1 Division of Medical Oncology, University of Udine, P. le S. M. Misericordia, Udine; 2 Department of Pathology, University of Udine, P. le S. M. Misericordia, Udine; 3 Division of Medical Oncology, S. Martino General Hospital, L. go R. Benzi 10, Genoa; 4 Division of Medical Oncology, Galliera General Hospital, Mura delle Cappuccine 14, Genoa, Italy
Received 25 June 2003; accepted 3 September 2003
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ABSTRACT |
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The level of the enzyme thymidylate synthase (TS) is known to inversely correlate with the clinical activity of 5-fluorouracil (FU) in advanced colorectal cancer patients. Since the correlation is not very strong, we have retrospectively analyzed the expression of E2F-1 in tumor samples or metastases from 25 patients with advanced colorectal cancer, homogeneously treated with an FU-based regimen. E2F-1 is a transcription factor regulating the expression of TS along with other crucial DNA synthesis related enzymes.
Materials and methods:
E2F-1 expression was analyzed by immunohistochemistry using the anti-E2F-1 monoclonal antibody KH95, scoring 2000 cells/case. Expression of TS was evaluated by immunohistochemistry using a rabbit anti-human polyclonal antibody.
Results:
The level of E2F-1 expression did not correlate with TS expression, although a trend for correlation between E2F-1 level and maximal tumor shrinkage was observed (r = 0.42; P = 0.054).
Conclusions:
In spite of previous reports demonstrating that E2F-1 quantified by rt-PCR and western blot correlates with TS and could be used as a predictor to select colorectal cancer patients more likely to respond to FU treatment, our data suggest that, under these experimental conditions, immunohistochemistry cannot be used for such selection.
Key words: colorectal cancer, E2F-1, immunohistochemistry, thymidylate synthase
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Introduction |
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Several molecules are under investigation as potential predictors for response to fluoropyrimidines. One of these is the transcription factor E2F-1, which controls the transcription of several genes encoding proteins involved in DNA synthesis, namely TS, dihydrofolate reductase, thymidine kinase, and ribonucleotide reductase (Figure 1) [57]. In vivo, a high correlation between E2F-1 and TS was recently shown in colorectal metastases to the lung and the liver, using quantitative rt-PCR [8].
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Materials and methods |
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TS immunohistochemical analysis
TS expression data were retrieved from previous analyses, performed as reported previously [3]. Briefly, tissue sections were heated in a microwave oven at 300 W for 10 min, cooled and stored in TBS at pH 7.6. To block non-specific binding of the primary antibody, a normal rabbit serum (DAKO X901; Dako, Glostrup, Denmark) dilution in TBS was used for 20 min. Sections were incubated with a rabbit polyclonal antibody to recombinant human TS (2 µg/ml) for 60 min in a humidified chamber at room temperature. Slides were then incubated with biotinylated swine anti-rabbit immunoglobulins for 20 min (DAKO-E353), followed by the avidinbiotinylated peroxidase complex for 30 min. After the color reaction product was developed with diaminobenzidine chromogen solution for 5 min, slides were counterstained with light hematoxylin for 1 min, dehydrated in a series of ethanols, cleared in xylene, and then examined under a light microscope. Only tumor cells with cytoplasmatic staining were counted as positive. TS expression was quantitated using a visual grading system based on the intensity of staining and classified into five groups from negative to very high intensity of staining. When heterogeneous levels of TS expression were found within a tumor (in multiple sections from different paraffin-embedded blocks of the same tumor), the level of TS expression of that lesion was defined according to the highest TS score.
E2F-1 expression
The expression of E2F-1 was retrospectively assessed by immunohistochemistry, following previously reported methods [9]. Briefly, sections were incubated in 0.01 M citrate buffer at pH 6.0 and heated twice in a microwave oven for 5 min per cycle. Staining was performed with the mouse anti-E2F-1 mAb KH95 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), used at 1:100 dilution. Reactivity was detected by an avidinbiotin immunoperoxidase detection system. Tonsil sections were processed in parallel, as a positive control. Counts were performed with a light microscope at 40x magnification, scoring 2000 epithelial cells/case. Only nuclear staining was considered positive, regardless of signal intensity. Tumor cells exhibiting brown reaction product were scored as positive, regardless of the staining intensity.
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Results |
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Discussion |
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In a search for easier methods of prediction of resistance or sensitivity, the immunohistochemical determination of E2F-1 seems particularly appealing.
Consistent with the biochemical cascade that leads to TS expression, a previous study showed that E2F-1 expression correlates with TS expression in colorectal cancer tissues, as determined by quantitative rt-PCR [8]. In addition, Banerjee et al. demonstrated that induced overexpression of E2F-1 in a human fibrosarcoma cell line results in increased levels of TS and resistance to FU [11]. Based on these studies, E2F-1 should be further investigated to confirm TS expression level in the selection of patients sensitive to FU.
The results of our study are negative both in terms of correlation between E2F-1 and TS, and prediction of treatment outcome. Aside from the small number of patients, there are at least two explanations for this potentially false-negative result. E2F-1 determination was done 2 years after the original TS determination and the effect of time could be substantial, as demonstrated for p53 [12]. As an alternative, immunohistochemistry does not discriminate between the acetylated and non-acetylated forms of E2F-1. As recently reported, E2F-1 activity is regulated by acetylation: the acetylated form of E2F-1 has an increased DNA-binding ability, a higher transactivation potential and an increased stability [13, 14]. The level of acetylated E2F-1 might correlate with TS and treatment outcome. Still, to quantitate the acetylated E2F-1, immunoblotting would be required, making this procedure time-consuming and expensive, and therefore not suitable for routine use.
In conclusion, in spite of the previously reported correlation between E2F-1 and TS expression by rt-PCR and western blot, suggesting E2F-1 as a predictor to select colorectal cancer patients more likely to respond to FU, our results suggest that under these experimental conditions immunohistochemistry may not be a reliable technique to assess E2F-1 level for such a selection.
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Acknowledgements |
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FOOTNOTES |
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REFERENCES |
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