protein. To assess reproducibility, frozen effluent samples from 7 patients were subjected to a COL-4 immunoassay after they had been stored for 3 years.
49
Farther investigations patients
in selected
Effluent fractionation Randomly selected effluent samples taken from 3 individuals with normal colonoscopic findings and low COL-4 effluent binding activity were subjected to a membrane enrichment procedure. This was achieved by ultracentrifugation on a 20 -40% discontinuous sucrose gradient [7,9]. The membrane and soluble factions were confirmed as such by light microscopy, assayeNd for protein and RIA was performed as described above.
Western blotting of effluent compared to tissue extracts Effluent samples with sufficient protein (> 1 mg/ml, i.e. conferring suitability for a Western blot experiment) were obtained from 3 patients and run on electrophoretic gels. The following were the respective colonoscopic diagnoses: 1 sigmoid adenocarcinoma, 1 large (3 cm) sigmoid tubulovillous adenoma, and 1 normalappearing mucosa. ‘For comparison of reactivity, tissue extracts [l] were prepared from corresponding samples taken from a cancer, a pool of fragments from tubulovillous adenomas and from normal colonic mucosa. These extracts were run on the same gels with the effluent samples, at identical protein concentrations. These were then transferred from the gels to nitrocellulose paper and reacted with COL-4 MAb in a sl;andard Western immunoblot technique as described [9]. Effect of polypecl:omy on COL-4 effluent binding In another patient, effluent was collected prior to colonoscopy, during which a polypectomy was performed. This patient later experienced a moderate delayed postpolypectomy bleed and was re-colonoscoped a week later. This provided an opportunity to compare the pre- and post-polypectomy effluent samples.
Comparison of COL-4 reactiuity in tissues and corresponding effluent sample Immunoperoxidase histochemistry by the ABC technique with the COL-4 MAb was performed [lo] on fixed tissue sections of colonoscopically biopsied lesions from another 3 study patients having colorectal neoplasia with known COL-4 effluent reactivity. Quantitation of CEA in effluent samples Quantitative levels of CEA were determined in the effluent samples of 36 patients with CRC, adenomas or a normal-appearing colonic mucosa. A combined poly- and monoclonal antibody ELISA, CEA-EIA (Abbott Diagnostics, Chicago, II), was used. Statistical methods Statistical analysis was by Student’s twotailed t-test. This study meets the established criteria of the George Washington University Committee on Human Research. Results Figure 1 is a scattergram of the COL-4 effluent binding values of all patients entered into the study according to their diagnosis. In patients with a past history of polyps, COL-4 activity was increased (P < 0.01) and a trend to lower values among those with a previous colonic resection for colorectal carcinoma was observed. No statistically significant difference was observed between the normal, adenoma and cancer groups. COL-4 binding reactivity was reproduced in the samples from 7 patients reassessed after prolonged storage with a correlation factor of R = 0.9 (data not shown). Figure 2 shows a representative COL-4 MAb binding curve of one of the 3 patients, for the membrane versus the soluble antigen fractionated by ultracentrifugation. The curves show the dominance of reactivity in the membrane-enriched over the non-membranebound fractions. On the same graph, the original (unfractionated) effluent sample is shown for comparison, with minimal binding. Binding curves in the other 2 patients were similar (data not shown).
50
COL-4 Mab REACTIVITY IN COLONIC EFFLUENTS AT 1.5 pG PROTEIN IN SOLID-PHASE RIA 14,000 .
12,ooa -
.
10,ooo F z
m ‘3, T F E a 0
.
a.cGG-
. :
l
.
6.000 -
.
:. . 4.ooo-
.
L :
! mo-&
f 1,000
:
-iI H ..
a-
:
.
!
:
E
r n
Multiple Polyps
History of Polyps
T c
-
Normal
Single POlYP
Ry$$m
l
Carcinoma
0
Fig. 1. COL-4 monoclonal antibody reactivity in colonic effluents at 1.5 pg protein in solid phase RIA. Scattergram of study patients grouped according to clinical findings. Horizontal lines denote arithmetic means. Binding in the history of polyps (adenomas) group is significant compared to normal (P < 0.01).
