Changes in glycosaminoglycan synthesis and in heparan sulfate deposition in human colorectal adenocarcinomas.

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

[The characteristics of bile acid excretion in patients with cancer of the large intestine]. / Osobennosti ékskretsii zhelchnykh kislot u bol'nykh rakom tolstoi kishki.

Marked difference in bile acids excretion (on the same diet) was observed between patients with large bowel cancer and healthy controls. The peak level of the bile acids in feces was registered in female patients 46-59 years of age suffering right-sided cancer. It is suggested that bile acid excretion in man is determined both by nutritional factors and hormonal-metabolic peculiarities of the body. High excretion of the bile acids is associated with risk for colon cancer.

Expression of the c-erbB-2 gene product (p185) at different stages of neoplastic progression in the colon.

The c-erbB-2 gene has been found amplified in a number of human adenocarcinomas leading to elevated levels of expression of the p185 protein product. Increased expression of this putative growth factor receptor has been reported to occur by molecular mechanism other than gene amplification and for this reason we have studied the expression of the p185 protein in normal colon and in lesions representing different stages of neoplastic progression. We report amplification of the c-erbB-2 gene in 3 of 44 colon carcinomas and 1 of 5 preneoplastic polyps studied. Confirmation of expression of the p185 protein product was established in Western blot analysis and by immunocytochemical staining of tissue sections. An extended study, involving adenomatous polyps and carcinomatous material in immunostaining, revealed detectable presence of the p185 protein in 20% of carcinomas, consistent with immunoprecipitation data derived using established cell lines. In contrast, a high percentage of polyps showed strong staining with both p185 antibodies used, indicating elevated levels of expression of the c-erbB-2 protein associated with preneoplastic lesions. Staining of normal human colon revealed a restricted localization of this putative receptor to cells on the luminal colonic surface, with no expression in cells of the crypt. Histologically normal mucosa, adjacent to the tumor, showed a more extensive distribution involving the crypt suggestive of a disturbance in the normal expression of c-erbB-2. These results indicate that elevated expression of the c-erbB-2 protein is associated with early stages of colonic neoplasia but do not establish it as a primary factor in these events. The occurrence of multiple copies of the c-erbB-2 in a percentage of colon lesions, however, suggests a possible role for this gene in some colon malignancies.

Histopathologic and flow cytometric analysis of adenomatous colonic polyps.

We evaluated the histopathology, DNA content, and proliferative activity of colonic polyps independently. Paraffin-embedded specimens were used as source material. In each case, additional sections were cut at 3 microns and stained with hematoxylin-eosin and trichrome for histopathologic analysis. For DNA analysis and measurement of proliferative activity, the polyp parts were dissected and the nonpolypoid tissue was discarded. The study was limited to those specimens that were received in our department in the years 1972 and 1977. Of the 104 polyps that were submitted for flow cytometric analysis, 36 could not be analyzed owing to excessive debris or insufficient nuclei. DNA aneuploidy was identified in 32% of the cases, with a higher value noted in larger polyps and in severely dysplastic polyps, but these values were not statistically significant. Multiple adenomas from the same patient often showed different DNA histograms. When analyzed according to the percentage of cells in S phase, no significant difference was found in proliferative activity of polyps according to DNA content or size of the polyps. These results suggest that the diagnostic significance of aneuploidy and proliferative activity in polyps must be interpreted with caution.

[Mucin histochemical changes in various colo-rectal polyps in relation to carcinoma of the large bowel].

210 various colo-rectal polyps including 46 inflammatory polyps, 21 juvenile polyps, 9 hyperplastic polyps, 65 tubular adenomas, 51 familial polyps, 11 villous adenomas, 7 adenomatous polyps with focal cancer, and 14 carcinoma of the large bowel were investigated by HE,HID-AB,PAT-KOH-PAS staining in order to study the mucin changes of these lesions. N-acetylated and C7,C9 O-acetylated sialomucin were mainly obtained in those adenomas with moderate and severe dysplasia (55-64.3%) and the proportion was even higher in cases of villous adenomas, familial polyps, adenomas with focal cancer and advanced carcinoma. These mucins might be assumed as a criteria in representing malignant transformation.

Mucin profiles and potential for malignancy of human colorectal adenomatous polyps.

