Brief Communication: Radioiodinated Antibody to Carcinoembryonic Antigen: Binding to Normal and Cancerous Human Colon in Vitro James
1,2,3
E. Coates,4 Maria Koch,4 Patricia F. Beaver,4 T. Alexander McPherson,4,6 and Anthony A. Noujaim
SUMMARY-Samples of tumor and normal mucosa from 32 patients with adenocarcinoma of the colorectum were examined for their capacity to bind radioiodinated antibody to caretnoembryonic antigen (anti-CEA) IgG. Twenty-three (72%) of the tumors bound significantly more antibody than the respective normal mucosa. The results indicate that radiolabeled anti-CEA may be useful in the in vivo localization of CEA-producing tumors and metastases in man, and may have application in vitro as a diagnostic marker of precancerous change in colorectal biopsies from patients at risk of developing colorectal cancer.-J Natl Cancer Inst 55: 25-27, 1975.
RADIOLABELED ANTIBODY TO CARCINOEMBRYONIC ANTIGEN (CEA) might be useful in the localization of tumors producing CEA (1). Several recent manuscripts have reported the successful localization of human colon tumors transplanted in hamsters (2-4) and nude mice (5) after the animals were given injections of radioiodinated antibody to CEA (antiCEA). However, binding of anti-CEA by normal human tissue (Le., normal human colon mucosa) is not assessed (6) when human colon tumor heterografts are used to study anti-CEA localization. In addition, only colon tumor heterografts below a certain size show increased uptake of radioiodinated anti-CEA, which suggests a loss or masking of CEA antigens with larger tumors or a blocking of injected antibody before contact with the tumor, perhaps by circulating CEA (2). As a preliminary to the use of radiolabeled anti-CEA IgG to localize colon tumors in vivo in man and to its use in vitro as a possible marker of precancerous change in patients at risk (e.g., patients with ulcerative colitis) , we have studied the in vitro binding of 125I-anti-CEA IgG to colon tumor tissue and normal colon mucosa taken from 32 patients at laparotomy. MATERIALS AND METHODS
Samples.-Tissue samples were obtained at the time of surgical resection from 32 patients with colorectal cancer. Tumor tissue was isolated and trimmed of normal and necrotic tissue; all tumors were adenocarcinomas. Normal mucosa, at least 10 em from the visible edge of the tumor, was dissected from underlying musculature. Control mucosa was obtained as rectal biopsy samples from 6 healthy volunteers. For 21 of the 32 patients, a sample of blood was taken within 9 days before or 5 days after surgery, and the plasma CEA concentration was determined by the Z-gel assay (7). 125[-anti-CEA IgG.-The anti-CEA used was the IgG fraction isolated from the serum of a goat immunized with purified CEA. This was labeled with 125 1 by the method of Greenwood et al. (8). Specific activities of different batches ranged from 10 to 30 p.Ci/ p.g. Assay procedure.-Tissue samples, treated individually, were blended in cold saline (1 :20 wt/vol) for 5 minutes in a Sorvall Omni-mixer homogenizer (setting 9.5). The homogenate was passed through an 88-p. sieve
5,7
and stored at - 80°C. When needed for assay, homogenates were thawed at 4° C, further diluted to 1: 80 (wt/vol) with cold saline, and centrifuged at 3,000xg for 15 minutes at 4° C. The supernatants were retained for subsequent determination of CEA concentration by direct Z-gel assay. The pellets were resuspended in saline, centrifuged as before, weighed, and resuspended I: 80 (wt Zvol in 0.05 M phosphate buffer (PB), pH 7.5. Triplicate 0.5-ml aliquots of the washed homogenates were incubated for I hour at 4° C with 125I-anti-CEA IgG (approximately 5 ng in 50 p.l PB containing 25 mg bovine serum albumirr/ml}. After incubation, the homogenates were centrifuged at 3,000Xg for 15 minutes at 4° C, washed twice with PB, and counted in an autogamma counter to determine the percentage of bound 125I-anti-CEA. For tissues from 3 patients, cell suspensions rather than homogenates were examined. Several steps were involved in the preparation of the cell suspensions: The tissues were first minced with scalpel blades in phosphate-buffered saline (PBS) containing 10 mM EDTA, then passed through an 88-p. sieve to remove cell aggregates and centrifuged at 600Xg for 20 minutes at 4° C. Lastly, the cell pellet was resuspended in sufficient PBS to give 7 X 105 cells/rnl. Examination of the suspensions by phase-contrast microscopy showed mainly single cells with a relatively small amount of debris. Binding of 125I-anti-CEA by triplicate 0.5-ml aliquots of the suspensions was determined as described for the homogenates. The specificity of the assay was checked by the preincubation of tissue homogenates with unlabeled antiCEA, normal human y-globulin, human serum albumin, or CEA, and then assessed for binding of 125I-anti-CEA. RESULTS
Text-figure 1 shows the distribution of binding values for the normal and cancerous colon samples from the 32 patients. The mean percentage binding for normal mucosa, 7.00± 1.16 (so), was significantly lower (P 13
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COATES, KOCH, BEAVER, MCPHERSON, AND NOUJAIM
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NORMAL TUMORS MUCOSAE
TEXT-FIGURE I.-Distribution of 1251-anti-CEA binding values for colorectal tumors and normal mucosae. suspensions of normal mucosal cells, 3.18 ± 0.63, was significantly lower (P
1,2,3
E. Coates,4 Maria Koch,4 Patricia F. Beaver,4 T. Alexander McPherson,4,6 and Anthony A. Noujaim
SUMMARY-Samples of tumor and normal mucosa from 32 patients with adenocarcinoma of the colorectum were examined for their capacity to bind radioiodinated antibody to caretnoembryonic antigen (anti-CEA) IgG. Twenty-three (72%) of the tumors bound significantly more antibody than the respective normal mucosa. The results indicate that radiolabeled anti-CEA may be useful in the in vivo localization of CEA-producing tumors and metastases in man, and may have application in vitro as a diagnostic marker of precancerous change in colorectal biopsies from patients at risk of developing colorectal cancer.-J Natl Cancer Inst 55: 25-27, 1975.
