Introduction
The efficacy of 5-fluorouracil (5FU)-based adjuvant chemotherapy and the associated survival benefit have been firmly established for patients with stage III colorectal cancer (CRC) [
1]. However, the use of adjuvant therapy for stage II colon cancer patients remains controversial [
2]. Typically, only high-risk stage II CRCs are treated. Hence, there is a need for predictive factors to support treatment decisions in this group of patients, the majority of whom will be cured by surgery alone [
3]. This is especially important because a wider implementation of prophylactic programs in recent years has resulted in an increased number of patients with stage B2 tumors.
5-Fluorouracil has been used to treat colorectal cancer patients for a long time [
4]. The target enzyme for 5FU is thymidylate synthase (TS). This enzyme catalyzes the conversion of deoxyuridine-5′monophosphate to deoxythymidine-5′-monophosphate and is therefore essential for DNA synthesis and repair. Resistance to 5FU-based treatment might depend both on the level of expression of TS and other enzymes involved in the metabolism of 5FU and on mechanisms involved in cell growth or apoptosis. p21
WAF1 is a multifunctional cell cycle-related protein that inhibits cyclin-dependent kinases (CDKs), which results in cell cycle arrest in the G1 phase [
5]. p21
WAF1 expression is known to be inversely related to cell proliferation and directly related to terminal differentiation [
6]. Recently, it has been shown that TS expression was upregulated in p21
WAF1-/- human colorectal cancer HCT116 cells, and TS promoter activity was downregulated by ectopic p21 expression [
7]. Furthermore, a CDK inhibitor reduced expression of TS in a dose-dependent manner, and the reduction resulted in enhancement of sensitivity to 5FU in cultured human colon cancer cells [
8]. Taken together with other reports [
9], this suggests a great importance of p21
WAF1 for in vitro response to chemotherapeutic agents and that the CDK inhibitor p21
WAF1 regulates thymineless stress-induced DNA damage. Hence, p21
WAF1 expression might have a predictive significance in 5FU-treated CRC patients. In several [
10‐
19] but not all [
20‐
24] previous studies, positive p21
WAF1 expression has been suggested as an indicator of good prognosis in patients with stage III/IV CRC. However, it is not known whether the same is true for stage B2 patients. Therefore, our purpose was to assess the influence of p21
WAF1 expression on disease-free survival (DFS) and overall survival (OS) of Astler–Coller stage B2 patients with CRC who either were or were not treated with 5FU-based adjuvant chemotherapy, and of stage C patients treated with adjuvant chemotherapy.
Discussion
We found p21
WAF1 nuclear expression in 60.4% CRCs which is comparable to the literature data (16–87%) [
11,
13‐
15,
28,
29]. The majority of reports (but not all) concerning the prognostic role of p21
WAF1 expression in CRCs indicate better OS and/or DFS for patients with tumors showing p21
WAF1 expression (Table
4). In some of these reports, p21
WAF1 expression was an independent prognostic factor for OS and/or DFS (Table
4). However, in those reports, patients with various stages of the disease who were subjected to different treatment protocols (surgery alone, surgery and adjuvant chemotherapy, radiochemotherapy) were often grouped together for the analysis, or no information on the mode of therapy is given. Thus, it is difficult to infer whether stage II patients with p21
WAF1-positive tumors may receive any benefit from adjuvant chemotherapy.
Table 4
Major reports on p21WAF1 expression and survival of patients with CRCs
| n = 39 | Metastatic CRC 5FU CHTH | 5FU responders had greater p21 expression |
Ropponen K.M, et al. [ 11] | n = 162 | 0–D incl. 62–B majority: surgery only; 22–CHTH | Better OS and RFS for patients with p21+ tumors; p21: independent prognostic parameter for OS and RFS (MA) |
| n = 191 | I–IV CHTH? | ↓p21 → poor OS and DFS (UA) |
Bukholm I.K., et al. [ 13] | n = 61 | B–D CHTH? | Low p21 → increased risk of metastases and death |
| n = 294 | I–IV incl. 90–II. surgery CHTH? | Better OS for patients with p21+ tumors; p21 independent prognostic parameter (MA) |
Holland T.A., et al. [ 15] | n = 126 | A–D RCHTH? | Better OS for patients with high p21 expression |
Pasz-Walczak G., et al. [ 16] | n = 122 | I–IV CHTH? | Better OS for patients with p21+ tumors (UA) |
| n = 460 | II (B)–105 III (C)–355, 5FU CHTH | No association of p21 expression with survival in all and in stage B tumors |
| n = 100 | T2–3, N0 rectum, surgery only | No association of p21 expression with survival |
Schwandner O., et al. [ 17] | n = 160 | I–III rectum, surgery, n = 69 surgery + CHTH + RT, n = 91 | Better RFS but not OS for patients with p21+ tumors; p21 independent prognostic parameter for RFS but not OS (MA) |
| n = 66 | T3–4, N0–2, M0–1 rectum, RCHTH | No prognostic significance of pretreatment p21 expression. Post-treatment increase of p21: shorter DFS |
| n = 184 | I–IV incl. 55-III, III: n = 32 -5FU, n = 23-5FU + RT | Better OS for patients with p21 + tumors (MA) |
| n = 211 | B–D incl. 83-B CHTH for C–D | Better OS for patients with high p21 expression; p21 independent prognostic parameter (MA) |
| n = 97 | Dukes B, C, surgery, CHTH? | No association of p21 expression with OS and DFS |
| n = 116 | II–III RCHTH | Better RFS and OS for patients with p21- tumors |
The benefit of adjuvant 5FU-based chemotherapy has been firmly established for patients with stage III CRC. However, in the stage B2 group, it is not known whether the survival benefit from chemotherapy is sufficient to outweigh the toxicity and cost of the treatment [
2]. Only one study [
20] has addressed the issue of the influence of 5FU-based adjuvant chemotherapy on the survival of patients with stage B2 CRC in relation to p21
WAF1 expression. In that study, pretreatment levels of p21
WAF1 were not related to survival of patients with stage II (and III) CRC treated with adjuvant chemotherapy. However, increased levels of p21
WAF1 were associated with the sensitivity of metastatic CRC to 5FU-based chemotherapy [
10].