The mucin histochemical and histologic features of 166 colorectal adenomatous polyps from 124 patients were studied. A majority of the polyps (62%) had a tubular growth pattern whereas 38% showed villous growth. Severe dysplasia was more frequently found in the latter group. A significant correlation (r = 0.27, P less than 0.001) was found between the severity of dysplasia and the size of the polyps. Moreover, the ratio between goblet and columnar cells was also found to decrease (P less than 0.0001) with the severity of dysplasia. Independent of the mucin stain used (periodic acid-Schiff, alcian blue, and high-iron diamine stains), mucin reactive cells were found to be negatively correlated (-0.17 less than r less than -0.44, P less than 0.01) with severity of dysplasia, especially in the tubular adenomas. These findings suggest that evaluation of mucin stain, related to dysplasia, may contribute to the assessment of premalignant and early malignant changes in adenomas of the colon.

Lectin histochemistry of colonic adenomas.

34 adenomas of the colon and adjacent flat mucosa were reexamined by morphological, histochemical and immunohistochemical means. Adenomas were grouped according to the degree of epithelial cell atypia: group 1 (dysplastic adenomas) showing mild and moderate dysplasia and group 2 (cancerous adenoma) with severe dysplasia and carcinoma. Morphological and secretive changes (hyperplasia with hypersialomucin secretion), considered typical of 'transitional mucosa', were constantly found in the adjacent and stalk mucosa. A panel of six lectins were tested using PAP and ABC system methods to compare amount and cytoplasmic localization of labelling sites with the morphological changes such as hyperplasia, dysplasia and carcinoma. All the tested lectins were reactive in up to 90% of adenomas as well as in adjacent mucosa. Positivity was unrelated to the severity of dysplasia and no preferential localization of labelled sites was shown in the adenoma groups. However, cytoplasmic distribution of reactivity was quite different in hyperplastic epithelium compared to dysplastic ones.

Distribution of tissue bile acids in the human alimentary tract and colon polyps.

To determine the characteristic distribution of tissue-bound bile acids in the human alimentary tract and colon polyps, we measured the concentration of bile acids in the mucosal tissues of the alimentary tract obtained at autopsy and polyps obtained by endoscopic polypectomy, using enzymatic fluorimetry and gas-liquid chromatography. The concentration of tissue-bound bile acid, especially chenodeoxycholic acid, was significantly higher in the ileum or ascending colon than in the other portions of the alimentary tract. The bile acid level of polyps was also higher in the ascending colon than in the other portions of the colon. These results suggest that the high concentration of tissue-bound bile acids is obtained at the site of absorption of bile acids in the alimentary tract.

DNA distribution pattern of the so-called severe dysplasias and small carcinomas of the colon and rectum and its possible significance in the tumor progression.

The DNA distribution pattern was determined by cytofluorometry in 25 cases of colorectal small carcinoma and the so-called severe dysplasia. The colorectal carcinoma and "severe dysplasia" consisted of four principal stemlines as to DNA ploidy: diploidy, aneuploidy, and their respective polyploidies. These patterns appeared in various combinations in individual neoplasms. DNA distribution of the severe dysplasia was diploid-predominant (11 cases) or aneuploid-predominant (three cases), usually showing mosaicism in various degrees with respective first order polyploidy. Similar DNA distribution patterns also were found in submucosally invasive small carcinomas. The neoplastic cell populations of a higher polyploidy (second or third order), however, occurred only in the submucosally invasive carcinomas (three cases) regardless of their basic ploidy. The mitotic index tended to be higher in the aneuploid-predominant tumors than in the diploid-predominant tumors. In the current observation, there was no significant correlation between the DNA distribution pattern and histologic type of the "dysplasia" or carcinoma. We found that most of the so-called severe dysplasias of the colon and rectum already gained definitive characteristic of carcinoma in the DNA pattern, i.e., ploidy heterogeneity. Therefore, they can be identified as intramucosal carcinomas, distinct from the normal epithelia and adenomas of the colon and rectum.

Beta-endorphin-like immunoreactivity in normal mucosa, muscle layer, adenocarcinoma, and polyp of the colon.

beta-Endorphin-like immunoreactivity was detected in the mucosa and muscle layer of normal colon, adenocarcinomas derived from the colon mucosa, and colon polyps which were histologically confirmed to be adenoma without a focus of carcinoma or with in situ carcinoma. The contents of beta-endorphin-like immunoreactivity in adenocarcinomatous tissue (11.94 +/- 1.77 pmol/g wet wt) and colon polyps without focus of carcinoma (10.71 +/- 1.50 pmol/g wet wt) were found to be significantly higher than those in the mucosal layer (6.86 +/- 0.64 pmol/g wet wt) and muscle layer (8.30 +/- 0.68 pmol/g wet wt) of normal colon. These data suggest that the production of beta-endorphin-like immunoreactivity is specifically increased in some adenocarcinomas and adenomatous polyps and may be related to the alteration of bowel habits. Gel exclusion chromatography of beta-endorphin-like immunoreactivity revealed three peaks corresponding to beta-endorphin, beta-lipotropin, and an immunoreactive form between the two. In the mucosal layer and muscle layer of the colon, a broad major peak was eluted at the position of beta-endorphin, and minor peaks were eluted at the position of beta-lipotropin and between beta-endorphin and beta-lipotropin. In adenocarcinoma and polyp, the peak size corresponding to authentic beta-lipotropin was greater than that of beta-endorphin. This study demonstrated that beta-endorphin-like immunoreactivity existed at a high concentration in some colon adenocarcinomas and polyps whose elution patterns were different from those of normal colon tissue.

