Predictive role of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer patients receiving adjuvant 5-fluorouracil.

OBJECTIVE: The combined assessment of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) gene expressions in metastatic colorectal cancer has been reported to be able to predict the efficacy of fluoropyrimidine-based chemotherapy. In order to evaluate the prognostic role in the adjuvant setting, we investigated the TS, DPD and TP expression in primary tumors of colorectal cancer patients treated with 5-fluorouracil (5-FU). METHODS: TS, DPD and TP expression levels were determined by immunohistochemistry in paraffin-embedded primary tumor tissues from 62 patients with Dukes' stage B and C colorectal cancers who underwent surgery and received adjuvant systemic chemotherapy with 5-FU. The median follow-up was 90 months (range 17-127). RESULTS: Dukes' stage C cancer and high TS expression were independent markers of poor prognosis for disease-free survival (DFS; p = 0.0009 and p = 0.007, respectively) and overall survival (OS; p = 0.0005 and p = 0.011, respectively). By multivariate analysis, patients with high DPD expression had significantly shorter DFS (p = 0.007) and OS (p = 0.005) compared to patients with low DPD expression. In the combined analysis of 2 markers, patients with low TS and low DPD had the best outcome in terms of DFS (p = 0.007) and OS (p = 0.03). The analysis of all 3 proteins showed that the patients with low expression of all 3 markers had significantly longer DFS (p = 0.04) and OS (p = 0.01) than patients with a high value of any one of the protein expressions. However, the joint analysis of 3 markers (group with TS-/DPD-/TP-) could not identify a subgroup of patients with a better prognosis compared to the analysis of 2 markers (group with TS-/DPD-). The analysis of Dukes' stage C cancer patients confirmed a significant benefit in terms of DFS and OS (p = 0.001 and p = 0.006, respectively) when all 3 markers had low expression. We also found a positive significant correlation between TS and TP protein expression (p = 0.033). CONCLUSIONS: This retrospective investigation suggests that the combined assessment of TS and DPD may be useful to evaluate the prognosis of patients with Dukes' B and C colon carcinoma receiving 5-FU adjuvant chemotherapy. The role of TP as a predictor for 5-FU-based therapy needs further investigations.

Predictive value of thymidylate synthase expression in resected metastases of colorectal cancer.

Recent investigations have focused on the prognostic value of thymidylate synthase (TS) assessment in metastases of colorectal carcinoma (CRC). In order to evaluate the prognostic impact of TS expression after resection of metastases of colorectal cancer followed by systemic adjuvant chemotherapy, we performed an immunohistochemical characterisation of TS in the primary tumours and in the corresponding radically resected hepatic and pulmonary metastases. An additional objective was to compare the levels of TS in primary and metastatic disease. TS expression was assessed by immunohistochemistry using the monoclonal antibody TS 106. The study population consisted of 60 patients: 48 underwent liver and 12 lung resection. All of them received adjuvant chemotherapy after metastasectomy according to the Mayo Clinic schedule. In the 49 evaluable primary tumours, TS score was high in 53% and low in 47% of patients, while in the 60 metastatic samples TS immunostaining was high in 33% and low in 67%. There was a significantly smaller number of high TS expressors in metastatic than in primary tumours (P<0.04). No correlation was observed between TS expression and the site of the metastasis. TS status did not significantly correlate with the median disease-free interval (DFI) after metastasectomy, although this parameter was longer for patients with low TS immunoreactivity in the resected metastases than for those with high TS lesions (19.6 versus 13.8 months). Patients with high TS levels, however, had a significantly shorter median overall survival (OS) (27.6 months) than those with low TS expression (36.3 months) (P<0.008). TS status in the resected metastases confirmed its independent prognostic value in the multivariate analysis and was the only prognostic marker of OS in the subgroup of patients with resected liver metastases. These results suggest that high TS levels in resected metastases of colorectal cancer are associated with a poor outcome after surgery and 5-FU adjuvant therapy; therefore, a prospective assessment of TS levels in resected colorectal metastases could be useful to define which patients will most likely benefit from 5-FU adjuvant therapy after metastasectomy. Chemotherapeutic agents that target TS may not be the appropriate adjuvant treatment after metastasectomy for patients with a high TS expression in the resected metastases of colorectal cancer.

Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells.

