© 2004 Foundation for Promotion of Cancer Research
Immunohistochemical Evaluation of Thymidylate Synthase (TS) and p16INK4a in Advanced Colorectal Cancer: Implication of TS Expression in 5-FU-based Adjuvant Chemotherapy
Departments of 1 Pathology and 2 Surgery, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
For reprints and all correspondence: Shingo Kamoshida, Department of Pathology, Fujita Health University School of Medicine, 1–98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470–1192, Japan. E-mail: skamo{at}fujita-hu.ac.jp
Received May 11, 2004; accepted August 16, 2004
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Abstract |
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 TOP Abstract INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Our previous analyses on the expression of thymidylate synthase (TS) and p16INK4a in colorectal cancer patients administered 5-fluorouracil (5-FU) pre-operatively demonstrated that a high level of TS expression was a predictor of 5-FU resistance, and that the combination of a low level of TS expression and induction of p16INK4a after chemotherapy implicated chemosensitivity. The present study aimed to assess the relationship between the biological behavior of advanced colorectal cancer treated post-operatively by 5-FU-based chemotherapy and the expression of TS and p16INK4a in primary tumors.
Methods: Formalin-fixed, paraffin-embedded specimens from 132 colorectal cancers (Dukes' B, 36 cases; Dukes' C, 60 cases; and Dukes' D, 36 cases) treated by 5-FU post-operatively were immunostained for TS and p16INK4a. Antigenicities were suitably retrieved.
Results: Primary tumors expressing high levels of TS in the Dukes' C group showed a significantly shorter recurrence-free interval (RFI) (P = 0.0002). The overall survival (OS) was shorter in high TS expressors than in low TS expressors (P = 0.001). A high level of TS expression also correlated with advanced Dukes' staging and the severity of nodal metastasis (Dukes' B versus Dukes' D, P = 0.001; Dukes' C versus Dukes' D, P = 0.008; N0 versus N2, P = 0.002; N1 versus N2, P = 0.03). p16INK4a expression was not correlated with the prognosis or clinicopathological features.
Conclusions: Appropriate immunohistochemical evaluation is essentially important. We suggest that, in the Dukes' C group, a 5-FU-based regimen can be chosen as a first-line chemotherapy for low TS expressors. TS-high cancer should be treated with anti-cancer agents acting through different mechanisms. Further research should be conducted on applying TS immunostaining to the treatment strategy.
Key Words: thymidylate synthase • p16INK4a • colorectal cancer • 5-fluorouracil • adjuvant chemotherapy
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INTRODUCTION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Colorectal cancer, one of the most common cancers in both men and women, is a major cause of morbidity and mortality in developed countries (1). Fifty percent of the patients will die within 5 years of diagnosis, usually as a result of metastatic disease. 5-Fluorouracil (5-FU)-based adjuvant chemotherapy has become the most widely used treatment, but with a response rate of
20% (2). Since alternative treatment options are available, the identification of patients unresponsive to 5-FU will allow them to receive other anti-cancer regimens, and thus to avoid unpleasant side effects. The active metabolite of 5-FU, fluorodeoxyuridine monophosphate (FdUMP), inhibits thymidylate synthase (TS), the enzyme essential for the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate in the DNA synthesis pathway (3). Multiple pre-clinical studies have suggested that high TS levels are closely associated with the 5-FU resistance of cancer cells (4,5). It has been reported that immunohistochemical expression of TS reflects the TS enzyme activity in colorectal tumors (6). The relationship between TS expression in primary colorectal cancer and the response to chemotherapy has thus been investigated extensively, but the studies have revealed conflicting results. Several investigators have shown that the TS expression in the primary colorectal tumor is of limited value in predicting the clinical response to 5-FU-based adjuvant chemotherapy (7–12). In contrast, several reports have documented the clinical relevance of low TS expression as a predictor of the response to 5-FU-based chemotherapy and the long survival of the patients with distant metastasis (13–15).
