A Cancer-Associated Mucin Alteration in Benign Colonic Polyps

GASTROENTEROLOGY 1982;82:664-72 A Cancer-Associated Mucin Alteration in Benign Colonic Polyps C. RICHARD BOLAND, CAROLYN K. MONTGOMERY, and YOUNG S. KIM Departments of Medicine and Pathology, Veterans Administration Medical Center and the University of California, San Francisco, California We have recently reported that the mucin in colon cancer is different from that in the normal colon, and this difference may be detected by selective Jectin binding characteristics. The Jectin Dolichos biflorus agglutinin [DBA) avidly binds to the mucin found in well-differentiated goblet cells in the normal colon, but does not bind to that in the majority of colonic cancers. Conversely, the Jectin peanut agglutinin (PNA) does not bind to the mucin from the normal colon, but avidly binds to that in colon cancer. Therefore, these two Jectins were used to determine if cancer-associated mucin were present in benign colonic polyps. Fluorescein isothiocyanate (FITC) conjugates of Dolichos biflorus agglutinin and peanut agglutinin were applied to fixed, unstained tissue sections of 56 colonic polyps and examined by fluorescent microscopy. Focal areas were found within the polyps in which FJTC-DBA labeling was diminished (compared with the normal colon) and labeling with FITC-PNA appeared. The findings are expressed as the percentage of glands per polyp labeled by FITC-PNA. The median JabeJing in 31 benign tubular adenomas was 7% of the glands per polyp. In 16 benign tubular adenomas 17 mm in diameter, only 2% of the glands per polyp were labeled, while in 15 benign tubular adenomas >7 mm in diameter, 24% of the glands per polyp were labeled. Carcinoma in situ or invasive carcinoma was found in nine adenomatous polyps, in Received July 24, 1981. Accepted November 11, 1981. Address requests for reprints to: C. Richard Boland, M.D., Gastrointestinal Research Laboratory, Veterans Administration Medical Center, 4150 Clement Street (151-M2), San Francisco, California 94121. This work was supported by National Institutes of Health Grant CA-14905 from the National Cancer Institute through the National Large Bowel Cancer Project and Training Grant F32-AM07007. Dr. Boland was the recipient of Research Fellowship Award F32- AM06327 from the National Institutes of Health. The authors thank R. Frautschi for preparation of the manuscript. 0 1982 by the American Gastroenterological Association 0016-5085/82/040664-09$02.50 which 41% of the adenomatous glands were labeled by FITC-PNA. Unexpectedly, in eight hyperplastic polyps, 89% of the glands were labeled by FITC- PNA. These results indicate that among neoplastic colonic polyps, cancer-associated mucin changes are present and are more widespread in larger tubu- Juar adenomas, villoglandular adenomas, and adenomas containing cancer. The presence of mucin that is bound by FJTC-PNA may be an indication of an increased risk of developing cancer within a colonic polyp. The finding of the cancer-associated mucin in hyperplastic polyps raises new questions about the pathogenesis of these benign, apparently nonneoplastic lesions. We have recently reported that colonic mucin undergoes an alteration in its carbohydrate structure during cellular differentiation and malignant transformation (1). These alterations were detected by the selective binding of lectins (proteins that bind to the carbohydrate portion of a glycoconjugate) to mucin. The lectin derived from DoJichos biflorus (DBA) binds to the mucin of the well-differentiated goblet cells found in the upper colonic crypt, but not to the goblet-cell mucin of the immature cells in the lower crypt. DoJichos biflorus agglutinin does not bind to the mucin secreted by the majority of colon cancers. The lectin derived from the peanut (PNA) does not bind to the goblet-cell mucin of any normal colonic mucosa, but binds to the mucin secreted by all of the colon cancer tissues studied. Peanut agglutinin also binds to the goblet-cell mucin of transitional mucosa, the benign but atypical colonic epithelium immediately adjacent to cancer. Peanut agglutinin has been reported to bind to the surfaces of certain malignant or undifferentiated cells, but not to their benign or differentiated counterparts in a number of experimental systems (2-7). This suggests that the appearance of receptors for PNA binding in glycoconjugates may be a common occurrence in malignant transformation.

