J. M. Ham

Urokinase-type plasminogen activator and its receptor in colorectal cancer: Independent prognostic factors of metastasis and cancer-specific survival and potential therapeutic targets

Urokinase‐type plasminogen activator (uPA) and its receptor (uPAR), plasminogen (Plg), and plasminogen activator inhibitors‐1 and ‐2 (PAI‐1 and PAI‐2) have been observed in many cancers and may contribute to progression and metastasis. In our study, we examined the expression of the 5 proteins by immunohistochemistry in 59 consecutive primary colorectal cancers (CRC) and correlated the protein expression with patient outcome. In addition, we determined the effect of down‐regulation of uPAR on the invasive/metastatic capability of CRC cells, by measuring antisense‐uPAR transfected HCT116 and control cell lines, in terms of uPAR expression, uPA‐binding activity, invasiveness through Matrigel in vitro and metastasis after cecal orthotopic implantation in nude mice in vivo. We found that higher expression of uPA or uPAR in primary tumor tissues was positively correlated with distant metastasis of CRC (Mann‐Whitney, p < 0.02) and negatively correlated with both patient overall survival (OS) and cancer‐specific survival (CSS; Cox model, p < 0.04). The prognostic value of uPA and uPAR for both OS and CSS was independent of other variables (multivariate Cox model, p < 0.007). Antisense‐uPAR transfected HCT116 cells, which expressed significantly lower levels of total cellular and cell surface uPAR proteins and uPA‐binding activity compared with either wild‐type or cells transfected with vector alone (Bonferroni, p < 0.05/3), consistently showed decreased invasiveness through Matrigel (Bonferroni, p < 0.05/3) and decreased metastasis formation in nude mice (Fisher, p < 0.05). Our data suggest that uPAR and uPA are independent prognostic factors in CRC; anti‐uPAR treatment, which affects both uPAR and uPA levels, may have potential for new treatment of the disease. Int. J. Cancer 89:431–439, 2000.

Higher expression of oncoproteins c-myc, c-erb B-2/neu, PCNA, and p53 in metastasizing colorectal cancer than in nonmetastasizing tumors.

AbstractBackground: Expression of individual oncogenes may predict outcome in patients with metastatic colorectal cancer (CRC). We studied the oncogene profile in the tumors of patients with CRC and assessed their value as predictors of liver metastases. Methods: The oncoproteins c-myc, c-erbB-2/neu (c-neu), PCNA and p53, were measured by immunohistochemistry in sections of metastasizing human CRC (n=34) and their liver secondaries as well as in sections of nonmetastasizing CRC (n=25). Results: The metastasizing primary CRC expressed proliferating-cell nuclear antigen (PCNA), c-neu, and c-myc at significantly higher levels than the nonmetastasizing primary cancer. p53 was also overexpressed in the metastatic group compared with the nonmetastasizing CRC, but this difference was not significant. The frequency of expression of all these markers was similar in the metastasizing primary CRC and the liver secondaries from the same patients. There was no correlation between the expression of the individual markers and histological grade, DNA ploidy, and subsequent local recurrence and lung metastasis and survival. However, when both groups were assessed together, positive expression of c-myc was more likely to occur in poorly differentiated tumors, whereas PCNA expression increased with more advanced Dukes stages. Conclusion: These results suggest that the overexpression of c-myc, c-neu, PCNA, and p53 may occur in CRC that are likely to metastasise to the liver.

Genetic Markers of Survival and Liver Recurrence after Resection of Liver Metastases from Colorectal Cancer