Our results indicate for the first time that p21
WAF1 expression in CRC tumor cells is an independent factor that is associated with favorable DFS and OS in patients with stage B2 tumors treated with 5FU-based adjuvant chemotherapy. Striking survival benefits were seen for stage B2 patients who received adjuvant chemotherapy compared with those who did not. Conversely, chemotherapy did not significantly influence DFS or OS of stage B2 patients with p21
WAF1-negative tumors. Rather, a statistically non-significant trend towards worse survival was observed for stage B2 patients with p21
WAF1-negative tumors treated with chemotherapy. The differences between our results and those of Watanabe et al. [
20] may be attributed to the different scoring systems and different cutoff points used for the interpretation of immunohistochemical staining, as well as to differences in immunohistochemical methods. The major advantage of tissue microarrays is that tens of cases can be processed in identical laboratory conditions which greatly improves the reproducibility of the immunohistochemical method. Prall et al. [
18] used tissue microarrays (
n = 184) and found better OS for combined group of stage I–IV patients with p21
WAF1-positive tumors. Our results (from the whole group) are well in accord with this report and give further support for the association of p21
WAF1 expression and longer survival of patients with stage C/III CRCs treated with adjuvant 5FU-based chemotherapy as has been reported previously (Table
4). One limitation of TMA technology is that “punched” cores from donor tissues may not always be representative of the entire tumor. In this report, we applied one core from carefully identified, histologically relatively homogenous area with the highest mitotic activity in the outer invasive zone of each CRC. Using this approach, we found 60.4% of p21
WAF1-positive CRCs which is within the range reported in the literature. Hoos et al. [
30] reported that correlations between phenotypes and clinical outcome were not significantly different between full sections and triplicate 0.6-mm core tissue microarrays. However, on the other hand, they were not significantly different when only one 0.6-mm core tissue microarray was used [
31]. The authors of the latter report conclude that tissue microarray “with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinico-pathological parameters, with no significant associated sampling bias.” So, careful sampling of the representative region of the tumor is regarded as the key step in the construction of tissue microarrays.
TS, the target enzyme for 5FU, is essential for DNA synthesis [
32], and it may function as an oncogene [
33]. Inhibition of TS induces apoptosis and cytotoxicity in human colorectal cancer cells [
7]. The importance of p21
WAF1 in the response of CRC to chemotherapeutic agents is supported by in vitro studies [
7‐
9]. It has been reported that p21
WAF1 (a CDK inhibitor) regulates thymineless stress-induced cytotoxicity of human colon carcinoma cell lines [
7]. Also, TS expression is mediated through the inhibition of CDK: TS expression was upregulated by the knockout of the p21
WAF1 gene in a CRC cell line [
8]. In addition, reduction of TS expression results in enhancement of the sensitivity to 5FU in human CRC cell lines [
8]. Our results are in line with other reports that show that p21
WAF1 is a critical mediator of the cytotoxic action of TS inhibitors in cultured human colorectal cancer cells [
7] and that CDK inhibitor enhances the sensitivity to 5FU in colorectal cancer cell lines [
8]. In fact, p21
WAF1 is required for maximal cytotoxicity induced by thymineless stress in colorectal cancer cells in culture [
7]. Poor survival of patients with p21
WAF1-negative tumors treated with adjuvant 5FU-based chemotherapy may perhaps be attributed to inherent resistance to 5FU. It has been shown that development of resistance to 5FU by colon cancer cell lines is associated with downregulation of the
CDKN1A gene, along with other genes engaged in DNA damage response/repair pathway [
34].
In summary, we found that p21WAF1 expression in CRC tumor cells identifies a subgroup of Astler–Coller stage B patients who would benefit significantly from 5FU-based chemotherapy and may therefore allow for better selection of patients for adjuvant chemotherapy. We believe that, in order to maximize the benefit of 5FU-based adjuvant therapy and to spare patients from unnecessary toxicity, stage B2 patients should be stratified according to p21WAF1 status. However, because this is a retrospective study, our results should be confirmed by further prospective randomized investigations.