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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.

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., (Copyright 2000 Wiley-Liss, Inc.)

This retrospective study was undertaken to investigate the expression of bcl-2 protein and messenger RNA in colorectal cancer (CRC). Immunohistochemical analysis using a monoclonal mouse antibody to the bcl-2 protein and in situ hybridization using a digoxigenin-labelled bcl-2 cRNA probe were carried out on formalin-fixed and paraffin-embedded specimens from 53 colorectal adenocarcinomas, 27 liver secondaries, and 60 adenomas with various degrees of dysplasia. Normal human tonsil sections were used as positive controls. Expression of bcl-2 protein and of messenger RNA was evaluated semiquantitatively. The expression of bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary CRC (moderate/severe dysplasia: Mann-Whitney U-test, p=0.0001; severe dysplasia/primary CRC: p=0.027), whereas the cellular expression of bcl-2 mRNA was gradually increased during the dysplasia/adenoma-carcinoma neoplastic sequence. These observations suggest that in a proportion of colorectal cancer cases, the bcl-2 proto-oncogene expression may be down-regulated at a post-transcriptional level., (Copyright 2000 John Wiley & Sons, Ltd.)

Genomic alterations at the long arm of chromosome 17, and in particular at the nm23 locus, are still controversial in colorectal cancer (CRC). Our aim was to investigate the possible relationship of loss of heterozygosity (LOH) and microsatellite instability (MI), at 4 microsatellite loci spanning the 17q21-23 region, to the risk of liver metastasis and nm23 protein expression. Genomic DNA extracted from 58 primary and 54 liver secondary formalin-fixed and paraffin-embedded CRCs was obtained from 82 patients. A fluorescent PCR coupled with an automated DNA sequencer was applied. Increasing fraction of loci showing LOH was positively associated with risk of liver metastases (logrank test for trend, p = 0.005); this remained independent after adjusting to T-stage (Cox regression, p = 0.022), N-stage (p = 0.007), or Dukes' stage (p = 0.012). Conversely, increasing frequency of MI was associated with a reduced risk of liver metastases in Dukes' B tumours (logrank test for trend, p = 0.032). When comparing 30 primary and matched liver secondary lesions, we found concordant genomic alteration in 72% (NME1) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity (chi2 test for trend, p = 0.024). Our findings suggest that genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via an unknown underlying mechanism.

Objective: To assess the significance of the expression of five protein markers (nm23, p53, c-erbB-2, u-PA, and VEGF) to the development of metastasis in colorectal cancer., Summary Background Data: The metastatic cascade is a complex multistep process involving several genetic alterations, angiogenesis activation, and tissue proteolysis. Although the prognosis of colorectal cancer depends on the stage of the tumor, the development of metastasis is difficult to predict., Methods: Paraffin-embedded specimens of 58 patients who underwent surgery for colorectal cancer were retrospectively analyzed by immunohistochemistry, and the coexpression of these protein markers was related to patient outcome., Results: The risk of developing liver secondaries was correlated with the expression of nm23 protein (p < 0.0001); this was also the case in those patients with Dukes' stage B showing positive nm23 immunostaining (p = 0.006). The determination of the number of positive markers or the cumulative intensity score did not improve the predictive value over and above that of nm23 protein alone., Conclusion: Expression of nm23 protein is correlated with the risk of developing liver metastasis. Its evaluation alone may help to determine which patients who have undergone apparently curative resection of a colorectal cancer have an increased risk of liver recurrence, especially those with Dukes' stage B tumors who might be considered for adjuvant chemotherapy.