p16INK4a protein, a product of the CDKN2/MTS1 gene located on chromosome 9 (9p21), is a member of the INK4 family of cyclin-dependent kinase (CDK) inhibitors (16). p16INK4a binds preferentially to CDK4 and CDK6, inhibiting their association with cyclin D1 and subsequent phosphorylation of the retinoblastoma tumor suppressor protein which allows the dissociation of E2F and subsequent transit to S phase. A high level of expression of p16INK4a results in G1 arrest, and inactivation of p16INK4a is associated with uncontrolled cell growth. These findings suggest that p16INK4a acts not only as a cell cycle regulator but also as a tumor suppressor (17–20). Inactivation of p16INK4a occurs in a significant proportion (18–53%) of colorectal cancer, preferentially through de novo methylation of its 5'-promoter-associated CpG island (21–24). The correlation between p16INK4a methylation and shorter survival in colorectal cancer has been reported, although this was statistically insignificant (22). Recently, Zhao et al. reported that strong immunostaining of p16INK4a was a predictor for a better prognosis of colorectal cancer patients (25). However, they described that the localization of p16INK4a was almost always ‘cytoplasmic’, which is inconsistent with most previous reports.
We recently assessed immunohistochemical expression of TS and p16INK4a in advanced colorectal cancer, treated pre-operatively with 5-FU derivatives (26,27). It was suggested that a high level of TS expression was a predictor of the resistance to 5-FU-based adjuvant chemotherapy, and that the combination of low expression of TS and induction of p16INK4a after chemotherapy was indicative of chemosensitivity. The present study was designed to investigate immunohistochemically the relationship between the prognosis of colorectal cancer patients receiving 5-FU-based adjuvant chemotherapy and the expression of TS and p16INK4a in primary tumors.
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SUBJECTS AND METHODS |
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PATIENT POPULATION AND SAMPLE PREPARATION
The study population consisted of 132 patients (80 men and 52 women; mean age 58.9 years with a range from 27 to 77 years; median 59), culled from the list of colorectal cancer patients in the Department of Surgery, Fujita Health University School of Medicine, Toyoake. All patients had undergone surgical resection of advanced colorectal cancer during the period from 1991 to 2002, and received 5-FU-based adjuvant chemotherapy post-operatively. In principle, we chose the patients with a known prognosis and a clinical follow-up for >5 years. A total of 35 patients who died in the period 1999–2002 are included in the study. The 5-FU-based regimens included tegafur and uracil (UFT) following 5-FU, 94 cases; UFT alone, 15 cases; 5-FU alone, nine cases; 5-FU by hepatic artery infusion (HAI), six cases, 1-hexylcarbamoyl-5-fluorouracil (HCFU), four cases; 5-FU with leucovolin, two cases; 5-FU with mitomycin C, one case; and 5-FU with radiation, one case. The clinicopathological features are summarized in Table 1. The histological grade, depth of invasion and lymph node metastasis were classified according to the TNM Classification of Malignant Tumors (28). Based upon the TNM classification, extranodal soft tissue cancer deposits were regarded as nodal metastasis. The recurrence-free interval (RFI) of the patients was calculated as the number of months from the date of primary tumor resection to the date of confirmation of recurrence. The overall survival (OS), the number of months from the date of primary tumor resection to death or the date of the last follow-up evaluation was available in 113 of the 132 patients. The present study was approved by the institutional ethical review board for human investigation at Fujita Health University on August 3, 2004.
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Surgically resected primary tumors were routinely fixed in 10% formalin and embedded in paraffin wax. Lymph node metastases from 58 patients in Dukes' C stage were also evaluated similarly. Sections were cut consecutively and mounted on aminopropyltriethoxysilane slides, and stained with hematoxylin and eosin (HE).
IMMUNOHISTOCHEMISTRY
The specimens examined (one block or two) contained tumor–non-tumor junction, invasive edge and viable tumor. For evaluation of the nodal metastasis, one block containing the largest lesion was selected. Paraffin sections were examined for the expression of TS and p16INK4a, by employing the universal immuno-enzyme polymer method (Histofine® Simple Stain MAX-PO; Nichirei, Tokyo, Japan). Endogenous peroxidase was inactivated by 0.03% hydrogen peroxide in methanol for 30 min. To retrieve the antigenicity, hydrated heating at 120°C was performed in a pressure cooker (Delicio 6L; T-FAL, Rumily, France) for 10 min. Optimal soaking solutions were selected for the respective markers determined by preliminary experiments; 1 mM EDTA solution, pH 8.0 for TS, and 10 mM citrate buffer, pH 7.0 for p16INK4a (26,27,29). After pressure cooking, the sections were left at room temperature to cool in the soaking solution for 30 min. The primary antibodies used included anti-TS (rabbit polyclonal; Taiho Pharmaceutical, Saitama, Japan) at a 1:200 dilution and anti-p16INK4a (mouse monoclonal, clone 6H12; Novocastra Laboratories, Newcastle, UK) at a 1:20 dilution. The reaction products were visualized in 50 mg/dl 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.003% hydrogen peroxide. Negative control studies were performed without applying the primary antibody. Sections known to be stained positively were included in each run as a positive control.
