Departments of 1 Medical Genetics, 2 Pathology and 6 Surgery, University Medical Center Groningen, Groningen; 3 Comprehensive Cancer Centre North Netherlands, Groningen, The Netherlands; 4 Department of Medical Oncology, University of Glasgow, Cancer Research UK Beatson Laboratories, Glasgow, UK; 5 Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands
* Correspondence to: Dr J. Plukker, Department of Surgery, UMCG, Hanzeplein 1, 9700RB, Groningen, The Netherlands. Tel: +31-50-3612317; Fax: +31-50-3614873; E-mail: j.th.plukker{at}chir.umcg.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and methods: TS and DPD protein expression was determined by immunohistochemical analysis using tissue microarrays of these colon tumours. Two hundred and twenty tumours had already been screened in a previous study for TP53 mutations.
Results: Low TS protein levels in primary stage III colon tumours appeared to be associated with mucinous histology and low DPD protein levels with young age at time of randomisation. Concordance between TS and DPD expression in primary and metastatic tumours was low. No associations were found between disease-free survival (DFS) and TS or DPD protein levels. When stratified by TP53 mutation status DFS did not differ with TS expression.
Conclusions: Expression of TS and DPD proteins is not predictive for survival in patients with stage III colon cancer treated adjuvantly with 5-FU regimens. TS protein levels did not alter the effect of TP53 mutation status.
Key words: colon cancer, dihydropyrimidine dehydrogenase, prediction 5-fluorouracil regimen, thymidylate synthase, TP53
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dihydropyrimidine dehydrogenase (DPD), the first and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism, is also important in the degradation and inactivation of 5-FU. DPD converts over 85% of clinically administered 5-FU into the inactive metabolite dihydrofluorouracil, a process that mainly occurs in the liver [5].
Different therapeutic strategies, including additional administration of leucovorin (5-formyltetrahydrofolate), have been explored to enhance the anticancer activity of 5-FU. Leucovorin increases the intracellular levels of reduced folate, which is necessary for optimal binding of FdUMP to TS, resulting in a prolonged inhibition of TS and subsequently of DNA synthesis [6]. Therefore, adding leucovorin to 5-FU is generally thought to improve the response to 5-FU and to prolong survival. TS protein also functions as an RNA-binding protein. It can bind to its own mRNA, that of c-myc and that of p53, thereby repressing translation [7
9
]. If FdUMP is bound to TS, no binding is possible to mRNA, resulting in an increase of TS, c-myc and p53 expression.
Literature is confusing regarding the prognostic or predictive value of TS and/or DPD. In the past, TS has been evaluated in patients who did not receive subsequent adjuvant chemotherapy. In such studies TS can be considered as prognostic and low TS values have been related to a longer survival [10]. However, when patients were adjuvantly treated with 5-FU-containing chemotherapy, high TS levels appeared to be predictive of a longer survival [10
13
], while others found no survival difference [14
16
]. The predictive value of TS in treatment of patients with advanced disease is more straightforward, since in most studies a low TS level was associated with a longer survival and/or a better response rate to 5-FU-containing therapy [17
19
]. However, this relationship appeared to be limited to TS levels determined in metastases, rather than in primary tumours [18
, 19
]. From these studies it can be concluded that the role of TS as a predictive factor is dependent on the type of disease to be treated with a 5-FU-containing regimen. Regarding the prognostic value of TS, data are relatively limited because of the low number of patients available for evaluation after receiving surgery alone. Regarding the role of DPD as a prognostic factor, the data are even more limited. Most studies focused on the role of DPD as a predictive marker for patients treated with a 5-FU-containing regimen. A low DPD level was generally related to either a longer survival or a better response rate [20
23
] to a 5-FU containing regimen. In combination, the predictive value of DPD was even better [12
, 23
]. In some studies DPD was not predictive or prognostic [24
, 25
], but in none of the reported studies was DPD a negative predictive parameter.
