BRACK A. BIVINS, M.D., WILLIAM R. MEEKER, JR., M.D., AND WARD 0. GRIFFEN, JR., M.D., PH.D.’ Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky 40506
M.D. AND JOSEPH C. PARKER, JR., M.D.
Department of Pathology, University of Kentucky Medical Center, Lexington, Kentucky 40506 Received November 8,1974
The demonstration of a tumor specific antigen (carcinoembryonic antigen) in 1965 raised the hope that the long-sought after “blood test for cancer” had been found [ 11, 121. Early optimistic reports suggested that the serum carcinoembryonic antigen (CEA) would be elevated or positive in as high as 90% of patients with colon cancer [9,24,27]. This original promise of CEA has not been fulfilled due to (1) a high incidence of false negatives in early lesions, (2) positive titers associated with malignancies outside the G.I. tract, (3) a significant number of false positives in patients with benign inflammatory disease [6, 9, 16, 20, 231. Despite these shortcomings, the CEA titer is elevated in over 70% of patients with carcinoma of the colon [5, 20, 251. During the past 5 years many investigators have tried to assess the role of CEA in cancer diagnosis, management and prognosis [2,20,21]. A number of studies have shown the plasma CEA may not be directly related to the size and extent of the colon cancer [ 1, 16,261. Recently we posed the question: Is the tumor associated with a high CEA titer histologically different from the tumor associated with a low CEA titer? In this study an attempt is made to answer that question by examining independently tumor ‘Address reprint requests to Dr. W. 0. Griffen, Jr., Department of Surgery, University of Kentucky Medical Center, Lexington, Ky 40506.
histology in 42 patients. Nine histologic variables were correlated with the serum CEA value. MATERIALS Eighty-two consecutive patients with carcinoma of the colon admitted to the University of Kentucky Medical Center were evaluated for inclusion in the study. There were three criteria for inclusion. First, the patient must have had a plasma CEA level drawn preoperatively. Second, the patient must have had adenocarcinoma of the colon. Finally, the operative specimen must have included sufficient material for a detailed histologic exam of primary tumor and lymph nodes. Twenty-two patients were excluded because the CEA level was not done or was done postoperatively. Four cases of squamous cell carcinoma were excluded. Fourteen caseshad inadequate tissue available for study usually due to far advanced disease with biopsy as the only procedure performed. Forty-two patients met all three criteria and are the basis for this study. METHOD The CEA titers were determined in the Surgery Department Cancer Research Laboratory at the University of Kentucky using the zirconyl phosphate gel technique developed by Hansen . A detailed description of our laboratory’s methods,
257 Copyright o 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 2 CEA, Mucus Production, and Fibrosis
TABLE 1 CEA, Dukes Stage, and Histologic Grade CEA level*
O-2.5 rig/ml >2.5 rig/ml Total patients Dukes Stage A B C Histologic grade Well differentiated Moderately differentiated Poorly differentiated
0 4 5
3 7 23
6 3 0
13 15 5
*All values show no significance (ns).
normals, standard curves, and comparisons with a reference laboratory (Hoffman LaRoche Laboratories, Nutley, NJ) have previously been reported . The upper limit of normal for CEA was established at 2.5 rig/ml. The pathologists who reviewed the surgical specimens were unaware of the CEA titers. The primary tumors were evaluated for Dukes Stage  and histologic grade as well as mucus production, necrosis, fibrosis, and vascular invasion. The lymph nodes were assessed for tumor invasion, nodal reaction, and lymphoplasmocytic infiltrates
P81. Following histologic examination, the data was separated into two groups: (A) those associated with a CEA titer of less than 2.5 rig/ml, and (B) those associated with a CEA titer of greater than 2.5 rig/ml. The two groups were then compared by chisquare analysis for statistically significant differences in each of the nine histologic criteria.
Total patients Mucus production 0 1 2 3 Fibrosis Present Absent
5 1 2 1
16 10 4 3
*All values show no significance (ns).
The histologic grade and CEA titer are outlined in Table 1. The tumors were graded as well, moderately, and poorly differentiated to reflect differences in organoid structure. The CEA level was independent of histologic grade. The tumors were arbitrarily classed as 0, 1,2, and 3 with respect to mucus production. As can be seen from Table 2, CEA levels do not correlate with mucus production. Fibrosis is included in Table 2 and was seen in all the tumors examined. Necrosis and vascular invasion are outlined in Table 3. Both are significantly associated with a CEA level of greater than 2.5 rig/ml. The p value for this correlation is less than 0.05. The lymph node data is outlined in Table 4. The relationship between serum CEA and lymph node metastases, lymphoplasmocytic infiltrates, or nodal reactions is not statistically significant. TABLE 3 CEA, Vascular Invasion, and Necrosis
RESULTS Of the 42 patients included in the study, 9 had CEA levels less than 2.5 rig/ml and 33 had CEA levels greater than 2.5 rig/ml. The comparison of Dukes Stage to CEA values is seenin Table 1. The CEA level was found to be statistically independent of Dukes Stage. As can be seen the vast majority of the patients in our serieshad advanced lesions.