The HE-stained and immunostained sections were reviewed independently by two investigators (S.K. and R.S.), without prior knowledge of the patients' outcome. For the evaluation of p16INK4a immunoreactivity, nuclear staining of the target cells was regarded as positive, regardless of cytoplasmic staining. As previously described (26,27), immunostained sections were classified into two groups based on the proportion of positively stained tumor cells in the lesion: low expression (negative or positive but in <30% of tumor cells) versus high expression (positive in 
30% of tumor cells). According to the principle of immunohistochemical evaluation using multiple clinical samples, the intensity of staining was not considered for the judgment of positivity.
STATISTICAL ANALYSIS
The association between the expression of TS and p16INK4a and clinicopathological features was analyzed by Fisher's exact probability test or the 
2 test. Categories consisting of <5% (i.e. 
7 cases) of the total number of cases were combined into appropriate categories (depth of invasion T2 and T3), united together with the other items (metastatic sites such as the bone, lymph node, peritoneum and mediastinum, and 5-FU-based regimens such as 5-FU by HAI, HCFU, 5-FU with leucovolin, 5-FU with mitomycin C and 5-FU with radiation) or excluded from analysis (G3 in histological grade). The TS expression in the primary and nodal metastatic lesions was analyzed by Fisher's exact probability test. The RFI and the OS curves were analyzed by the Kaplan–Meier method. The associations of prognostic variables with the patients' prognosis were evaluated using the log-rank test for univariate analysis and the Cox proportional hazard model for multivariate analysis. The statistical significance was defined as P < 0.05.
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RESULTS |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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The expression of TS was low in 90 tumors (68%) and high in 42 (32%), and p16INK4a expression was low in 87 tumors (66%) and high in 45 (34%). TS immunoreactivity was invariably demonstrated in the cytoplasm of cancer cells. TS was expressed in non-neoplastic crypt epithelial cells, germinal center lymphocytes, plasma cells, endothelial cells, fibroblasts and smooth muscle cells, even when the cancer cells were TS negative. p16INK4a usually showed simultaneous distribution in both the nuclei and cytoplasm. Cytoplasmic expression of p16INK4a without nuclear staining was observed focally in 16 lesions. p16INK4a expression was seen occasionally in part of non-neoplastic crypt epithelial cells, and also in macrophages, fibroblasts and endothelial cells. p16INK4a occasionally showed predominant positivity in the invasive component of the tumor, while TS scarcely demonstrated such an intratumor heterogeneity of expression. The cancer tissues expressing high levels of both TS and p16INK4a frequently showed a reciprocal antigen localization pattern: in other words, the cancer cells expressing TS were p16INK4a negative, and vice versa (Fig. 1).
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The significant relationship between the expression of TS and p16INK4a and clinicopathological features are summarized in Table 1. In patients with Dukes' stage D or with N2 lymph nodal metastasis, the cancer showed significantly higher levels of TS expression in comparison with the patients in other stages. High levels of TS expression were observed in eight (19%) of 43 tumors without lymph node metastasis (N0), 14 (27%) of 51 with metastasis in 1–3 lymph nodes (N1) and 20 (53%) of 38 with metastasis in four or more lymph nodes (N2) (N0 versus N2, P = 0.002; N1 versus N2, P = 0.03). A high level of TS expression was seen in six (17%) of 36 Dukes' B tumors, 16 (27%) of 60 Dukes' C tumors and 20 (56%) of 36 Dukes' D tumors (Dukes' B versus Dukes' D, P = 0.001; Dukes' C versus Dukes' D, P = 0.008). There was no significant association of TS expression with the patient's age, gender, tumor location, histological grade, depth of invasion, synchronous or metachronous metastasis, site of metastasis and regimens administered. No correlation was found between the expression of p16INK4a and any of the clinicopathological parameters.