April 1982 CANCER-ASSOCIATED MUCIN 665 Adenomatous polyps are benign epithelial neoplasms commonly found in the human colon. A full spectrum of cytologic atypia may be found within adenomatous polyps-from well-differentiated epithelial cells to focal nests of carcinoma. This observation has led some investigators to suggest that many or most colon cancers arise within previously benign polyps (8). However, this has been a controversial concept (9). Carcinoma is found in less than 1% of adenomatous polyps <l cm in diameter, but occurs in 9.5% of polyps 1-2 cm in diameter and 46% of polyps >2 cm in diameter (10). In addition, cancer is more likely to be found in villous adenomas than in tubular adenomas (10). On the other hand, hyperplastic polyps are felt to be nonneoplastic, remain small, and never contain foci of cancer (11). Therefore, we studied the lectin binding characteristics of the goblet-cell mucin in a series of 56 colonic polyps in an attempt to determine whether the cancer-associated mucin were present in benign but possibly premalignant colonic lesions. We also developed a method for quantitating the appearance of the mucin, and studied polyps of various sizes from each pathological category to determine if the amount of the altered mucin would correlate with the likelihood of finding cancer in the lesion. Materials and Methods Lectins Two fluorescein isothiocyanate (FITC) conjugated lectins were used to label the tissue sections (Table 1). Lyophilized preparations of FITC-DBA and FITC-PNA (Vector Laboratories, Burlingame, Calif.) were reconstituted in phosphate-buffered saline to a concentration of 0.2 mg protein/ml solution for use. To document specific carbohydrate binding by the lectins, a solution of 0.2 M N- acetyl-galactosamine (GalNAc) was used to inhibit the binding of DBA, and 0.3 M galactose (Gal) was used to inhibit the binding of PNA to glycoconjugates on the tissue section. Tissue Samples Fifty-six polyps were obtained from surgically resected colons or colonoscopic polypectomies (Table 2). Five-micron tissue sections were cut and stained with hematoxylin and eosin, as well as periodic acid-schiff for pathological diagnosis, and adjacent serial sections were cut for the fluorescent microscopic studies. Thirty-one of the polyps were benign tubular adenomas ranging from 2 to 30 mm in diameter. The median size was 7 mm, of which 16 were 57 mm and 15 were >i mm in diameter. Twenty-seven of the polyps were pedunculated, four were sessile. Thirteen of the benign tubular adenomas came from colons which also contained carcinoma (diameters: Z-30 mm, median 7 mm). One benign tubular adenoma came from a patient with quiescent chronic ulcerative colitis. None of the polyps came from patients with a familial polyposis syndrome. Eight of the polyps were benign villoglandular adenomas ranging from 7.5 to 35 mm in diameter (median = 14.5 mm). One of these polyps (diameter = 35 mm) came from a colon in which carcinoma was also present. Six of the villoglandular adenomas were sessile and two were pedunculated. None of these polyps came from colons in which ulcerative colitis or familial polyposis was present. Nine of the polyps contained either carcinoma in situ (n = 7) or invasive carcinoma confined to the head of the polyp (n = 2). These polyps ranged from 4 to 40 mm in diameter (median = 12 mm). Five were tubular adenomas and four were villoglandular adenomas. Two of these polyps came from colons in which another sessile, invasive carcinoma was simultaneously present. All nine of the polyps that contained cancer were pedunculated. None of the polyps containing cancer came from patients with ulcerative colitis or familial polyposis. The remaining eight were hyperplastic polyps ranging from 3 to 9 mm in diameter (median = 4 mm). Two came from colons that also contained carcinoma. Seven were sessile and one was pedunculated. None came from patients with ulcerative colitis or familial polyposis. The tissue diagnoses were determined independently after the fluorescent microscopic studies were completed, and each polyp was categorized as a tubular adenoma, a villoglandular adenoma (>50% villous), an adenoma containing cancer (either tubular or villoglandular adenomas included), or a hyperplastic polyp. By their nature, most adenomatous polyps contain at least mild dysplasia (12), and since degrees of dysplasia are subjective, no attempt was made to distinguish between mild and moderate dysplasia. Severe dysplasia may be equated with carcinoma in situ (12), and such polyps were grouped together with polyps containing invasive carcinoma (in which the cancer penetrates the muscularis mucosae). Table 1. FITC-Lectins Used for Study Preferred colonic Lectin Sugar specificity mucin binding Dolichos biflorus agglutinin (DBA) Peanut agglutinin WA1 GalNAc GaleGalNAc, or Gal Mucin from welldifferentiated goblet cells Cancer; transitional mucosa Tissue Preparation The tissues were obtained from the operating room or endoscopic unit and immediately placed in either a modified Bouin s solution (750 ml aqueous saturated picric acid, 250 ml 38% formaldehyde, and 10 ml glacial acetic acid) or 3% formaldehyde. The tissues were fixed for 2-14 h, serially dehydrated, embedded in paraffin, and 5-pm sections were cut and mounted on glass slides.

