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Mutations of the TP53 and Ki- ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki- ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3–144 months) whether or not p53 protein expression, TP53 mutation and K- ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5–8 of the TP53 gene and in codons 12 and 13 of the K- ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki- ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P =0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours ( P =0.010). The presence or absence of mutations in Ki- ras did not correlate with prognosis ( P =0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR)=2.16, 95% confidence interval (CI) 1.12–4.11, P =0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.

Interferon-alpha (IFN-alpha) enhances the activity of the 5-fluorouracil (5-FU) prodrug 5'-deoxy-5-fluorouridine (5'-dFUrd) in colorectal cancer cells in vitro by upregulating the enzyme pyrimidine nucleoside phosphorylase (PNPase), which is responsible for converting 5'-dFUrd to 5-FU. We examined whether such enhancement also occurs in vivo using human colorectal xenografts in nude mice. The Co-115 line has high basal levels of PNPase and the enzyme level was increased in tumours from mice treated for 3 weeks with 50,000 IU/day (5 days/week) of IFN-alpha A/D. The prodrug 5'-dFUrd (200 mg/day, 5 days/week) had a much greater antitumour activity than 5-FU had when it was used at an approximately equitoxic dose (20 mg/day, 5 days/week). However, because of the high activity of 5'/dFUrd as a single agent, no enhancement by IFN-alpha A/D was observed. Studies on xenografts of WiDr cells indicated that this line is much less sensitive to 5'-dFUrd. However, treatment of animals with IFN-alpha A/D at doses of 75,000 IU/day or 150,000 IU/day resulted in significant inhibition of WiDr tumour growth. Combination treatment with 75 mg/kg/day or 150 mg/kg/day of 5'-dFUrd resulted in enhanced antitumour activity, particularly at the higher dose of IFN-alpha A/D. Synergy of this drug combination was confirmed by isobologram analysis. Analysis of PNPase levels in WiDr tumours, excised from mice treated with IFN-alpha A/D, demonstrated that the enzyme activity was increased by IFN-alpha in a dose-dependent manner. Slight increases were also seen in normal liver and intestine from the same animals. Our results indicate that modulation of converting enzymes for anticancer prodrugs by cytokines could be a novel therapeutic strategy for combination therapy of colorectal cancer.

The biological activity of 5'-deoxy-5-fluorouridine (5'-dFUrd) depends upon intracellular enzymatic cleavage by pyrimidine phosphorylase to form 5-fluorouracil (5-FU). Interferon-alpha 2a (IFN-alpha) effect was analysed alone and combined with 5-FU or 5'-dFUrd, on proliferation inhibition of eight human colorectal cancer cell lines. The toxicity of 5-FU was enhanced by IFN-alpha in only one line (SW-480). In contrast, interactive enhancement of IFN-alpha was observed with 5'-dFUrd in five lines (WiDr, HT-29, 513, SW-480 and Co-115). In each of the lines showing potentiation by IFN/5'dFUrd but not by IFN/5-FU, cytoplasmic pyrimidine phosphorylase activity was increased after 5 days' incubation with IFN-alpha in a dose-dependent manner. Two lines (LISP-1 and SW-620) showed no potentiation of either 5-FU or 5'-dFUrd toxicity by IFN-alpha, and no change in pyrimidine phosphorylase activity. Potentiation of 5'-dFUrd effect by IFN-alpha may thus be explained by an enhancement of its conversion to 5-FU through stimulation of pyrimidine phosphorylase activity.