Figure 2 shows the relationship between the expression of TS and p16INK4a in primary tumors and the RFI. TS expression was closely correlated with the RFI. The RFI was significantly shorter in patients with a high level of TS expression than in low TS expressors (P < 0.0001). Figure 3 demonstrates the relationship between TS expression and the RFI, according to Dukes' staging. In the Dukes' C group, a high level of TS expression exhibited a significant correlation with a short RFI (P = 0.0002; Fig. 1A). In Fig. 4, a representative TS-negative tumor showing a long RFI is shown. In the Dukes' B group, there was no such association between TS expression and recurrence. The expression of p16INK4a was unrelated to the recurrence. When the expression pattern of TS and p16INK4a was combined, no specific association was demonstrated in any phenotype, including TS-low/p16INK4a-low, TS-low/p16INK4a-high, TS-high/p16INK4a-low and TS-high/p16INK4a-high.
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TS expression was low in 38 (66%) and high in 20 (34%) of the 58 nodal metastatic lesions in the Dukes' C group. TS expression in the nodal metastatic lesions was compared with that in the primary tumors, as follows: 48 (83%) lesions revealed comparable TS expression, while nodal TS expression was lower in two (3%) and higher in eight (14%). Figure 5 illustrates the relationship between TS expression in the nodal metastatic lesions and the RFI. The RFI tended to be shorter in the TS-high group than in the TS-low group, though the difference was not significant (P = 0.08). A representative metastatic lesion with a high level of TS expression is demonstrated in Fig. 6.
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The relationship between the expression of TS and p16INK4a in the primary tumors and the OS is demonstrated in Fig. 7. Patients with a low level of TS expression exhibited a significantly better OS than high TS expressors (P = 0.001). Regarding p16INK4a expression, no significant correlation was noted.
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In the multivariate Cox analysis, TS expression in the primary tumors remained as a predictive value for the RFI and the OS (P = 0.002 and P = 0.005, respectively) (Table 2). The independence was kept when the analysis was limited to the Dukes' C group (RFI, P = 0.002). p16INK4a expression revealed no prognostic significance. Dukes' staging was a significant predictor for the OS (P = 0.002).
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The expression of TS in the primary tumors and the tumor recurrence or the OS of the patients was significantly correlated, even when eight cases treated with HCFU, 5-FU with leucovolin, 5-FU with mitomycin C or 5-FU with radiation were excluded from the analysis (data not shown).
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DISCUSSION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Our previous immunohistochemical analysis of TS and p16INK4a in 37 colorectal cancers administrated 5-FU derivatives pre-operatively demonstrated that high levels of TS expression were observed in 71% of the non-responders, but none in the responders (26). The TS-low/p16INK4a-high phenotype was seen in 83% of the responders but only in 3% of the non-responders, and the responders commonly showed the induced expression of p16INK4a after pre-operative chemotherapy (27). These results indicated that high TS expression was a predictor of the resistance to 5-FU-based neoadjuvant chemotherapy, and that the combination of low expression of TS and induction of p16INK4a following chemotherapy was indicative, but unfortunately not predictive, of the sensitivity to 5-FU derivatives in colorectal cancers. In the present study, we performed the immunohistochemical analysis on the expression of TS and p16INK4a in the primary lesion of advanced colorectal cancer treated post-operatively with 5-FU-based adjuvant chemotherapy.
The high level of expression of TS in the primary colorectal cancer was suggestive of a short RFI in the Dukes' C group. In general, Dukes' C tumors should be a suitable target of 5-FU-based adjuvant chemotherapy (30). Berglund et al. (10) and Cascinu et al. (31) reported that high levels of TS expression significantly predicted a short period of recurrence after 5-FU-based adjuvant chemotherapy, but others failed to demonstrate such a significant relationship (7–9,11,12). TS expression was correlated between the primary and nodal metastatic lesions. In the Dukes' C group, the RFI tended to be shorter in high TS expressors, although this difference was not significant statistically. Unlike our results, a previous study using 42 paired samples showed significant discordance (36%) of TS expression between the primary tumors and the nodal metastatic lesions (32). No predictive significance of TS expression was detected in the Dukes' B group. At the present time, no sufficient prognostic marker of high-risk Dukes' B colorectal cancer is thus available (33).