666 BOLAND ET AL. GASTROENTEROLOGY Vol. 82, No. 4 Table 2. Colonic Polvos Studied Histoloav Benign tubular adenomas 57 mm >7 mm Benign villoglandular adenoma Adenoma containing carcinoma Tubular adenoma Villoglandular adenoma Carcinoma in situ Invasive carcinoma Hyperplastic polyps 31 8 9 8 - Diameter Gross morphology Host colon n Range [mm) Mean lmml Pedunculated Sessile Cancer No cancer 16 15 5 4 2 2-30 2-7 7.5-30 7.5-35 4-40 5-13 4-40 4-40 12-13 3-9 7 27 4 13 18 5 11 14.5 2 6 1 7 12 9 0 2 7 15.5 12.5 4 1 7 2 6 - - Totals 56 39 17 18 38 Fluorescent Microscopy The paraffin-embedded tissue sections were serially rehydrated by two 2-min washes in xylene and 2-min washes in loo%, 95%, 80%, and 70% ethanol followed by three 2-min washes in phosphate-buffered saline [PBS) at ph 7.4. A solution of FITC-lectin was prepared in 0.05 M PBS, ph 7.4, at a concentration of 0.2 mg protein/ml solution. Fifty microliters of FITC-lectin were applied to the hydrated tissue sections, which were then incubated for 20 min, and the excess unbound FITC-lectin was rinsed off with a 5-min wash in PBS. The tissue sections were mounted in Gelvitol (Monsanto, St. Louis, MO.), ph 8, and covered with a glass coverslip. The lectin specificity was confirmed by incubating 50 ~1 of FITC-lectin together with 50 ~1 of the specific inhibitory sugar before the application to the tissue section. Sugar specificity was demonstrated by the abolition of specific fluorescence on the tissue section. The prepared slides were examined with a Zeiss epifluorescent microscope. The epithelial glands in colonic polyps secrete mucus into the goblet-cell vacuoles which may be labeled by the appropriate FITC-lectin (1). The presence or absence of fluorescence in the mucin vacuoles of each gland was noted with each of the lectins, and the entire tissue section or at least 200 randomly selected glands [in the larger polyps) were counted. The percentage of positively labeled glands per polyp was recorded. The slides were examined immediately after preparation, stored at 4 C, and reread within 3 days; the counts were then averaged. Student s t-test was used for statistical analysis. Results Labeling of goblet-cell glycoconjugates with FITC-DBA was seen in every polyp. In general, labeling with FITC-PNA was found only in focal areas within the polyps (Figures l-5). Focal areas were seen in which the labeling with FITC-DBA was diminished or absent. The areas that showed reduced labeling with FITC-DBA showed a concomitant appearance of labeling with FITC-PNA; that is, FITC-PNA labeled in the same areas in which labeling by FITC-DBA was absent. Since the remaining portions of the polyp did not label with FITC-PNA, the polyps were scored as the percentage of glands per polyp that were labeled by FITC-PNA. In each case, labeling with FITC-DBA was totally abolished by coincubation with 0.2 M GalNAc; labeling with FITC-PNA was totally abolished with 0.3 M Gal. There was no difference in labeling regardless of the fixative used. In each case, the repeat count of the tissue sections gave results within 5% of the initial reading. Among the 31 benign tubular adenomas a range of OS-80% of the glands was labeled, with a median labeling score of 7% (Figure 6). The median size of the benign tubular adenomas was 7 mm. Among the 16 polyps 17 mm in size, a median labeling score of 2% was found, and 7 of 16 showed 0% labeling [Figure 7). Among the 15 benign tubular adenomas >7 mm in diameter, a median labeling score of 24% was found, and none showed 0% labeling. The difference in labeling between the larger and smaller tubular adenomas was not statistically significant. Among the 18 benign tubular adenomas from colons free of cancer, the labeling ranged from 0% to 68%, median labeling being 6%. The median size in this group was 7 mm. Among the 13 benign tubular adenomas from colons that also contained cancer, the labeling ranged from 0% to 80%, the median labeling being 18%. The median size in this group was 8 mm. The differences in labeling between polyps from colons that did or did not contain cancer was not statistically significant. No difference was found between pedunculated and sessile polyps. Among the eight benign villoglandular adenomas, the labeling ranged from 3% to 71%, the median labeling being 26%. Although a trend toward increased labeling was seen in villoglandular adeno-