Since TP53 plays a crucial role in the cellular response to 5-FU-induced DNA damage and TP53 is found mutated in 50% of colorectal cancers, loss of functional p53 might reduce the response to 5-FU [26
, 27
]. Therefore, TS might play a pivotal role in regulating cell cycle checkpoints and apoptosis through regulation of p53 expression and other cell cycle-related proteins in response to 5-FU treatment. Suppression of p53 by TS may abrogate G1 phase arrest and subsequently DNA repair. It is still not clear whether this response to 5-FU regulated by TS is TP53 status dependent. Chu et al. [7
, 8
] showed in a series of studies that TS not only showed autoregulation of its translation, but also that p53 plays an important role in the regulation of translation of TS protein. This effect was dependent on the TP53 status, mutated or not. In a previous study, we found that presence of TP53 mutation in colon tumours was associated with a shorter disease-free survival (DFS) (P = 0.009). Therefore, we included the TP53 mutation status in our analysis.
In this study we wanted to (semi-)quantitatively investigate TS and DPD protein levels by immunohistochemical analysis of primary tumours and lymph node metastases from stage III colon cancer patients adjuvantly treated with 5-FU-based chemotherapy, on tissue microarrays. We analysed a homogenous group of patients treated with either 5-FUlevamisole or 5-FUlevamisoleleucovorin. Since leucovorin did not add to the outcome of treatment, these groups of patients can be considered as equivalent and form a large homogenous group suitable for evaluation of predictive parameters [28]. In addition we determined whether the use of leucovorin and TP53 mutation status of the primary tumour, both thought to be functionally linked to TS activity, altered the effect of TS protein level.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For all patients, clinical and tumour characteristics were derived from the clinical database maintained at the Comprehensive Cancer Centre North Netherlands. The mean age of all patients at the time of randomisation was 58.7 years (range 26.275.8). Two hundred and thirteen patients (54.5%) were males. One hundred and eighty-two tumours (46.5%) were right-sided, i.e. proximal of the splenic flexure. With regard to histological grade, 17.5% of the tumours were well differentiated, 57.3% moderately differentiated and 25.2% were poorly differentiated. According to the tumournodemetastasis (TNM) staging system, 13.6% of tumours were in stages T1 and T2 and 86.4% in stages T3 or T4. Furthermore, 74.2% of the resection specimens belonged to stage N1, while 101 (25.8%) were staged N2 [29]. Two hundred and twenty tumours had previously been screened for TP53 mutations using denaturing gradient gel electrophoresis (exons 48) and DNA sequencing [30
]. Of these, 116 showed presence of a TP53 mutation (Westra JL, Schaapveld M, Hollema H et al. J Clin Oncol 2005: 23; in press).
Tissue microarrays
Representative tumour tissue from all included patients were selected using haematoxylin and eosin-stained slides and arrayed into five tissue microarrays, constructed according to standard protocols [31]. Briefly, three 0.6-mm tissue cores were taken from representative regions of each tumour, using the manual tissue arrayer (MTA-I) from Beecher Instruments (Sun Prairie, WI, USA) and these were put into a standard-size recipient paraffin block, the tissue microarray block. Each array contained 322 tissue cores, representing 94 tumour samples in triplicate; 10 internal controls (i.e. the same on all five arrays) in duplicate; and 10 normal tissues in duplicate. In total, five arrays were made; four with primary tumour samples and one with matching lymph node metastases. Four-micrometre sections were cut from each array block, using the paraffin tape-transfer system with adhesive-coated slides from Instrumedics (Hackensack, NJ, USA).
Immunohistochemical analysis of TS and DPD
TS and DPD staining was performed as reported by Van Triest et al. [32] and Huang et al. [33
], respectively. Briefly, the sections were deparaffinised and rehydrated. Antigen retrieval was performed by heating the slides in a microwave oven at 750 W for 10 min in a 10 mM sodium citrate buffer (pH 6.0), followed by cooling to room temperature for 20 min. Subsequently the sections were washed, blocked and incubated for 1 h with the primary polyclonal TS (diluted at 1:100) or DPD (rabbit IgG, diluted at 1:500) antibody. They were then incubated for 1 h with the secondary antibody (biotinylated goat anti-rabbit IgG, diluted 1:300), followed by incubation with the horseradish peroxidase-labelled avidinbiotin complex for 30 min. The antibody binding was visualised with 3-amino-9-ethylcarbazole (AEC). Lastly, the sections were counterstained with haematoxylin.
The immunostaining sections of TS and DPD were scored using a visual grading system of four categories based on intensity (0 = no expression, 1 = low expression, 2 = moderate expression, 3 = strong expression). The score was determined by the highest degree of staining seen in the tumour tissue cores. Lymph node metastases were also put in triplicate on the tissue microarray to keep tissue loss as low as possible in view of intratumoural variation as well. Scoring was done the same way for primary tumour as for nodal metastases, using the average of the three samples.