The expression of TS was also closely correlated with the OS of the patients. The OS was significantly shorter in patients with high levels of TS expression than in low TS expressors. So far, investigators have suggested that the TS level in the primary colorectal cancer is not significantly predictive of the clinical response to 5-FU-based adjuvant chemotherapy and the OS (7–12). We believe that such a discrepancy is due largely to the technical aspect of immunostaining, including the method of antigen retrieval. Hydrated heating is a useful and powerful pre-treatment for retrieving the antigenicity of a variety of tissue antigens, being the key step in reproducible and reliable immunohistochemical staining (34). The effect on antigen retrieval is dependent upon the soaking solution and the method of heating. We reported that the best condition of antigen retrieval for TS localization in archival paraffin sections was pressure cooking in 1 mM EDTA solution, pH 8.0, for 10 min (29). From a technical point of view, Paradiso et al. employed microwaving in 10 mM citrate buffer, pH 6.0 (8), a condition of low reliability, we believe. Microwaving was employed but soaking solution was not specified in two reports (9,32). No pre-treatments for antigen retrieval were performed in the remaining reports (10–12). We often experience difficulty in suitably demonstrating TS when the immunostaining is performed under these non-suitable conditions.
It is of note that a high level of TS expression was seen frequently in primary tumors of the advanced stage of Dukes' classification (Dukes' D) and in tumors with extensive lymph nodal metastasis (N2). The cancer cells expressing high levels of TS may show a high potential of both hematogeneous and lymphatic metastases. Our previous analysis on the expression of TS and p16INK4a in resected colorectal cancers administered 5-FU pre-operatively failed to show a correlation between the antigen expression and clinicopathological parameters (27). Lack of correlation may have resulted from a limited number of patients analyzed, and also probably from considerable tumor shrinkage (possible down-staging) by the neoadjuvant chemotherapy in the responders. Johnston et al. (35) and Edler et al. (6) reported a significantly high level of TS expression in colorectal cancer in advanced Dukes' stage, but a recent report by Berglund et al. failed to detect a significant difference (10). To our knowledge, there have been no reports concerning the correlation between high levels of TS expression and the degree of nodal metastasis.
No correlation was observed between p16INK4a expression and the prognosis or clinicopathological features of colorectal cancer. Methylation of the p16INK4a gene has been observed in short-term survivors of colorectal cancer, although the difference was not statistically significant (22). Recently, Zhao et al. reported strong immunostaining of p16INK4a as a predictor for a better prognosis of colorectal cancer (25). However, they described that the localization of p16INK4a was almost always ‘cytoplasmic’, being inconsistent with most other reports. Hibi et al. failed to show a significant difference between p16INK4a methylation and clinicopathological features, such as the patient's age, gender, tumor location, lymph node metastasis, depth of invasion and Dukes' staging (24). Our previous study demonstrated that 5-FU-related induction of p16INK4a in TS-low tumors suggested the efficacy of 5-FU-based chemotherapy, though it was not predictable. We have examined the immunohistochemical expression of the Polycomb group oncogene Bmi-1, which acts as a transcriptional repressor of the INK4a locus (36), in biopsy and resected specimens of advanced colorectal cancer, treated pre-operatively with 5-FU derivatives. The expression of Bmi-1, however, was not predictive of the effect of 5-FU treatment (data not shown).
Tailor-made chemotherapy on the basis of TS expression is attractive. The situation is similar to hormone therapy based on estrogen or progesterone receptor determination or trastuzumab administration based on HER2 expression in breast cancer (38). In conclusion, the results of both univariate and multivariate analyses demonstrated that a high level of TS expression was an important predictor of unfavorable RFI and OS for colorectal cancer patients treated with 5-FU-based adjuvant chemotherapy. We believe that in high TS expressors, 5-FU administration is not suitable and chemotherapeutic agents other than 5-FU or a humanized monoclonal antibody cetuximab against the epidermal growth factor receptor (EGFR) should be chosen (38,39). In order to give a definitive answer as to whether or not TS expression can be used as a predictive marker for a routine use, however, randomized prospective and retrospective studies must be conducted using a large number of cases, employing our highly sensitive immunostaining sequence. Finally, the identification of factors inducing p16INK4a expression after 5-FU treatment may contribute to accurate selection of colorectal cancer responsive to 5-FU-based chemotherapy.
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References |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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