RADIOLABELED ANTIBODY TO CARCINOEMBRYONIC ANTIGEN (CEA) might be useful in the localization of tumors producing CEA (1). Several recent manuscripts have reported the successful localization of human colon tumors transplanted in hamsters (2-4) and nude mice (5) after the animals were given injections of radioiodinated antibody to CEA (antiCEA). However, binding of anti-CEA by normal human tissue (Le., normal human colon mucosa) is not assessed (6) when human colon tumor heterografts are used to study anti-CEA localization. In addition, only colon tumor heterografts below a certain size show increased uptake of radioiodinated anti-CEA, which suggests a loss or masking of CEA antigens with larger tumors or a blocking of injected antibody before contact with the tumor, perhaps by circulating CEA (2). As a preliminary to the use of radiolabeled anti-CEA IgG to localize colon tumors in vivo in man and to its use in vitro as a possible marker of precancerous change in patients at risk (e.g., patients with ulcerative colitis) , we have studied the in vitro binding of 125I-anti-CEA IgG to colon tumor tissue and normal colon mucosa taken from 32 patients at laparotomy. MATERIALS AND METHODS
Samples.-Tissue samples were obtained at the time of surgical resection from 32 patients with colorectal cancer. Tumor tissue was isolated and trimmed of normal and necrotic tissue; all tumors were adenocarcinomas. Normal mucosa, at least 10 em from the visible edge of the tumor, was dissected from underlying musculature. Control mucosa was obtained as rectal biopsy samples from 6 healthy volunteers. For 21 of the 32 patients, a sample of blood was taken within 9 days before or 5 days after surgery, and the plasma CEA concentration was determined by the Z-gel assay (7). 125[-anti-CEA IgG.-The anti-CEA used was the IgG fraction isolated from the serum of a goat immunized with purified CEA. This was labeled with 125 1 by the method of Greenwood et al. (8). Specific activities of different batches ranged from 10 to 30 p.Ci/ p.g. Assay procedure.-Tissue samples, treated individually, were blended in cold saline (1 :20 wt/vol) for 5 minutes in a Sorvall Omni-mixer homogenizer (setting 9.5). The homogenate was passed through an 88-p. sieve
5,7
and stored at - 80°C. When needed for assay, homogenates were thawed at 4° C, further diluted to 1: 80 (wt/vol) with cold saline, and centrifuged at 3,000xg for 15 minutes at 4° C. The supernatants were retained for subsequent determination of CEA concentration by direct Z-gel assay. The pellets were resuspended in saline, centrifuged as before, weighed, and resuspended I: 80 (wt Zvol in 0.05 M phosphate buffer (PB), pH 7.5. Triplicate 0.5-ml aliquots of the washed homogenates were incubated for I hour at 4° C with 125I-anti-CEA IgG (approximately 5 ng in 50 p.l PB containing 25 mg bovine serum albumirr/ml}. After incubation, the homogenates were centrifuged at 3,000Xg for 15 minutes at 4° C, washed twice with PB, and counted in an autogamma counter to determine the percentage of bound 125I-anti-CEA. For tissues from 3 patients, cell suspensions rather than homogenates were examined. Several steps were involved in the preparation of the cell suspensions: The tissues were first minced with scalpel blades in phosphate-buffered saline (PBS) containing 10 mM EDTA, then passed through an 88-p. sieve to remove cell aggregates and centrifuged at 600Xg for 20 minutes at 4° C. Lastly, the cell pellet was resuspended in sufficient PBS to give 7 X 105 cells/rnl. Examination of the suspensions by phase-contrast microscopy showed mainly single cells with a relatively small amount of debris. Binding of 125I-anti-CEA by triplicate 0.5-ml aliquots of the suspensions was determined as described for the homogenates. The specificity of the assay was checked by the preincubation of tissue homogenates with unlabeled antiCEA, normal human y-globulin, human serum albumin, or CEA, and then assessed for binding of 125I-anti-CEA. RESULTS
Text-figure 1 shows the distribution of binding values for the normal and cancerous colon samples from the 32 patients. The mean percentage binding for normal mucosa, 7.00± 1.16 (so), was significantly lower (P 13
.... •
12 w uI II
.;
26
COATES, KOCH, BEAVER, MCPHERSON, AND NOUJAIM
r•
~ eX
~IO eX
9 10 N 8 ~ 7 ~
-
6 5
4
3
.... ~
!• •
f
fI'
• I
fit
•
NORMAL TUMORS MUCOSAE
TEXT-FIGURE I.-Distribution of 1251-anti-CEA binding values for colorectal tumors and normal mucosae. suspensions of normal mucosal cells, 3.18 ± 0.63, was significantly lower (P