Scoring was performed by two experienced observers without knowledge of the clinical outcome for the patients. For discordant cases the two observers reached consensus. Categories 0 and 1 were considered to represent low expression, and categories 2 and 3 to represent high expression (Table 1).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interpretable results for TS could be obtained in 90% (352 of 391) of the tumour samples. For DPD analysis, 87% (341 of 391) of the tumour samples were informative. Fourteen per cent (48 of 352) of the included samples showed low TS expression (combined categories 0 and 1) and 86% (304 of 352) of the primary tumour samples showed high TS expression (combined categories 2 and 3). For DPD, 26% (88 of 341) of the tumour samples presented with low expression, and 74% showed high expression. Results of immunohistochemical analyses for TS and DPD in combination with clinicopathological features are shown in Table 1. Low levels of TS protein were associated with mucinous histology (P = 0.04) and low levels of DPD protein were associated with younger age at time of randomisation (P = 0.01; low versus high DPD expression, 56.2 and 59.6 years, respectively). No associations with TS or DPD were found for any of the other clinicopathological characteristics.
Survival related to TS or DPD protein levels in primary tumours
No significant associations were found between TS or DPD expression levels and DFS (P = 0.17, Figure 1A; and P = 0.09, Figure 1B, respectively). Furthermore, we did not observe any combined effect of TS and DPD in relation to DFS (P = 0.19).
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In searching associations between clinical parameters and TS and DPD protein levels, an association between TS and mucinous histology was noted. For DPD, an association was found with the mean age at time of randomisation. Patients with a low intratumoural DPD protein expression were in general 3 years younger than those with a high intratumoural DPD expression (56.2 and 59.6 years, respectively). This raises the question of whether the suggested association of low DPD expression with longer survival [21
, 22
] is due to the low DPD level proper or to other factors associated with younger age.
We did not find any prognostic significance of TS expression. This is in agreement with several adjuvant studies [1416
]. Our study also showed no prognostic relevance of intratumoural DPD expression in adjuvantly treated patients with stage III colon cancer. This is in agreement with results published by Kornmann et al. [24
] and Ikeguchi et al. [25
], but is in contradiction to results published by others [21
23
]. A combined TS and DPD effect was not found in our study, probably due to many subgroups and the relatively large group of patients with high TS expression. We might exclude the possible existence of a strong combined effect, since it would have been detected even with small numbers.
Why are the results of the studies mentioned so different? The effect of adjuvant chemotherapy in stage III colon cancer is limited to 10% of the treated patients and only in these 10% is a predictive parameter of value, because the other patients would already have been cured by surgery alone. This may lead to a lack of statistical power to determine whether TS or DPD are predictive for survival of patients receiving adjuvant therapy. For treatment of patients with advanced disease the group to be evaluated is different, because all patients have cancer that is sensitive or resistant to chemotherapy. A partial explanation may be found in the way TS and DPD protein levels were determined. Another technical explanation might be the difference in antibodies used in the different reported studies, monoclonal (TS106 or RTSMA2) or polyclonal, as in our study, to recognise different epitopes [32
]. A clinical explanation might be the selection of patients that are very heterogeneous in the different studies, with respect to both tumour stage and tumour location (in colon or rectum), and with respect to treatment regimens (5-FU-based or other; bolus or infusional administration), in adjuvant settings or related to advanced disease. Comparison of results of the reported studies is therefore very difficult.
As mentioned, TP53 plays a central role in cell cycle control and apoptosis, and also seems to be important in the response to 5-FU-induced DNA damage under TS inhibition [26, 27
]. As 220 of our tumours had already been screened for TP53 mutations, we were able to investigate a possible combined effect of TP53 and TS. TS expression levels did not change the adverse prognostic effect of TP53 that we found earlier (Westra et al.). This absence of a TS effect does not suggest an interaction between TS and TP53, which is in line with results of others [34
, 35
], who found TS inhibition in response to 5-FU to be independent of TP53 status. It should be noted, however, that the group with low intratumoural TS expression is small and that analysis therefore lacks the power to confirm the hypothesis that the highest response to 5-FU, implying a longer survival, would be seen in the p53 wild-type cases in the low TS expressing group.
