Comparative localization of cathepsin B protein and activity in colorectal cancer.

Cathepsin B is a lysosomal cysteine proteinase that may participate in cancer progression. We compared localization of its protein and activity during progression of human colorectal cancer. In adenomas and carcinomas, protein expression and, particularly, activity were elevated compared with those in normal colorectal mucosa. In normal mucosa, cathepsin B protein expression was moderate in stroma and variable in epithelium, whereas activity was mainly present in distinct areas of stroma directly underneath the surface of the colon and in epithelium at the surface of the colon. Stroma in adenomas and carcinomas contained moderate to high protein levels but little activity except for areas of angiogenesis, inflammation, and necrosis, in which activity was high. In adenomas and the majority of well-differentiated carcinomas and moderately differentiated carcinomas, cathepsin B protein and activity were found in granular form in the epithelium, close to the basement membrane. Protein and activity levels were low and diffusely distributed in cancer cells in the remainder of the well-differentiated and moderately differentiated carcinomas and in all poorly differentiated carcinomas. Invasive fronts in most cancers contained moderate protein levels but high activity. We conclude that (a) activity localization is essential to understand the role of cathepsin B in cancer progression, and (b) cathepsin B activity in human colon is associated with invasion of cancer cells, endothelial cells, and inflammatory cells, and in cell death, both apoptotic and necrotic.

Differential diagnosis of chronic pancreatitis and pancreatic cancer in brush cytology specimens.

Discrimination between chronic pancreatitis and pancreatic carcinoma can be complicated, particularly in brush cytology specimens. Previous studies have shown that the oxygen insensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase activity based on neotetrazolium reduction can be used for discriminating malignant cells from nonmalignant cells. In the present study, we investigated the value of the assay for differential diagnosis between the two pancreatic diseases. Oxygen insensitivity in ductal epithelial cells in normal human pancreas, chronic pancreatitis, and pancreatic carcinoma was determined by quantitative image analysis in sections of biopsies and in brush cytology preparations. In sections, the reaction in the absence of oxygen was a proper reflection of glucose-6-phosphate dehydrogenase activity, whereas in the presence of oxygen only malignant cells showed a significant reaction. Of 39 brush cytology specimens, diagnosis of all 11 cases of pancreatitis and 28 cases of cancer with the oxygen insensitivity test were in agreement with independent measures of chronic pancreatitis and cancer. The oxygen insensitivity test is a simple and valuable tool in addition to conventional pathology for differential diagnosis between pancreatitis and pancreatic cancer, both in biopsies and in brush cytology specimens.

Prognostic estimation of survival of colorectal cancer patients with the quantitative histochemical assay of G6PDH activity and the multiparameter classification program CLASSIF1.

Prognosis of colorectal cancer patients that show similar histopathology may vary substantially. An attempt was made to improve prognosis by the self-learning classification program CLASSIF1, based on automated multiparameter analysis of quantitative histochemical and clinical parameters of 64 colorectal carcinomas and adjacent normal mucosae. The histochemical parameters applied were the oxygen-insensitivity assay of glucose-6-phosphate dehydrogenase (G6PDH) activity, a valid discriminator between normal and cancerous mucosae, and related parameters CuZn- and Mn-superoxide dismutase (SOD) levels, and lipid peroxidation (LPO) capacity. Data were processed on the basis of a postoperative follow-up of minimally 32 and maximally 56 months. CLASSIF1 selected the parameters oxygen insensitivity of G6PDH activity, CuZn-SOD and Mn-SOD levels, LPO capacity, lymph node metastasis, Dukes' stage, and age for the highest prognostic value. On the basis of these selected parameters, CLASSIF1 correctly predicted favorable outcome in 100% of the surviving patients and fatal outcome in 64% of the deceased patients. G6PDH activity appeared to be the major information carrier for CLASSIF1. On the basis of G6PDH activity parameters alone, 96% of the surviving patients and 55% of the deceased patients were correctly classified. In comparison, estimation of prognosis on the basis of Dukes' stage alone resulted in 71% correctly classified surviving patients and 61% of patients who died. It is concluded that the self-learning classification program CLASSIF1, on the basis of quantitative histochemical and clinical parameters, is the best prognostic estimator for colon cancer patients yet available.

Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity for the discrimination between nonmalignant and malignant cells.

