Carcinoembryonic Antigen Immunoreactivity Patterns in Colorectal Cancer: Correlation with Morphologic Parameters

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1 Carcinoembryonic Antigen Immunoreactivity Patterns in Colorectal Cancer: Correlation with Morphologic Parameters Simun Andelinovic, MD; Jerolim Bakotin, MD; Zeljko Dujic, MD; Deny Andelinovic, MD; Robert Stojan, MD From the Department of Pathology and Cytology (Drs. S. Andelinovic, Bakotin, Stojan), Department of Physiology (Dr. Dujic), Clinical Hospital Firule Split and Medical Faculty, Zagreb - Study in Split, and Department of Dermatology (Dr. D. Andelinovic), Clinical Hospital Firule, Spinciceva, Split, Yugoslavia. Address reprint requests and correspondence to Dr. Andelinovic: Department of Pathology, Clinical Hospital Firule, Spincicka 1, Split, Yugoslavia. Accepted for publication 5 November The expression of carcinoembryonic antigen (CE A) was analyzed immunohistochemically in 100 colorectal cancer specimens. The tumors were divided into three groups, according to the predominant staining which was either apical, cytoplasmic or stromal. CE A localization was apical in 26 cases, cytoplasmic in 48 and stromal in 26 cases. Overall, well differentiated tumors showed apical CE A, whereas the poorly differentiated tumors showed stromal staining. Significant correlation was noted between the s and histological grading ( P < 0.001), nuclear grading ( P < 0.001), mucin synthesis ( P < 0.01), blood vessels, lymphatic vessels and peripheral nerve invasion ( P < 0.01), and lymph node metastasis ( P < 0.01). was not correlated with patients age, sex or gross appearance of tumors and their site of origin, nor the Dukes classification. It is concluded that the CEA immunoreactivity may provide data that are relevant for the prognosis of the neoplastic disease. S Andelinovic, J Bakotin, Z Dujic, D Andelinovic, R Stojan, Carcinoembryonic Antigen Immunoreactivity Patterns in Colorectal Cancer: Correlation with Morphologic Parameters. 1991; 11(5): Carcinoembryonic antigen (CEA) is the most frequent clinically investigated antigen of colorectal cancer. Since its first isolation from the fetal tissues and from the serum of the patients with colorectal cancer by Gold and Freedman [1], it wasshown that both normal colonic and malignant epithelial cells are able to produce CEA [2,3]. However, Ahnen et al [4] found a polar apical distribution of CEA in normal colonic epithelium using immunoelectronmicroscopy, whereas malignant epithelium was characterized by a loss of polarity in correlating with the grade of anaplasia. The prognostic value of tissue CEA immunoreactivity in colorectal cancer was confirmed later by other authors [5,6], The presence of CEA immunoreactivity in surrounding stroma of the malignant cells from the poorly differentiated tumors has been suggested as an important factor in increasing plasma CEA levels [5]. CEA-negative and CEA-positive tumors with membranous immunoreactivity were reported in the group of poorly differentiated tumors and were correlated with patient survival [6]. In the present study we analyzed CEA s of immunoreactivity using polyclonal anti-cea antibody in 100 colorectal cancer specimens. CEA immunoreactivity was correlated with some important gross (tumor size, macroscopic appearance and localization) and histologic parameters (tumor histological and nuclear grading, Dukes classification, ability of mucin synthesis, blood and lymph vessel and peripheral nerve invasion, and lymph node metastasis).