We previously reported increased expression of p27Kip1 in a series of human breast cancer cell lines, as compared to cell lines established from normal mammary epithelial cells. These data were surprising because this protein exerts a growth-inhibitory function in normal cells, and p27Kip1 has been proposed as a candidate tumor suppressor gene. A possible explanation for the paradoxical increase in p27Kip1 in the breast cancer cell lines is that they had become refractory to the inhibitory effects of this protein. To address this question, here, we transfected the MCF7 breast cancer cell line and the MCF10F nontumorigenic mammary epithelial cell line with a vector containing the p27Kip1 cDNA to obtain derivatives that express increased levels of p27Kip1. The increased expression of p27Kip1 in both of these cell lines was associated with lengthening of the G1 phase, an increase in the doubling time, a decreased saturation density, and a decreased plating efficiency. In the MCF7 cells, anchorage-independent growth and in vivo tumorigenicity were also suppressed. These effects were associated with decreased cyclin E-associated in vitro kinase activity in both cell lines. The increased expression of p27Kip1 was associated with a decreased level of expression of cyclin D1 in the MCF10F cells but an increased level of the cyclin D1 protein in the MCF7 cell line. Both derivatives expressed slightly increased levels of the cyclin E protein. Thus, breast cancer cells are still responsive to p27Kip1-mediated inhibition of cell growth despite the high basal level of this protein. These results suggest that therapeutic strategies that further increase the level of expression of p27Kip1 or mimic its activity might be useful in cancer therapy.

Localization and expression of p27KIP1 in multistage colorectal carcinogenesis.

The cyclin-dependent kinase inhibitor p27Kip1 can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. However, our laboratory found that a variety of human cancer cell lines express relatively high levels of this protein and that this is often associated with increased expression of cyclin D1 or cyclin E. Therefore, in the present study we analyzed by immunohistochemistry the expression of p27Kip1 in a series of human tissue samples representing various stages of colon carcinogenesis, using 20 samples of normal colon mucosa, 20 hyperplastic polyps, 19 samples of adenomatous polyps, and 40 samples of various types of colorectal carcinomas. Parallel immunostaining was done for cyclin D1 and also for Ki67 to evaluate cell proliferation. An additional 17 human colon carcinoma samples, together with paired adjacent normal mucosa samples, were analyzed for levels of expression of the p27Kip1 protein by Western blot analysis, and 7 of these pairs of samples were examined by Northern blot analysis for levels of p27Kip1 mRNA. We did not find a positive or negative correlation between p27Kip1 expression and cell proliferation in the normal mucosa and tumor samples. There was, however, an inverse correlation between p27Kip1 and Ki67 expression in the lymphoid follicles present in the colonic mucosa. There was no evidence for a consistent increase or decrease in p27Kip1 expression in the mucosal cells during colon carcinogenesis, because the mean values for percentage p27Kip1-positive cells were similar in the normal mucosa, adenomatous polyps, and carcinoma samples. This is in contrast to Ki67 and cyclin D1 expression, which did show significant increases in mean values with tumor development. A subset (35%) of the carcinomas displayed diffuse cytoplasmic staining, in addition to nuclear staining, for p27Kip1, and in these cases the percentage of cells that were positive for p27Kip1 was higher than in cases that had only nuclear staining. There was a significant correlation between p27Kip1 expression and tumor grade; ie., well and moderately differentiated carcinomas had high p27Kip1 expression, whereas poorly differentiated carcinomas had lower expression. The Western blot analysis data on p27Kip1 expression confirmed this correlation. Comparisons of Northern and Western blots did not show a correlation between the level of p27Kip1 mRNA and the corresponding protein, a finding consistent with evidence that the p27Kip1 protein is regulated mainly via a posttranscriptional mechanism. The immunostaining studies revealed a significant correlation between high p27Kip1 protein expression and high cyclin D1 expression in the adenomatous polyps and in the subset of carcinomas that had only nuclear p27Kip1 expression. This may reflect the existence of a homeostatic feedback mechanism that is lost in the high-grade carcinomas that express low levels of p27Kip1.

P-170 Glycoprotein (MDR) and p53 Expression in Breast Cancer.