CEA level* Total patients Vascular invasion Present Absent Necrosis Present Absent
*All values significant at p 2.5 rig/ml Total patients Lymph node reaction Lymphocytic predominance Germinal center predominance Lymphocytic depletion Unstimulated Lymphoplasmocytic infiltrate 0 1 2 3 Lymph node metastases Present Absent
5 0 0
17 2 5
0 4 5 0
2 20 10 1
*All values show no significance (ns). The presence of lymph node metastases correlated with CEA levels approaches significance.
COMMENT Relatively little has been written about the histology of tumors associated with a high CEA. Most investigators have related plasma CEA to the Dukes Stage for the tumor [l, 21, 251. Plasma CEA is generally accepted to be elevatated in 40% Dukes A; 65% Dukes B; 75% Dukes C; and 90% Dukes D [ 1, 211. The cooperative study found a highly significant correlation (p < 0.01) between increasing extent of local disease as evidenced by Dukes Stage and plasma CEA [ 11.This is at variance with our data in that Dukes Stage was not related to plasma CEA, but the distribution of positive CEA titers in this series (100% Dukes A; 63% Dukes B; and 82% Dukes C) is skewed by the 100% positives in the few early lesions and may account for this statistical dicrepancy. Holyoke, using the TMN system of staging, also, did not find a significant correlation between plasma CEA and stage of disease [ 161. Most investigators have reported that advanced tumors tend to have higher CEA values [l, 16,261. Studies of tumor grade and CEA have not
been particularly productive. Laurence reported that neither cell type nor differentiation influences CEA . One study using immunofluorescent techniques found tumor CEA to be abundant in well-differentiated tumors . In this study there was no correlation between plasma CEA and tumor grade. Mucus production and fibrosis were evaluated as part of the histologic review and were also found not to be significantly related to the plasma CEA level. A better correlation of plasma CEA with the lymph node analysis was anticipated. The lymph node reaction has been described recently as a histologic index to prognosis  but in this study it did not correlate well with plasma CEA. The relationship between CEA titer and the incidence of lymph node metastases approaches significance, and it might be argued that a larger series would show a correlation between these two parameters. Most investigators have indicated that nodal involvement is associated with high CEA titers [ 16,261. Vascular invasion and necrosis were found to be significantly related to plasma CEA. Vascular invasion as a determinant of plasma CEA is supported by current research. The CEA is a cell surface antigen bound to the glycocalyx (luminal surface) of the tumor cell and can be visualized by immunofluorescent techniques [7, 9, 13, 14, 201. Studies on the metabolism of the glycocalyx suggest that the carbohydrate layer may have a half-life as short as 24 hr [lo, 191. Immunofluorescent studies have demonstrated CEA within the lumina of the tumor acini suggesting that it may be secreted by the tumor cell . Thus the antigen may be involved in a dynamic process of recycling or secretion. It is certainly outside the plasma membrane where it could be easily washed off by exposure to blood or lymph . This would provide a morphologic explanation for the observation that patients with very high CEA levels have advanced disease and a poor prognosis because extensive vascular invasion has already occurred.
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The high incidence of false positive CEA values in benign inflammatory disease may be related to the association of CEA and tumor necrosis. As mentioned before CEA may be seenin the lumina as a secretion, it is also seen in association with intraluminal cell debris . Cellular necrosis liberates glycoproteins that may be identified as CEA at the ultrastructural level . The CEA has recently been described as a normal component of plasma and colonic mucosa although in very small amounts [3, 41. Elevated CEA titers have been reported in association with a large number of benign conditions including ulcerative colitis, diverticulitis, Crohn’s disease,and pancreatitis [6, 18, 20, 23, 291. These diseaseshave in common an increased turnover of cells associated with acute inflammation and netrosis. It would follow that necrosis or cell death would liberate CEA from the cell surface as part of the disintegrative process. Thus sufficient necrosis could contribute to the elevated CEA levels seen in these diseases. In summary, vascular invasion and tumor necrosis are significantly associated with elevated CEA levels and a rational explanation for these findings may be found in work on CEA identification on cell surfaces. Necrosis seems especially attractive in explaing the elevated CEA titers seen in benign inflammatory disease. REFERENCES 1. A Joint National Cancer Institute of Canada/ American Cancer Society Investigation. A collaborative study of a test for carcinoembryonic antigen (CEA) in the sera of patients with carcinoma of the colon and rectum. Canada Med. Assn. J. 107:25, 1972. 2. Bivins, B. A., and Meeker, W. R. Post-operative
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