April 1982 CANCER-ASSOCIATED MUCIN 667 Figure 1. Periodic acid-schiff stain of a gland within an adenomatous colonic polyp (tubular adenoma). The goblet-cell mucin appears black. (x IX). mas (as seen in the larger tubular adenomas and in adenomas obtained from colons that contained cancer), the difference was not statistically significant (0.10 < p < 0.20). When all villoglandular adenomas were considered together (including eight benign adenomas and the four adenomas that contained cancer), a median labeling score of 37% was found, which is significantly different from the group of benign tubular adenomas (p < 0.05). Among the nine polyps containing cancer, the labeling ranged from 1% to 95% with a median score of 41%. (With the exception of one polyp that had only 1% labeling, a range of 22%-95% was observed.) The labeling of this group of malignant polyps was significantly different from the group of benign tubular adenomas (p < 0.02). The glands that labeled with FITC-PNA in the benign polyps tended to occur in focal clusters rather than to be randomly distributed throughout the polyp. In the malignant polyps, the focus of cancer always showed positive labeling of its secreted glycoconjugates, and in each case showed clustering of positively labeled adenomatous glands around the periphery of the cancer. Among the eight hyperplastic polyps, the labeling ranged from 45$, to 100% with a median labeling score of 89%, and no differences were seen whether the polyp was pedunculated, or obtained from a colon that contained cancer. Discussion Our studies demonstrate that benign colonic polyps contain foci in which the labeling of the goblet-cell mucin by FITC-DBA is diminished in concert with an appearance of labeling by FITC- PNA. The altered mucin appears in focal clusters of glands within the polyps. The percentage of glands per polyp containing this mucin is higher in those lesions that are more likely to develop cancer. When taken in conjuction with our previous work in which PNA selectively bound to the mucin found in colon cancer but did not bind to the goblet-cell mucin of the normal colon, one may conclude that some benign colonic polyps synthesize mucin with the same lectin binding characteristics as those seen in cancer. The observation that incremental differences in labeling scores are seen between smaller vs. larger tubular adenomas and between tubular vs. villoglandular adenomas correlates well with the in-

668 BOLAND ET AL. GASTROENTEROLOGY Vol. 82, No. 4 Figure 2. FITC-PNA labeled section of a gland within an adenomatous colonic polyp (tubular adenoma) demonstrating the presence of speckled fluorescence (white) within the goblet-cell mucin. (X 125). creased likelihood of finding cancer in larger and villous polyps. A wide range of labeling was seen within the various histologic categories, making this technique impractical for clinical diagnosis at this time. The wide range of labeling probably reflects the focal nature of this change within adenomatous polyps. Small tubular adenomas are less likely to contain a focus of carcinoma, and would be less likely to contain evidence of early malignant transformation. Nonetheless, a 5-pm histologic section is only 0.1% of the diameter of a 5-mm polyp, and most of the polyp is not examined. Therefore, in some of the smaller benign tubular adenomas that were heavily labeled by FITC-PNA, it is possible that higher degrees of dysplasia or carcinoma in situ might be found in adjacent sections upon careful histologic examination of serial sections. Mucin that is bound by PNA appears to be a colon cancer-associated mucin (1). The mucin molecule consists of a central protein core with oligosaccharide side chains (13). Lectins bind to the exposed, nonreducing termini of specific carbohydrate structures. By incubating the FITC-lectins (DBA and PNA), in the presence of a carbohydrate (GalNAc and Gal, respectively) that is critical for lectin binding, labeling of the tissue section is inhibited. Thus, the specificity of the lectin binding is documented, and the presence or absence of specific carbohydrate structures is indicated. Peanut agglutinin possesses a relatively low affinity for exposed Gal residues, but has a 50-fold higher affinity for the disaccharide