An immunohistochemical comparative analysis of P-170 glycoprotein and p53 was performed in 37 breast cancer patients who underwent curative resection without preoperative chemotherapy. The antibodies utilized were C-219 and JSB-1 for P-gp and DO-7 for p53. Positive cytoplasmic and membrance positivity for P-gp was found in 22 (59.45%) of the 37 tumor specimens. In comparing tissue immunoreactivity by the specific antibodies, 15 (40.54%) samples showed immunostaining for C-219, 12 (32.43%) for JSB-1, and 5 (23%) for both. P-gp expression was not statistically related with the clinicopathological variables analyzed, that is, age, TNM stage, histologic type, lymph node involvement, tumor diameter, and hormone receptor status. p53 overexpression was observed in 22 (59.45%) of the 37 tumor samples analyzed. There was a significant correlation between p53 overexpression and P-gp positive immunostaining (p = .007). p53 was also significantly correlated with TNM stage (p < .05) and lymph node involvement (p < .02). Our results demonstrated that the three distinct patterns of reactivity with the two antibodies result from the combined expression of each of the three P-gp isoforms. An immunohistochemical analysis with different antibodies may be used to determine and correlate the expression pattern of P-gp isoforms with response to chemotherapy.

Disorders in cell circuitry associated with multistage carcinogenesis: exploitable targets for cancer prevention and therapy.

The development of a malignant tumor involves the progressive acquisition of mutations and epigenetic abnormalities in multiple genes that have highly diverse functions. Some of these genes code for pathways of signal transduction that mediate the action of growth factors. The enzyme protein kinase C plays an important role in these events and in the process of tumor promotion. Therefore, we examined the effects of three inhibitors of protein kinase C, CGP 41251, RO 31-8220, and calphostin C, on human glioblastoma cells. These compounds inhibited growth and induced apoptosis; these activities were associated with a decrease in the level of CDC2 and cyclin B1/CDC2-associated kinase activity. This may explain why the treated cells accumulated in G2-M. In a separate series of studies, we examined abnormalities in cell cycle control genes in human cancer. We have found that cyclin D1 is frequently overexpressed in a variety of human cancers. Mechanistic studies indicate that cyclin D1 can play a critical role in carcinogenesis because: overexpression enhances cell transformation and tumorigenesis; introduction of an antisense cyclin D1 cDNA into either human esophageal or colon cancer cells reverts their malignant phenotype; and overexpression of cyclin D1 can enhance the amplification of other genes. The latter finding suggests that cyclin D1 can enhance genomic instability and, thereby, the process of tumor progression. Therefore, inhibitors of the function of cyclin D1 may be useful in both cancer chemoprevention and therapy. We obtained evidence for the existence of homeostatic feedback loops between cyclins D1 or E and the cell cycle inhibitory protein p27Kip1. On the basis of these and other findings, we hypothesize that, because of their disordered circuitry, cancer cells suffer from "gene addiction" and "gene hypersensitivity," disorders that might be exploited in both cancer prevention and therapy.

Deregulated expression of p27(Kip1) in human breast cancers.

Protein complexes composed of cyclins and cyclin-dependent kinases control the orderly progression of mammalian cells through the cell cycle. The p27(Kip1) protein belongs to a family of cyclin-dependent kinase-inhibitory proteins that are negative regulators of cell cycle progression and have been proposed as candidate tumor suppressor genes. However, the p27(Kip1) gene is only rarely mutated in human primary breast carcinomas and breast cancer cell lines. To further address the role of p27(Kip1) in the development of human tumors, we determined by Western blot analysis the levels of expression of the p27(Kip1) protein in a series of human cancer cell lines and found that this protein is expressed at high levels in many of these cell lines, even during exponential growth. The levels of p27(Kip1) were significantly associated with the levels of cyclins D1 and E. In contrast to the high level of p27(Kip1) in breast cancer cell lines, three cell lines established from normal mammary epithelium expressed low levels of this protein. Cell synchronization studies demonstrated deregulation of the expression of p27(Kip1) throughout the cell cycle in two breast cancer cell lines but normal regulation in a normal mammary epithelial cell line. Immunohistochemical studies on p27(Kip1) expression in 52 primary human breast cancers indicated that this protein was also expressed at relatively high levels in 44% of the tumor samples, but it was barely detectable or undetectable in the remaining 56% of the samples. Additional studies are required to determine why some breast cancer cells express relatively high levels of p27(Kip1) despite its known role as an inhibitor of cell cycle progression.