The trial in which our patients participated did not show a survival benefit when leucovorin was added to the combination of 5-FU and levamisole [28]. We found no effect of leucovorin on DFS of patients stratified by TS expression in their primary tumours.
When comparing expression levels of TS in primary tumours and lymph nodes, a low concordance of 59% was found, almost equal to the value expected by chance. This agrees with results reported by others [18, 36
]. We could not confirm that high TS levels in metastases predict for non-responsiveness to 5-FU, which normally leads to shorter survival [18
, 20
].
In conclusion, in this group of stage III colon cancer patients treated adjuvantly with an 5-FU-containing regimen, expression of TS and DPD proteins are not predictive for survival of patients. Concordance between TS and DPD protein levels determined in primary tumours and in the matching lymph node metastases was low. TS protein levels did not alter the effect of either adding leucovorin to the adjuvant treatment or the effect of TP53 mutation status.
![]() |
Acknowledgements |
---|
Received for publication March 18, 2005. Revision received June 6, 2005. Accepted for publication June 7, 2005.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. International Multicentre Pooled Analysis of Colon Cancer Trials (IMPACT) Investigators. Efficacy of adjuvant fluorouracil and folinic acid in colon cancer. Lancet 1995; 345: 939944.[CrossRef][ISI][Medline]
3. Pinedo HM, Peters GF. Fluorouracil: biochemistry and pharmacology. J Clin Oncol 1988; 6: 16531664.
4. Aschele C, Lonardi S, Monfardini S. Thymidylate synthase expression as a predictor of clinical response to fluoropyrimidine-based chemotherapy in advanced colorectal cancer. Cancer Treat Rev 2002; 28: 2747.[CrossRef][ISI][Medline]
5. Lu ZH, Zhang R, Diasio RB. Purification and characterization of dihydropyrimidine dehydrogenase from human liver. J Biol Chem 1992; 267: 1710217109.
6. Wright JE, Dreyfuss A, el-Magharbel I et al. Selective expansion of 5,10-methylenetetrahydrofolate pools and modulation of 5-fluorouracil antitumor activity by leucovorin in vivo. Cancer Res 1989; 49: 25922596.[Abstract]
7. Chu E, Takechi T, Jones KL et al. Thymidylate synthase binds to c-myc RNA in human colon cancer cells in vitro. Mol Cell Biol 1995; 15: 179185.[Abstract]
8. Chu E, Copur SM, Ju J et al. Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex. Mol Cell Biol 1999; 96: 15821594.
9. Ju J, Pedersen-Lane J, Maley F, Chu E. Regulation of p53 expression by thymidylate synthase. Proc Natl Acad Sci USA 1999; 96: 37693774.
10. Johnston PG, Fisher ER, Rockette HE et al. The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer. J Clin Oncol 1994; 12: 26402647.
11. Takenoue T, Nagawa H, Matsuda K et al. Relation between thymidylate synthase expression and survival in colon carcinoma, and determination of appropriate application of 5-fluorouracil by immunohistochemical method. Ann Surg Oncol 2000; 7: 193198.
12. Edler D, Glimelius B, Hallstrom M et al. Thymidylate synthase expression in colorectal cancer: A prognostic and predictive marker of benefit from adjuvant fluorouracil-based chemotherapy. J Clin Oncol 2002; 20: 17211728.
13. Kornmann M, Schwabe W, Sander S et al. Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression levels: predictors for survival in colorectal cancer patients receiving adjuvant 5-fluorouracil. Clin Cancer Res 2003; 9: 41164124.
14. Yamachika T, Nakanishi H, Inada K et al. A new prognostic factor for colorectal carcinoma, thymidylate synthase and its therapeutic significance. Cancer 1998; 82: 7077.[CrossRef][ISI][Medline]
15. Tomiak A, Vincent M, Earle CC et al. Thymidylate synthase expression in stage II and III colon cancer: a retrospective review. Am J Clin Oncol 2001; 24: 597602.[CrossRef][ISI][Medline]
16. Nanni O, Volpi A, Frassineti GL et al. Role of biological markers in the clinical outcome of colon cancer. Br J Cancer 2002; 87: 868875.[CrossRef][ISI][Medline]
17. Paradiso A, Simone G, Petroni S et al. Thymidilate synthase and p53 primary tumour expression as predictive factors for advanced colorectal cancer patients. Br J Cancer 2000; 82: 560567.[CrossRef][ISI][Medline]
18. Aschele C, Debernardis D, Tunesi G et al. Thymidylate synthase protein expression in primary colorectal cancer compared with the corresponding distant metastases and relationship with the clinical response to 5-fluorouracil. Clin Cancer Res 2000; 6: 47974802.