A significant number of patients with liver metastases from colorectal cancer (CRC) achieve 5-year survival after liver resection. Increased expression of genetic markers in the primary tumor are known to predict outcome after colonic resection, but the predictive value of such markers after resection of hepatic metastases is unknown. The objective of this study was to evaluate whether DNA content and multiple genetic markers, separately or expressed together, can predict patient outcome (liver recurrence and survival) after resection of hepatic metastases. We studied the paraffin-embedded liver tissue of 71 consecutive patients who had undergone a potentially curative resection of hepatic metastases from CRC. Using DNA flow cytometry and immunohistochemical staining techniques we determined the DNA content and the level of co-expression of seven tumor-associated proteins: proliferating cellular nuclear antigen (PCNA), epidermal growth factor receptor (EGFr), p53, c-erbB-2, H-ras, c-myc, and nm23. Three endpoints (liver recurrence, cancer specific, overall survival) were correlated with these tumor markers. The 5-year overall survival of the group was 31.2%. There was no correlation detected between the DNA aneuploidy and overall or cancer-specific survival. Similarly, expression of the individual tumor-associated proteins did not predict survival. Patients whose tumors co-expressed multiple markers had survivals similar to those whose tumors expressed fewer markers. However, a significant difference in hepatic recurrence was found between the p53-positive and p53-negative patients (p= 0.007), with marker-negative tumors having decreased recurrence. In conclusion, this study demonstrates that the DNA content and genetic markers c-myc, c-erbB-2, EGFr, H-ras, p53, PCNA, and nm23 do not predict survival after potentially curative resection of hepatic metastases from CRC. However, the immunoreactivity of p53 may be an important marker of local recurrence in the liver, which may be useful if re-resection of metastatic liver tumors is considered a viable management option in this disease.

DNA flow-cytometric analysis in colorectal cancer: A comparison of metastasizing and non-metastasizing tumours

The most common cause of death in patients with colorectal cancer is metastatic liver disease. In order to identify patients at a high risk of developing hepatic secondaries from colorectal cancers, DNA content was measured in metastasizing colorectal primaries (Group I, n= 32) as well as in their subsequently resected liver secondaries and in sections of non‐metastasizing colorectal cancers (Group II, n= 25). A modified interpretation system involving both a DNA index and percentage of cycling cells (those in S and G2 + M phases) was developed. DNA content was measured in paraffin‐embedded sections by flow cytometry using internal controls (human peripheral blood mononuclear cells) and non‐malignant tissue controls (19 patients with diverticular disease). In Group I there were significantly more tumours with both abnormal ploidy (aneuploid or abnormal tetraploid peak) and > 15% cycling cells compared with Group II (Chi‐squared; P= 0.034). The combination of abnormal ploidy and > 15% cycling cells was superior to Dukes’ classification for identifying metastasizing tumours (Logistic Regression; P= 0.047). However, it was not possible to discriminate between the two groups using either DNA ploidy or the percentage of cycling cells alone. The metastasizing colorectal cancers exhibited similar DNA ploidy characteristics and had a similar percentage of cycling cells compared with their liver metastases. These results suggest that tumour DNA ploidy plus the percentage of cycling cells may predict the development of liver metastases and thus survival in patients with colorectal cancer.

Effect of cholestasis produced by temporary bile-duct occlusion on hepatic glutathione and γ-glutamyl transferase levels in the pig

Important factors in drug metabolite‐induced hepatocellular injury are the production of toxic intermediaries by biotransformation processes and the protective capacity of hepatic detoxification systems. In the present study, the hepatic total glutathione (reduced glutathione plus oxidized glutathione), and γ‐glutamyl transferase (γGT) levels, which are important components of a hepatic detoxification system, were measured in pigs before, during, and after bile‐duct occlusion (BDO). The hepatic reduced glutathione (GSH) level fell significantly in the group with BDO compared with a sham operated control group. However, there were no significant changes in the hepatic oxidized glutathione (GSSG) and γGT levels in the two groups. It was also demonstrated that the hepatic GSH level returned to normal 1 week after the BDO was relieved.

THE BILIARY EXCRETION OF SULFBROMOPHTHALEIN IN THE PIG

The characteristics of the hepatic metabolism of Sulfbromophthalein (BSP) have not been described previously for the pig. This is an important deficiency, since the pig is particularly suitable for studies of hepatic physiology and pharmacology which might apply to man. The aim of these experiments was to establish the pattern of serum clearance and biliary excretion of BSP and to determine that dose which would produce a maximal concentration in bile. A dose response and pattern of biliary excretion of BSP was studied at three dose levels administered either as a single bolus of a continuous infusion. All experiments were performed in conscious, conditioned pigs. The patterns of serum clearance and biliary excretion were found to be similar to other laboratory animals and to man. Maximary biliary concentration of BSP was achieved by a single bolus of 5-9 mumol/kg or a constant infusion of 0-59 mumol/kg/min. At these dose levels no significant alteration in bile flow was demonstrated nor was there any correlation between bile flow and BSP excretion. Supra-maximal doses produced a significant increase in bile flow and with these doses there was a significant positive correlation between bile flow and BSP excretion.