April 1982 CANCER-ASSOCIATED MUCIN 669 F&U re 3. FITC-PNA Iabeied section of a gland within an adenomatous colonic polyp (tubular adenoma) demonstrating intense label ling of the goblet-cell mucin (X 125). Uniform fluorescence of the goblet-cell mucin as shown here, and speckled labeling of the mucin as seen in Figure 2 are both considered equally positive glands. group Gal=GalNAc, and a 12,500-fold higher affinity for T antigen (14). One possible explanation for the binding of PNA to the mucin in colon cancer is the presence of incompletely glycosylated mucin glycoprotein in the malignant colon (1). The presence of similar mucin in benign colonic polyps suggests that the biochemical characteristics of malignancy may antedate the morphologic appearance of cancer in premalignant colonic epithelium. Numerous abnormalities in the biologic activity of colonic polyps have been reported by other laboratories. The epithelial cells of colonic adenomas are morphologically undifferentiated by light and electron microscopic analyses (15,16). Deoxyribonucleic acid synthesis and cell division continue into the upper half of the epithelial crypt, indicating an abnormality in the control of normal proliferation and differentiation in adenomatous polyps (17,181. Using the specific erythrocyte adherence assay, ABO blood group activity which is not seen in the normal human colon (but present in fetal colon) has been reported to appear in adenocarcinomas and neoplastic polyps of the distal colon, suggesting that cryptic or hidden blood group antigens tend to appear in both benign and malignant colonic neoplasms (19). Alterations in the histochemical staining properties of mucin have been reported in transitional mucosa (20) and benign colonic polyps (21,22). Indirect immunofluorescence studies have suggested the presence of common antigenic components in the mutinous glycoproteins of colon cancer, transitional mucosa, and benign colonic polyps. Normal colonic mucosa has not been studied with these antisera for comparison, however, and not all neoplastic mucosa was labeled by the antisera used in this work (23). The histochemical staining characteristics of the mucin in cancer and adenomatous polyps of the colon have been heterogeneous, and the relationship between the changes found in benign vs. malignant neoplasia remains unclear. Our findings indicate a similarity in the structure of mucin found in malignant and premalignant colonic epithelium, demonstrate stepwise quantitative changes among various subgroups of polyps, and identify a common carbohydrate structure in the mucin such as the one that is bound by PNA. Our most problematic observation was finding a high degree of FITC-PNA labeling in the mucin of hyperplastic polyps. Although a long controversy has existed over whether adenomatous polyps are

670 BOLAND ET AL. GASTROENTEROLOGY Vol. 82. No. 4 Figure 4. Hematoxylin and eosin stain of a hyperplastic polyp demonstrating the typical serrated appearance of the hyperplastic gland. The mucin is unstained and appears white. (X 125). precursors of colon cancer (8,9), it is generally agreed that hyperplastic polyps are not neoplastic and do not give rise to carcinoma (11,24). Light and electron microscopic studies reveal the epithelial cells of hyperplastic polyps to be morphologically well differentiated, and [sh]thymidine incorporation studies demonstrate that ceil replication is confined to a proliferative zone in the lower epithelial crypt (17,25). It has been proposed that hyperplastic polyps arise from a failure of the normal detachment of mature epithelial cells (15,16,25). However, there is a paucity of data on the pathogenesis of hyperplastic polyps. These polyps are found primarily in the sigmoid colon and rectum, and rarely grow larger than 1 cm in diameter (26). They are often found clustering around carcinoma of the colon, and have been termed satellite lesions. Hyperplastic polyps tend to be distributed in the distal colon in a manner similar to the distribution of colon cancer. Hyperplastic polyps occur more frequently in colons that contain adenomas and carcinoma than in those free of such lesions (27). This suggests the possibility that hyperplastic polyps, although not themselves neoplastic. may be caused by the same factors that produce colonic neoplasia. Our data suggest that it may be possible to produce cancer-associated alterations in glycoprotein synthesis in colonic mucosa without producing malignant behavior. Hyperplastic polyps could result from mucosa that has received a potentially carcinogenic

April 1982 CANCER-ASSOCIATED MUCIN 671 Figure 5. FITC-PNA labeled section of a hyperplastic polyp demonstrating three glands that are positively labeled by fluorescent (X 125). let :tin FITC-PNA Labeling: Colonic Polyps FIX-PNA Labeling: Subsets of Tubular Adenomas 89% s-%n Bmlgn A*.nom.to * yperpl,*tlc I b!m rib- wyp8 WYPS.d. Wll.* g*nd hr ConUlnlnp tn=q m=31, **ma.. E.Klnom. I =),, G, Figure 6. Mean percentage of glands per polyp labeled by FITC- PNA according to pathological diagnosis. Figure 7. Mean percentage of glands per polyp labeled by F ITC- PNA among the subsets of tubular adenomas.

672 BOLAND ET AL. GASTROENTEROLOGY Vol. 82, No. 4 stimulus, but has resisted malignant transformation. At present, this hypothesis awaits supporting experimental evidence. In conclusion, we have reported the appearance of cancer-associated mucin in benign colonic polyps. Increased numbers of mucus secreting glands are affected in polyps removed from colons that also contain cancer, larger tubular adenomas, villoglandular adenomas, and adenomas containing carcinoma. The affected glands tend to occur in focal groups within benign polyps and cluster around the focus of carcinoma in malignant polyps. The altered mucin appears to express an antigenic structure usually hidden or cryptic in normal human tissues. Cancerassociated mucin is also found in hyperplastic polyps, raising new questions concerning the pathogenesis of these nonneoplastic colonic polyps. References 1 Boland CR, Montgomery CM, Kim YS. 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