2 Material and Methods The material was composed of 100 patients with primary colorectal adenocarcinoma treated by surgical resection in the Department of Surgery of the Clinical Hospital Split from Fifty-five of the patients were men (mean age, 63.7 years) and 45 were women (mean age, 64.9 years). Tumor localization was as follows: 6 tumors arose in cecum, 7 in the ascending colon, 3 in the hepatic flexure, 9 in the transverse colon, 3 in the splenic flexure, 6 in the descending colon, 35 in the sigmoid, 3 in the rectosigmoid junction, 27 in the rectum and1 in the anal region. The tumors were, according to their macroscopic appearance, described as exophytic (N = 28), ulcero-exophytic (N = 26) and ulcerated and/or infiltrative (N = 46) [7]. Modified Dukes classification [8] was used for histological examination. There were 27 tumors in Dukes class A (tumors not invading through the entire thickness of the muscularis propria), 28 in class B (tumors that invaded the entire thickness of the muscularis propria to or through the serosa, whenever it existed), 38 in class C (tumors with one or more metastatic lymph nodes, irrespective of the extent of penetration through the bowel wall), and 7 in class D (tumors that had spread to a continuous organ or metastasized to distant sites). Histological differentiation of the tumors was performed using criteria defined by Blekinsopp et al [9] and Morson and Sobin [10]. There were 10 tumors in grade I (well differentiated), 47 in grade II (moderatelydifferentiated) and 43 in grade III (poorly differentiated). In addition, tumors were classified according to their ability to synthesize mucin as mucin-secreting (N = 81) and non-mucin-secreting (N = 19). Tissue specimens obtained from the resected colorectal region were fixed in 10% formaldehyde and embedded in paraffin. All serial sections were stained with hematoxylin and eosin, PAS-alsian blue for mucin detection and Verhoef s method for elastic fibers detection. Tumor cell nuclei were graded as well differentiated (N = 3), moderately differentiated (N = 37), and poorly differentiated (N = 60) [11,12]. Tumor invasion in the blood and lymph vessels and peripheral nerves was classified as present (invasion of one or more blood vessels or lymph vessels or peripheral nerves) or absent (no invasion) (N = 27). The invasion of the tumor cells in blood and lymph vessels, peripheral nerves and in the regional lymph nodes was shown either with hematoxylin and eosin staining, Verhoef s method or CEA immunoreactivity. Spread of the tumor in the regional lymph nodes was classified as present (N = 43) or absent (N = 57). Immunoperoxidase staining was performed according to Hamada et al [5]. Polyclonal anti-cea rabbit antibodies and swine anti-rabbit antibodies and the peroxidase-antiperoxidase (PAP) complex were obtained from Dakopatts, Copenhagen, Denmark. CEA immunoreactivity was graded according to the combination of criteria defined by Wiggers et al [6] and Hamada et al [5]. Tumors were classified as negative if less than 80% of the individual tumor cells displayed immunoreactivity, or as positive if more than 80% of the tumor cells had CEA expression [6]. All 100 specimens examined in this study were positive. Cellular CEA localization was classified into three groups according to the predominant staining using the method of Hamada et al [5], as I (apical type CEA is restricted mainly to the apical border of the malignant glands) (Figure 1), II (cytoplasmic CEA is present also in the cytoplasm and on the basolateral surfaces as fine granules) (Figure 2) and III (stromal CEA is present not only intracellularly but distributed diffusely in the surrounding stroma adjacent to the basal membrane of the malignant glands) (Figure 3). We found 26 tumors in I (apical), 48 in II (cytoplasmic) and 26 in III (stromal). The data were analyzed by the chi-square and Student s t test. Figure 1. Pattern I - CEA is localized predominantly on the apical border of the malignant glands. (anti-; original magnification, x 250.)