Proliferative activity of rectal mucosa and soluble fecal bile acids in patients with normal colons and in patients with colonic polyps or cancer.

Rectal biopsies and fecal collections were obtained from a consecutive series of 34 outpatients prior to colonoscopy at a gastroenterology clinic. Subsequently, 14 were found to have no colonic pathology, 13 had adenomatous polyps, (3 of those had a previous history of colon cancer) and 7 were diagnosed with colon cancer. In confirmation of earlier studies the tritiated thymidine labelling index was higher in patients with tumors than in those without pathology (7.9% vs. 5.8% with P = 0.06). The patients with colonic tumors also had significantly higher levels of deoxycholic acid (P = 0.01) and lithocholic acid (P = 0.005) in the aqueous extract of their feces. This study shows that these biochemical measures may indicate colon cancer risk.

Plasminogen activators and tumor development in the human colon: activity levels in normal mucosa, adenomatous polyps, and adenocarcinomas.

Malignant changes are often accompanied by alterations in activity and composition of the plasminogen activators (PA). To study the relationship between PA expression and the development of colorectal cancer, we determined urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in normal mucosa (n = 80), adenomatous polyps (n = 76), and adenocarcinomas (n = 71) of the colon. Tissues obtained from surgical resection or polypectomy were analyzed for t-PA and u-PA activity in a specific enzymatic assay using plasminogen, a chromogenic substrate, and selective quenching with monospecific antibodies to both activators. The plasminogen activator activities were found to be changed in adenocarcinomas as compared to normal mucosa. The relative contribution of u-PA (expressed as percentage of u-PA) was raised from 6 to 50% for, respectively, normal mucosa and adenocarcinoma. This change could be attributed to a 3-fold decrease in t-PA activity and a 5-fold increase in u-PA activity in the carcinomas. Adenomatous polyps as a group showed percentages of u-PA [20.2 +/- 1.3 (SE)] which were intermediate as well as significantly different (P less than 0.001) from those of normal mucosa and adenocarcinomas. This observation was strengthened by a gradual rise in the relative contribution of u-PA in four resection specimens containing both adenomatous polyps and adenocarcinomas. Zymography showed the presence of minor quantities of PA-PA inhibitor complexes in the tissue extracts studied. The present study shows that the sequence of normal mucosa-adenomatous polyp-adenocarcinoma in the colon is associated with a parallel change in plasminogen activator activity. Thus, change in the regulation of plasminogen activator activity is an early event in the development of colorectal cancer.

Ferritin immunohistochemical localization in normal and neoplastic colonic mucosa.

High levels of ferritin have been detected in serum and tumoral extracts of gastrointestinal neoplasms. However, its histological localization is not well known. An immunoperoxidase technique (PAP) was used for detecting ferritin in 30 colorectal carcinomas, 20 polyps and 8 cases of non-neoplastic mucosae. Ferritin staining was detected in stromal cells (98%) much more than in epithelial cells (21%). Connective cells were positive in 5 cases of normal mucosae (62%), 19 polyps (95%) and all carcinomas (100%). The number of positive cells gradually rose from normal mucosa to carcinoma with an intermediate score in adenomas. However, no relation could be found between the stromal ferritin score and dysplasia in polyps. Likewise, no relation was found between the stromal ferritin score and the differentiation grade, invasion or metastases in carcinomas. The positive epithelial pattern seen in 12 cases (21%) suggests non-specific staining due to passive diffusion from the stroma. Thus, these immunohistochemical findings suggest that in colonic neoplasms, ferritin could be a tumor marker produced mainly by stromal cell reaction more than by the epithelial cells.

DNA in situ sensitivity to denaturation: a new parameter for flow cytometry of normal human colonic epithelium and colon carcinoma.

Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.

[A study of tumor-associated cellular proteins from carcinoma and adenoma of the human colon].