We review here the oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity to detect cancer cells. This inexpensive and rapid assay can be performed within half an hour. Discrimination between cancerous and noncancerous cells is based on a combination of elevated G6PDH activity, decreased superoxide dismutase (SOD) activity, and decreased lipid peroxidation in cancer cells. The test discriminates between adenomas and carcinomas of the colon with a certainty of >80% and has a high prognostic value for survival of colon cancer patients. Pancreatitis and pancreatic cancer are discriminated with a certainty of 100%. Therefore, the test can be applied by pathologists to provide additional information in difficult cases of diagnosis of cancer and for prognosis.

Development of oxygen insensitivity of the quantitative histochemical assay of G6PDH activity during colorectal carcinogenesis.

The effects of oxygen on the quantitative histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium reduction were studied in the different stages of carcinogenesis in the colon. Normal and hyperplastic epithelium, mucosae of patients with active Crohn's disease, and adenomas and adenocarcinomas of the colon were used. Epithelium of normal and inflamed mucosa, and hyperplastic epithelium, showed a residual G6PDH activity (RA) in oxygen that was always less than 20 per cent of the activity in the absence of oxygen. In adenomas and in dysplastic epithelia adjacent to carcinomas, the RA was significantly higher than that in normal epithelium, but significantly lower than that in adenocarcinomas. The RA of adenomas never exceeded 35 per cent. The RA of adenocarcinomas was on average 53 per cent and always higher than 20 per cent. When 35 per cent was used as a cut-off level, the sensitivity of RA to diagnose malignancy was 96.5 per cent. In a parallel study, a mouse model was used in which colon carcinomas and their precursors were induced chemically. Development of oxygen insensitivity during chemically induced carcinogenesis showed a pattern similar to that observed in the human. In conclusion, the test to determine RA is a useful tool for the detection of malignant mucosa in the colon. The test is particularly helpful in addition to histopathology for the detection of small lesions and the early stages of cancer.

Expression of CuZn- and Mn-superoxide dismutase in human colorectal neoplasms.

Decreased intracellular SOD protein levels and activity have been related with malignancy in the past. To investigate their relevance in the carcinogenetic process in the colon, we determined quantitatively CuZn-SOD and Mn-SOD levels and total SOD activity by histochemical means in human normal colorectal mucosa, adenomas, and carcinomas. Protein levels and activity were significantly decreased in carcinomas. CuZn-SOD protein levels, but not Mn-SOD levels or total SOD activity were related with differentiation grade and to a lesser extent with Dukes stage. Moderately differentiated carcinomas and Dukes stage A carcinomas showed lowest levels. Some carcinomas expressed elevated levels of CuZn-SOD and this was an indication of poor survival. It is concluded that decreased SOD expression is not a prognostic marker and seems to be a secondary phenomenon rather than directly linked with the carcinogenetic process.

Locoregional therapy with polyethylene-glycol-modified interleukin-2 of an intradermally growing hepatocellular carcinoma in the guinea pig induces T-cell-mediated antitumor activity.

Therapy with repeated intratumoral and perilymphatic administration of relatively low doses of polyethylene-glycol(PEG)-modified interleukin-2 (IL-2) in the syngeneic guinea pig line 10 (L10) hepatocarcinoma results in significant local tumor growth inhibition and a delay in development of regional lymph node metastases of more than 3 weeks when compared to controls. Occasionally animals are cured of tumor. The mechanism of this antitumor activity was studied. The antitumor activity of locoregionally administered PEG-IL-2 was abrogated by pretreatment with polyclonal anti-thymocyte serum, indicating that the observed tumor growth inhibition was a T-cell-mediated phenomenon. Besides the locoregional tumor growth inhibition, a systemic effect was recorded as the growth of a second tumor cell inoculum at the contralateral side was inhibited as well. Furthermore, those animals cured after PEG-IL-2 therapy developed specific immunity against the L10 tumor and this immunity could be transferred to naive animals by spleen cells. Immunohistological observations of the tumor site revealed a slight increase of helper and cytotoxic T cell subpopulations after PEG-IL-2 therapy. More pronounced, however, was the rise in number of eosinophilic granulocytes present in the stroma surrounding the tumor cells. Involvement of cytotoxic cells in the antitumor effects of PEG-IL-2 could not be demonstrated: regional lymph node cells and spleen cells obtained immediately after therapy (day 15) or on day 21 showed no cytotoxic activity in vitro against L10, K562, Daudi and line 1 (L1) target cells. In conclusion, locoregional therapy with PEG-IL-2 induced a a systemic T-cell-mediated antitumor response. As no cytotoxic T cell activity was measured, however, the underlying mechanism is most likely a T-helper response. Eosinophils at the tumor site may be tumoricidal but further experiments must reveal the role of these cells in the PEG-IL-2-induced tumor regression.