3 Figure 2. Pattern II - CEA is localized in the cytoplasm and on the apical and basolateral surfaces of the malignant cells. In the malignant glands CEA-positive necrotic material can be seen. (anti-; original magnification, x 150.) Figure 3. Pattern III - CEA is present not only intracellularly but also can be found diffusely in the surrounding stroma. (anti-; original magnification, x 150.) Results Age and CEA Pattern All 100 colorectal adenocarcinomas examined were positive for CEA. For statistical analysis, patients were grouped based on their age (less than 60 years and more than 60 years). No significant relationship was noted between the distribution of CEA and age groups. Apical, cytoplasmic or stromal types of CEA localization were observed in each of the age groups. However, the majority of the examined colorectal cancer specimens were from the older patients (64 cases in age range). Cellular distribution of CEA was not related to sex of the patients. Tumor Site of Origin and CEA Pattern Seventy-four percent of all adenocarcinomas were present at the left side, while 26% of the cases were present on the right side. No statistical relationship was noted between the immunohis-tochemical grading and tumor localization. Tumor Gross Appearance and CEA Pattern The tumor exophytic gross appearance was observed more frequently in the group of patients with apical CEA, whereas ulcerated and/ or infiltrative tumors were present in the subjects with stromal CEA.

4 However, no statistically significant relationship between the CEA and tumor gross appearance was found. The apical, cytoplasmic or stromal CEA can be present in every tumor irrespective of its gross appearance. Tumor Size and CEA Pattern The mean diameter of tumor with apical type of immunoreactivity was 4.9 ± 2.7 cm, cytoplasmic was 4.4 ±1.9 cm, and stromal was 4.1 ± 1.9 cm. No statistical difference between the three groups was noted. Tumor Differentiation and CEA Pattern Table 1 shows the relationship between the CEA and tumor differentiation. In the group of patients with apical, 9 (34.6%) had well-differentiated cancer and 17 (65.4%) had moderately differentiated cancer. None of the tumors with this staining was classified as poorly differentiated. In the group ofpatients with cytoplasmic CE A, 26 (54.2%) had moderately differentiated cancer and 21 (43.8%) had poorly differentiated cancer. Only one patient had a well-differentiated cancer. The stromal immunohistochemical CEA staining was present in the poorly (22 cases %) and moderately (4 cases %) differentiated cases of cancer. There was a statistical difference between well-differentiated tumors with apical or cytoplasmic CEA and poorly differentiated tumors with stromal CEA (P< 0.001). Tumor Nuclear Grading and CEA Pattern Table 2 shows the relationship between the CEA and tumor nuclear grading. Well-differentiated nuclei (grade I) were present in tumors with apical or cytoplasmic CEA s, whereas poorly differentiated nuclei (grade III) were present predominantly in tumors with stromal or cytoplasmic CEA s (P < 0.001). Moderately differentiated nuclei were found in the patients with apical or cytoplasmic CEA s (34 out of 37 cases). Dukes Classification and CEA Pattern Table 3 shows the relationship between the and Dukes classification. Apical CEA was present in 12 patients with Dukes A (46.2%), 8 patients with Dukes B (30.8%) and 6 patients with Dukes C (23.1%). The cytoplasmic CEA was proportionally present in all Dukes classes. The stromal CEA was predominantly present in Dukes C (50.6%) and B (23.1%). Four patients with Dukes A had stromal immunohistochemical grading. There was no significant relationship between the and Dukes classification. Tumor Mucin Synthesis and CEA Pattern Synthesis of mucin was evident in the majority of examined colorectal cancer cases (81%). We found a significant relationship between s and ability to synthesize mucin (P< 0.01). All 26 tumors with apical CEA distribution produced mucin, whereas cytoplasmic CEA tumors had producing (81.2%) and nonproducing (18.8%) tumors, as was evident in stromal tumors (61.5% producing and 38.5% non-producing). Loss of mucin synthesis was present in cytoplasmic or stromal CEA s, which are characteristic for moderately or poorly differentiated tumors. Regional Lymph Node Metastasis and CEA Pattern Tables 4 and 5 show the relationship between the and the presence of regional lymph node metastasesand the number of lymph nodes with metastases. A significant relationship between the CEA grading and the ability of tumor to spread in the regional lymph nodes was noted (P< 0.01) (Table 4). Apical tumors rarely metastasized (23.1%) whereas poorly differentiated stromal tumors had high metastasizing capability (65.4%). The average number of regional lymph nodes with metastases increased in relation to the CEA s. Apical had an average 1.7 ± 0.8 lymph nodes with metastases, cytoplasmic had 2.8 ± 2.5 and stromal had 3.3 ± 4.8 (Table 5).