19. Johnston PG, Benson 3rd AB, Catalano P et al. Thymidylate synthase protein expression in primary colorectal cancer: lack of correlation with outcome and response to fluorouracil in metastatic disease sites. J Clin Oncol 2003; 21: 815819.
20. Salonga D, Danenberg KD, Johnson M et al. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 2000; 6: 13221327.
21. Tokunaga Y, Nakagami M, Tokuka A, Ohsumi K. Prognostic effects of dihydropyrimidine dehydrogenase in advanced colorectal cancer after surgery: immunohistochemistry using a new monoclonal antibody. Anticancer Res 2003; 23: 17631768.[ISI][Medline]
22. Tsuji T, Sawai T, Takeshita H et al. Tumor dihydropyrimidine dehydrogenase in stage II and III colorectal cancer: low level expression is a beneficial marker in oral-adjuvant chemotherapy, but is also a predictor for poor prognosis in patients treated with curative surgery alone. Cancer Lett 2004; 204: 97104.[CrossRef][ISI][Medline]
23. Ichikawa W, Uetake H, Shirota Y et al. Combination of dihydropyrimidine dehydrogenase and thymidylate synthase gene expressions in primary tumors as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer. Clin Cancer Res 2003; 9: 786791.
24. Kornmann M, Link KH, Galuba I et al. Association of time to recurrence with thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression in stage II and III colorectal cancer. J Gastrointest Surg 2002; 6: 331337.[CrossRef][ISI][Medline]
25. Ikeguchi M, Makino M, Kaibara N. Thymidine phosphorylase and dihydropyrimidine dehydrogenase activity in colorectal carcinoma and patients prognosis. Langenbecks Arch Surg 2002; 387: 240245.[CrossRef][ISI][Medline]
26. Lenz HJ, Hayashi K, Salonga D et al. p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival. Clin Cancer Res 1998; 4: 12431250.[Abstract]
27. Longley DB, Boyer J, Allen WL et al. The role of thymidylate synthase induction in modulating p53 regulated gene expression in response to 5-fluorouracil and antifolates. Cancer Res 2002; 62: 26442649.
28. Bleeker WA, Mulder NH, Hermans J et al. The addition of low-dose leucovorin to the combination of 5-fluorouracil-levamisole does not improve survival in the adjuvant treatment of Dukes' C colon cancer. Ann Oncol 2000; 11: 547552.[Abstract]
29. Sobin LH, Wittekind Ch (eds). UICC International Union Against Cancer TNM Classification of Malignant Tumors, 6th edition. New York: Wiley-Liss 2002.
30. Hayes VM, Bleeker W, Verlind E et al. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue. Diagn Mol Pathol 1999; 8: 210.[CrossRef][ISI][Medline]
31. Hoos A, Cordon-Cardo C. Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations. Lab Invest 2001; 81: 13311338.[ISI][Medline]
32. Van Triest B, Loftus BM, Pinedo HM et al. Thymidylate synthase expression in patients with colorectal carcinoma using a polyclonal thymidylate synthase antibody in comparison to the TS 106 monoclonal antibody. J Histochem Cytochem 2000; 48: 755760.
33. Huang CL, Yokomise H, Kobayashi S et al. Intratumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase in non-small cell lung cancer patients treated with 5-FU-based chemotherapy. Int J Oncol 2000; 17: 4754.[ISI][Medline]
34. Backus HH, Wouters D, Ferreira CG et al. Thymidylate synthase inhibition triggers apoptosis via caspases-8 and -9 in both wild-type and mutant p53 colon cancer cell lines. Eur J Cancer 2003; 39: 13101317.[CrossRef][ISI][Medline]
35. Longley DB, Latif T, Boyer J et al. The interaction of thymidylate synthase expression with p53-regulated signaling pathways in tumor cells. Semin Oncol 2003; 30 (Suppl 6): 39.[Medline]
36. Marsh S, McKay JA, Curran S et al. Primary colorectal tumour is not an accurate predictor of thymidylate synthase in lymph node metastasis. Oncol Rep 2002; 9: 231234.[ISI][Medline]
|