0
1.5
Antigen
0.3
0.15
0.06 0.030.015
Dilution (pG/well)
Fig. 2. The effect of membrane,fractionation on COL4 binding curves. The fractions are derived by ultracentrifugation of effluent from a normal patient. The original level of binding (WJ is shown starting at 1.5 pg protein of antigen with serial dilutions on a log scale (abscissa), contrasted with the membrane-bound (0) and soluble (A) fractions at identical protein concentrations. There is an approximate 5-fold enrichment of the CEA in the membrane-associated fraction.
The Western immunoblot preparations show that, at equal protein concentrations, similar reactivity is seen in the effluents and the corresponding tissue extracts (Fig. 3). Furthermore, in these selected specimens, there is increased activity in the effluent from a cancer-bearing patient and in extracts derived from cancerous tissue. This contrasts with the lesser activity observed in the pooled adenoma extract and the effluent from a patient with a large adenoma. There was no binding activity apparent in the normal preparations. The antigen precipitated in all these preparations is bound by COL-4 and has a M, of approximately 180 000, corresponding to that of the CEA molecule for which COL-4 is specific. There was no decline in reactivity between the preand post-polypectomy effluent samples in the polypectomized patient studied (data not shown). In the immunohistochemically stained tissue
51
IMMUNODETECMlON OF CEA BY WESTERN BLOTTING IN HUMAN COLON EXTRACTS AND EFFLUENTS USING MAB COL-4
- 200
-
-
92 6643-
- 25.71 ,;S3 I
of CEA by Western blotting in human colon extracts and effluents using monoclonal antibody Fig. 3. lmmunodetection COL-4. Autoradiograph of 3 extracts (left box) of colon tissue after immunoblotting with COL-4 MAb detected by 1251IgG goat anti-mouse antibody. Auto-radiographs of effluent samples are depicted in the right box. Position of relative molecular weight markers are shown between the boxes.
specimens, COL-4 reacted positively with lesions removed at colonoscopy, staining the luminal surfaces of neoplastic cells and luminal debris. Tissue reaction from a carcinomatous Table 1. COL-4 MA.b activity in effluent of 3 patients as compared to their tssue sections. Pathology
R1.A effluent reactivity
lmmunoperoxidase tissue staining
(cpm) Tubulovillous Adenoma (1 cm) Tubulovillous Adenoma (3 cm) Polypoid Carcinoma (1 cm)
2865
++
4538
++
2524
+++
RIA effluent activity (COL-4 MAb counts/min/l.5 g protein) compared to corresponding tissue staining intensity by immunoperoxidase technique. No correlation was demonstrated.
polyp was more intense than that from a large (3 cm) tubulovillous adenoma. There was no correlation between the RIA effluent activity and the staining intensity of lesions removed at colonoscopy (Table I). As can be seen in Table II, the CEA-EIA effluent assay shows the positive correlation Table II. samples.
Quantitative
CEA assay in colonic effluent
Group (n)
Mean CEA ng/ml f SD.
CEA ng/mg protein l S.D.
Cancer (8) Adenoma (14) Normal (14)
1133 zt 875’ 581 f 653 459 * 602
1027 zt 512 1784 zt 2449 1136 zt 1470
CEA assay, CEA-EIA (Abbott Diagnostics, Chicago, II). n, number of patients. ‘Denotes statistical significance between cancer and the normal group at P < 0.05. S.D., standard deviation.