5 Table 1. Tumor differentiation and CEA. Tumor differentiation Well Moderate Poor Total Apical Cytoplasmic Stromal Total P < Table 2. Neclear differentiation and CEA. Tumor differentiation Well Moderate Poor Total Apical Cytoplasmic Stromal Total P < Table 3. Dukes classification and CEA. Dukes classification A B C D Total Apical Cytoplasmic Stromal Total NS Tumor Blood Vessels, Lymph Vessels or Peripheral Nerve Invasion and CEA Pattern Tumors with apical CEA invaded primarily lymph vessels (38.5%), whereas blood vessels or lymph vessels and peripheral nerves were rarely invaded (3.8% each) (Table 6). The cytoplasmic CEA most frequently invaded lymph vessels (37.5%), followed by lymph vessels and peripheral nerves (16.6%) and lymph and blood vessels (8.3%) invasion. Stromal CEA tumor hadthe highest invading capability: lymph vessels (42.3%), blood vessels, lymph vessels and peripheral nerves (26.9%) and lymph vessels and peripheral nerves (11.5%). However, combined lymph vessel, blood vessel and peripheral nerve invasion was noted only for stromal tumors. Table 4. Regional lymph node metastasizing and CEA. Regional lymph node metastases Yes No Total Apical Cytoplasmic Stromal Total P < 0.001

6 Table 5. Averagenumber of lymph nodes with metastates and CEA. Tumors with metastases Lymph nodes with metastases X (SD) Apical (0.8) Cytoplasmic (2.5) Stromal (4.8) Total NS Table 6. Tissue CEA and tumor blood vessel and/or lymph vessel and/or peripheral nerve invasion. Tumor invasion CEA grade Apical Cytoplasmic Stromal Total Blood vessel Lymph vessel Peripheral nerve Lymph vessel and peripheral nerve Blood vessel and peripheral nerve Lymph and blood vessel Lymph, blood vessel and peripheral nerve No invasion Total Discussion In this study we analyzed the relationship between CEA immunoreactivity s and some important morphologic parameters of colorectal cancer. All examined cancer specimens of the large bowel and rectum showed cell immunoreactivity with specific polyclonal anti-carcinoembryonic antigen antibody. As was previously reported by others [4-6], intra- and extracellular CEA localization was found to be more sensitive than the intensity of CEA immunoreactivity. Three types of CEA cellular localization were defined by Hamada et al [5] (apical, cytoplasmic, and stromal), and in the present study we were able to confirm tneir categorization. However, Ahnen et al [4] and Wiggers et al[6] described the of CEA immunoreactivity in terms of apical/cytoplasmic or membranous localization in tumor cells. Our study confirms the observations of others [4-6] that the of CEA immunoreactivity is closely related to the grade of histological differentiation. In addition to histologic differentiation, we related CEA immunoreactivity s with other parameters. We found that CEA expression reflects the degree of cancer cell nuclear differentiation. Poorly differentiated cancer cell nuclei were found in tumors with predominantly stromal CEA localization. In our study we had 27% cases in Dukes A, 28% in Dukes B, 38% in Dukes C, and 7% in Dukes D. We found no significant difference between CEA immunoreactivity and Dukes classification. However, we found subgroups of patients within Dukes classification that could have better or worse prognosis. In Dukes grade A there were 4 patients with stromal CEA with potentially more aggressive behavior and in Dukes grade C there were 6 patients with apical CEA type which is less aggressive. We suggest that immunohistochemical CEA staining is independent but a complementary prognostic factor todukes classification in colorectal cancer patients. Regional lymph node metastasesis the most important prognostic factor in survival of colorectal cancer patients [ I ]. We found that tumor metastasizing capability is related to the CEA immunoreactivity: the apical CEA rarely had regional lymph node metastaseswhereas the stromal was more frequently associated with metastases. In addition, the number of lymph nodes with metastaseswas also related to the CEA immunoreactivity (stromal had higher number of lymph nodes with metastases). We found that the size of the tumor is inversely related to the CEA immunoreactivity, as was previously