In order to detect tumor-associated cellular proteins and to obtain information pertaining to the theory of an adenoma-carcinoma sequence, cellular proteins from mucosa, carcinoma, adenoma, carcinoma in adenoma, and polypoid carcinoma of the human colon were analyzed by two-dimensional (isoelectric focusing-SDS PAGE) electrophoresis. The results revealed the presence of about 300 spots with pIs from 5.5 to 8.5 and MWs from 20,000 to 200,000 in all of the five tissue types. The vast majority of them were common to these five tissue types. However, there were nine spots which differed between mucosa and carcinoma of the colon: Three spots (82/6.3, 65/8.2, 56/8.1, MW X 10(-3)/pI) were detected often in carcinoma but seldom in mucosa, and four spots (72/8.2, 72/8.5, 61/7.5, 38/6.5) were increased in amount in carcinoma. These seven spots could be the tumor-associated cellular proteins. The remaining two spots (31/7.2, 28/6.5), which were decreased in amount in carcinoma, were considered to be normal colon-associated cellular proteins. The three spots 82/6.3, 65/8.2 and 56/8.1 were also detected in adenoma, carcinoma in adenoma, and polypoid carcinoma. The four spots 72/8.2, 72/8.5, 61/7.5 and 38/6.5 were increased in amount in these three tissue types and spots 31/7.2 and 28/6.5 were decreased. The above results might provide evidence, though indirect, for the theory of an adenoma-carcinoma sequence.

Hyperplastic polyposis of the colorectum and adenocarcinoma in a 24-year-old man.

Hyperplastic polyps in the large intestine are considered common after the age of 40, devoid of neoplastic transformation capability, and have a well-defined histological picture. Nevertheless, there exists evidence that hyperplastic polyps can present with adenomatous areas, eventually leading to carcinoma. There has been a suggestion in certain cases that a transformational sequence might exist in the genesis of colorectal cancer: hyperplastic polyp-adenoma-adenocarcinoma. Adenocarcinoma resulting from a pure hyperplastic polyp has also been described. These data have generated reconsideration of the significance of hyperplastic polyps in relation to cancer. In this paper we present a 24-year-old man with adenocarcinoma of the colon who underwent a right hemicolectomy. Twenty-eight hyperplastic polyps were found in the surgical specimen. Two of these polyps had adenomatous areas. The patient died 18 months after the surgical resection.

Correlation of nuclear ploidy with histology in adenomatous polyps of colon.

Histological sections of adenomatous polyps of the colon showing carcinoma were studied by video image analysis. Nuclear DNA content and morphology were measured in regions identified as either dysplasia, carcinoma confined to the mucosa, or carcinoma invading the muscularis mucosa. Where carcinoma was present, areas of dysplasia in the same polyp were found to have similar distributions of nuclear DNA content and size, supporting the notion that adenomatous polyps becomes cancer. The method can be used to detect those regions in sections of adenomatous polyps with the most severe nuclear abnormality.

Polyps of the colon in juvenile and young patients: histological type and C.E.A. content in relation to follow-up.

31 colonic polyps in 21 patients between 2 and 30 years of age removed by endoscopy were studied. The site, size, cytohistological characteristic and tissue CEA content of these polyps were evaluated. 58% of the polyps were located in the rectum and sigmoid, the remaining 42% were in the descending colon. At histological examination 13 polyps proved to be neoplastic, 3 of which had severe dysplasia. In 3 cases of juvenile hyperplastic hamartomatous polyps and in 5 cases of inflammatory polyps the tissue C.E.A. was low; in 13 neoplastic polyps it was in direct correlation with the degree of dysplasia. Moreover in 10 patients with neoplastic polyps, 5 presented further adenomas at follow-up, and in 3 of them the C.E.A. tissue content of the first polyps obtained was medium to high. The occurrence in young patients of polyps of the colon with C.E.A. content medium to high must be followed by endoscopic examination at close intervals.

Mucin histochemistry in colonic polyps and cancer.

Mucins are the predominant glycoproteins found in gastrointestinal epithelia, and their structures differ according to the location in the GI tract and the state of cellular differentiation. Different forms of mucin are secreted in colonic polyps and cancers compared to those found in the normal colon. This paper reviews the methods available to probe mucin structure and the state of knowledge regarding the structures of neoplasia-associated mucins. Many of the assumptions made in the interpretation of classical histochemical stains on tissue sections have recently been questioned, and new insight into mucin structure has been gained from the development of newer methodologies such as lectin histochemistry and immunocytochemistry that use well-defined monoclonal antibodies.