52
between CRC and normal groups for quantitative CEA, expressed in ng/ml (means 1133 f 875 vs. 459 f 602 CEA ng/ml res., P < 0.05). However, when CEA is standardized for protein content of effluent as was done in the COL-4 qualitative assay, the correlation is not maintained. The difference between the protein in effluent samples from patients with cancer or normal colonoscopic examinations was not significant (1.15 f 2.03 vs. 1.28* 1.00 mg/ml respectively, P-0.9). Discnssion This study demonstrates than an MAb can specifically detect CEA in colonic effluent with acceptable reproducibility of binding after storage. Using MAbs to detect CEA in cofonic effluent we have shown that quantitative CEA levels in colonic washings uncorrected for protein correlate with the presence of CRC regardless of location. However, in contrast with the work of Winawer et al. [2] and in agreement with that of Vellacott et al. [3] we find that levels of CEA in lavage fluid standardized for protein does not correlate with malignancy. Furthermore, in this study, qualitative levels of CEA in effluent samples using COL-4 MAb also fails to differentiate between cancer and normal groups. We do however confirm an earlier report showing that CEA is detectable in material from subjects with a macroscopically normal colon [l l] and work showing that CEA positive ceils in the colonic effluent may be likewise found [12]. The CEA-EIA assay, with CEA content expressed in ng/ml gave the only correlation with malignancy. The CEA, as expressed in ng/mg of protein, did not reflect this correlation, despite the fact that the mean protein content of the normal and cancer effluent samples was similar. The numbers of patients are too small to support the use of this assay as a diagnostic aid when examining colonic effluent material, but the potential should be evaluated. We have not performed these quantitative estimations in patients with a past history of adenomas because of the cost of the
quantitative assay (as compared to the COL-4 qualitative assay) and are therefore unable to confirm the increased qualitative CEA levels observed in this particular group of patients. Dominant CEA reactivity resides in the membrane-bound fraction of the effluent, as shown by the ultracentrifugation data. This confirms recent data showing that fetal CEA exists as a membrane-bound form [13]. The major source for the CEA reactivity in colonic effluent may be the region of neoplastic predisposition rather than from the neoplasm alone. This supposition is supported by the failure of lavage CEA activity to decline in a post-polypectomy effluent sample (as seen in the polypectomized patient). This phenomenon has been previously reported [3]. The lack of correlation between the CEA effluent binding and the COL-4 immunohistochemistry in the actual lesion suggests that it is unlikely that the CEA originates from the neoplasm alone. These data are in agreement with the observation that the CEA content of neoplasttc gastrointestinal lesions does not correlate with plasma CEA levels 1141. The similarity in reactivity between effluent and actual tissue extracts as demonstrated in the Western immunoblot experiment is striking. The observation that an effluent sample (representative of a large mucosal area) may be as antigenically reactive as an extract derived from a biopsy sample is encouraging evidence for the further investigation of the use of effluent material for screening or diagnosis of CRC as has been realized recently using genetic markers in fetes [6]. However, in comparing relative reactivity, it should be noted that the protein standard creates a bias that results in increased effluent binding in effluent samples. This is because much of the protein in biopsy material may come from noncancerous, non-epithelial tissue, whilst effluent appears to have a more substantial membranederived protein content where the predominant CEA activity resides (Fig. 2). The two published studies have reported a statistical correlation between the amount of CEA in colonoscopically or sigmoidoscopically
53
obtained lavage material and the presence of large colonic polyp:j and CRC [2] and colonic but not rectal cancer in the other [3]. Most of our patients had Dukes A stage disease (50%) as compared to later stages which may explain the discrepancy which exists when comparing our data to that of earlier studies. In those studies, colonic lavage fluid was collected via sigmoidoscopy and saline lavage at a predetermined insertion length in the left colon [2], or by aspirating fluid directed at the lesion of interest during colonoscopy [3]. This type of fluid collection is expensive, invasive, requires specialized medical personnel and causes some patient discomfort. Patients might more readily accept a simpler and cheaper collection of colonic effluent fluid, obtainable without endoscopy and yielding similar information. Furthermore, the acceptable reproducibility after prolonged storage of effluent samples in this study confirms our previous findings in which a different TAM was employed [15]. Effluent samples may be suitable for the detection of dysplasia-associated antigens or other TAM’s [1,6] in patients at high risk for colorectal cancer. Such an approach may be beneficial in the follow-up of individuals with longstanding ulcerative colitis. A diagnostic profile that include,s MAb testing for TAM’s in effluent may enhance the diagnostic yield in the search for early colonic neoplasia [2]. Such markers should not be components of normal gastrointestinal secretions, as is the CA19-9 defined antigen, which is found in normal pancreatic and biliary secretions in high quantity [16] and which we have shown to have no predictive value in identifying patients with using effluent as test colorectal neoplasia, material [ 15,161.
Alexis Shelokov for their assistance in reviewing the manuscript; J. Peacock and J.P. Henry for performing the CEA assays; G. Friedman and C. Halperin for typographical support. This paper is dedicated to the memory of David Shuman. References 1
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We thank A.P. ljpence for expert technical assistance, M.L. Aure (B.S.M.T.) and Dr. D. Wilkinson for their patience and permission to University use The George Washington Medical Center Clinical Laboratory facility; Prof. Paul Rozen, Dr. R. Dubrow and Dr.
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