The function and prognostic significance of the nm23 gene is controversial in colorectal cancer (CRC). The aim of this study was to determine if nm23 protein expression correlated with the subsequent development of liver metastasis. Paraffin-embedded sections of 30 metastasizing CRC primaries and their subsequently resected liver secondaries were compared with those of 28 nonmetastasizing CRCs, 20 adenomas, and 20 cases of normal colonic mucosa. Expression of nm23 protein, assayed by immunohistochemistry, was measured using a standard semiquantitative scaling system and compared with a microcomputerbased color video image analysis (VIA). There was good correlation between color VIA and semiquantitative evaluation of nm23 immunoreactivity, confirming the validity of quantitative analysis (Pearson's r = 0.88; p < 0.001). Metastasizing CRC primaries and secondaries overexpressed nm23 protein when compared with the other clinical groups, particularly nonmetastasizing CRC (Student's t-test, p < 0.001). Furthermore, more nm23 immunoreactivity was associated with a higher risk of death from CRC (log-rank test, p = 0.002). These results suggest that overexpression of nm23 is highly associated with liver metastases from CRC and reduced survival.

We undertook a retrospective study to investigate the correlation between the expression of vascular endothelial growth factor (VEGF) and urokinase-type plasminogen activator (u-PA) proteins with progression of colorectal carcinoma (CRC). Immunohistochemical analyses using antibodies against VEGF and u-PA were carried out on archival specimens of 58 human colon carcinomas, 30 liver secondaries and 20 adenomas. Expression of VEGF was significantly reduced in the metastatic liver tumours compared with primary ones (Wilcoxon test, P = 0.002), suggesting VEGF activity to be secondarily down-regulated once the tumour cells reach the hepatic parenchyma. There was no strong evidence from our data that the level of VEGF or u-PA in the primary tumour could predict risk of liver metastasis or survival duration. VEGF and u-PA expression were positively correlated in primary CRC suggesting that both proteins may interact in vivo (chi-square test, P = 0.019) in tumour progression.

We undertook a study to analyze the expression of urokinase-type plasminogen activator (u-PA) protein in colorectal cancer (CRC) and to compare it with c-erbB-2 (HER2/neu) and nm23 protein expression. Paraffin-embedded specimens from 58 patients with CRC were retrospectively collected. Immunohistochemical staining of u-PA, c-erbB-2, and nm23 was quantitatively evaluated using a color video image analysis (color VIA) technique. No correlation was found between u-PA expression and tumor stage, age, sex, or tumor site. Although there was no evidence from our data that the level of u-PA in the primary tumors could predict risk of liver metastasis or survival duration, CRC showing overexpression of u-PA (above 85 pixels) had a worse prognosis (P = 0.013). There were significant positive correlations among all three u-PA, c-erbB-2, and nm23 proteins (u-PA vs. c-erbB-2, P = 0.003; u-PA vs. nm23, P < 0.001; c-erbB-2 vs. nm23, P = 0.001), suggesting that, in vivo, all proteins interact or are similarly regulated.

We have previously shown that c-erbB-2 oncoprotein encoded by the erbB-2 gene is overexpressed in human colorectal cancers that metastasis compared to those that are cured by surgery. To determine whether c-erbB-2 is also differentially expressed in vivo in metastasising and non-metastasising tumours, we developed models of colorectal cancer growth in nude mice. Human colon cancer cell lines, HCT116, KM12SM, LIM1215 and SW480, were injected into the caecum after characterising their morphology, doubling time, DNA flow-cytometry and expression of c-erbB-2. Six weeks later, xenografted tissues were fixed for histological analysis and detection of c-erbB-2 by immunohistochemistry, 78% (21/27) of mice developed caecal cancers. The caecal tumours derived from HCT116, KM12SM or LIM1215 were highly metastatic; 67 to 100% of them had liver metastases and lymph node involvement and 33 to 75% had lung tumours. Most of the tumours were c-erbB-2-positive. In contrast SW480 caecal tumours had only 33% lymph node involvement, but not liver or lung metastases. Only one SW480 caecal tumour and one lymph node metastasis expressed c-erbB-2. C-erB-2 was more frequently expressed in xenografted tissues in colon cancer primaries and secondaries of the highly metastatic cells (HCT116, KM12SM and LIM1215) compared to the cells (SW480) giving predominantly local growth. Our results suggest that c-erbB-2 gene may play an important role in the development of metastasis from colorectal cancer.