7 reported by Wolkmark et al [14] for relation between size and regional lymph node metastases. In 1932 Dukes reported also that there was inversai correlation between the size of the tumor and the depth of penetration [15]. In our study ceilular CEA localization was not related with age of the patients, tumor gross appearance, tumor localization, or sex distribution. Mucin production was, however, significantly related with the CEA immunoreactivity s. All cancer specimens classified as apical produced mucin, whereas the majority of stromal tumors were lacking mucin production. Cohen et al [16] and Mascarel et al [17] in their studies also suggested the prognostic value of mucin production in colorectal cancer patients. The prognostic significance of the invasion of blood vessels, lymph vessels and peripheral nerves has been previously emphasized [7]. Our results suggest that, with increase in the immunohistochemical grading, there is high ability of tumor to invade surrounding structures. In conclusion, we think that immunohis-tochemis-try provides some important additional information in the diagnosis of the colorectal cancer patients and that this method should complement Dukes criteria in the prognostic evaluation. References 1. Gold P, Freedman SO. Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption technique. J Exp Med 1965;121: Huitric E, Leumoniec R, Burtin P, et al. An optical and ultrastructural study of the localization of carcinoem-bryonic antigen (CEA) in normal and cancerous human rectocolic mucosa. Lab Invest 1976;34: Wagener C, Czaszar H, Totovic V, et al. A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue. Histochemistry 1978;58: Ahnen DJ, Nakane PK, Brown WR. Ultrastructural localization of carcinoembryonic antigen in normal and colon cancer: abnormal distribution of CEA on the surface of colon cancer cells. Cancer 1982;49: Hamada Y, Yamamura M, Hioki K, et al. Immunohis-tochemical study of carcinoembryonic antigen in patients with colonic cancer: correlation with plasma carcinoembryonic levels. Cancer 1985;55: Wiggers T, Arends JW, Verstijenen C, et al. Prognostic significance of CEA immunoreactivity s in large bowel carcinoma tissue. Br J Cancer 1986;54: Brandao O, Sobrinho-Simoes MA, Serrao D, et al. Prognosis in colorectal cancer. Path Res Pract 1985;198: Dukes CE. The pathology of the rectal cancer. In: Smithers DW, Dukes CA, eds. Cancer of the rectum. Edinburgh: Churchill and Livingstone, 1960: Blekinsopp WK, Stewart-Brown S, Blenkovsky L, et al. Histopathology reporting in large bowel cancer. J Clin Pathol 1981;34: Morson BC, Sobin LM. Histological typing of intestinal tumors. Geneva: WHO, Fisher ER, Gregorio RM, Fisher B. The pathology of invasive breast cancers: a syllabus derived from the findings of the National Surgical Adjuvant Breast Project (Protocol No. 4). Cancer 1975;36: Lozowski MS, Mishriki Y, Chao S, et al. Estrogen receptor determination in fine needle aspirates of the breast. Acta Cytologica 1987;31: Dukes CE, Bussey HJR. The spread of the rectal cancer and its effect on prognosis. Br J Cancer 1958;12: Wolmark N, Cruz I, Redmond CK, et al. Tumor size and regional lymph node metastasesin colorectal cancer: a preliminary analysis from the NSABP clinical trials. Cancer 1983;51: Dukes CE. The classification of cancer of the rectum. J Pathol Bacteriol 1932;35: Cohen AM, Wood WC, Gunderson LL, et al. Pathological studies in rectal cancer. Cancer 1980;45: Mascarel A, Coindre JM, Mascarel I, et al. The prognostic significance of specific histological features of carcinoma of the colon and rectum. Surg Gynecol Obstet 1981;153:511-4.