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Thymidylate Synthase Expression: An Independent Prognostic Factor for Local Recurrence, Distant Metastasis, Disease-free and Overall Survival in Rectal Cancer¹

Department of Surgery, South Hospital, S-11883 Stockholm, Sweden [D. E., I. M.]; Department of Oncology, Karolinska Hospital, S-171 76 Stockholm, Sweden [M. H., P. R., H. B.]; and Department of Oncology, Queens University of Belfast, Belfast, BT97AB Northern Ireland [P. G. J.]

	ABSTRACT

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Several studies have suggested that the intratumoral level of thymidylate synthase (TS) in colorectal tumors correlates with survival. We have studied the correlation between TS expression in primary rectal cancer and locoregional recurrence, distant metastases, and survival.

TS enzyme levels were evaluated immunohistochemically using the specific monoclonal antibody TS 106 in paraffin-embedded tumors from 243 patients who had undergone primary surgery for rectal cancer during the years 1980–1993. All patients were included in prospective randomized trials aimed at determining the clinical value of a short preoperative course of local radiation therapy (five doses of 5 Gy each).

With a median follow-up of 94 months (range, 43–202 months), it was observed by multivariate analysis that Dukes’ stage and TS expression were independent prognostic markers of locoregional recurrence (P < 0.001 and P = 0.038, respectively) distant metastasis (P < 0.001 and P = 0.011, respectively) disease-free survival (P < 0.001 and 0.014, respectively), and overall survival (P < 0.001 and 0.020, respectively). By multivariate analysis, preoperative irradiation therapy showed a borderline improvement in locoregional recurrence (P = 0.051). No other factors, such as age, sex, differentiation of the tumor, or p53 expression, were noted to be independent prognostic factors for clinical outcome in these patients.

We concluded that the intratumoral expression of TS in primary rectal cancer is an independent prognostic factor for locoregional recurrence, distant metastases, disease-free survival, and overall survival. Patients with low intratumoral TS expression had a significantly better outcome than those with high TS expression.

	INTRODUCTION

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Several studies have suggested that intratumoral levels of TS,³ which plays a key role in the biosynthesis of DNA, correlate with the clinical course in patients with colorectal cancer (1, 2, 3, 4, 5, 6) . Moreover, the enzyme is considered to be an important target for 5-FU, which is the most widely used chemotherapeutic agent against this tumor disease. Therefore, it is possible that the intratumoral concentration of TS may act as an indicator of clinical response to 5-FU-based chemotherapy.

Intratumoral levels of TS can be measured using different techniques (2 , 7, 8, 9, 10) . Johnston et al. (1) have earlier reported that TS expression in rectal tumors may be of prognostic value in a manner independent of Dukes’ stage and that TS expression is also predictive for response to 5-FU-based therapy.

Rectal adenocarcinoma is a major health problem in the Western world, with about 36,000 new cases every year in the United States (11) and about 70,000 in Europe (12) . It represents a severe problem due to the high risk of locoregional recurrence and development of distant metastasis. The reported local recurrence rate varies from 4 to 40% (13, 14, 15, 16, 17, 18, 19, 20) . Recent prospective randomized trials have shown that a course of preoperative local irradiation shortly before surgery may significantly reduce the local recurrence rate and possibly also prolong survival (15, 16, 17, 18) . The primary aim of the present investigation was to assess whether intratumoral levels of TS, as determined immunohistochemically with TS 106 monoclonal antibodies, may predict for the risk of locoregional recurrence, distant metastasis, disease-free survival, and overall survival following surgery for rectal cancer. We also wished to establish the possible prognostic value of intratumoral expression of p53. The examined tumors were obtained from patients who were included in two prospective randomized trials during the years 1980–1993 aimed at determining the clinical value of preoperative irradiation (15, 16) .

	PATIENTS AND METHODS

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Patients.
The primary tumors of 243 patients with rectal cancer have been examined. The patients were included in multicenter trials in Stockholm, Sweden, aimed at determining the clinical value of preoperative irradiation. Following a biopsy confirming the diagnosis of rectal adenocarcinoma, patients without signs of distant metastasis were randomized to either preoperative irradiation followed by surgery or surgery only.

In the first trial, which included a total of 849 patients during the years 1980–1987, large radiation fields were used (15) . In the second trial, which included a total of 557 patients during the years 1987–1993, relatively small radiation fields were used (16) .

In the present study, 114 patients were obtained from the first trial (consecutively curatively operated at one hospital) and 129 from the second trial (consecutively curatively operated at three hospitals).

The median age of the patients was 69 years (range, 24–84 years), and median follow-up was 94 months (range, 43–202 months). The characteristics of patients and tumors are presented in Table 1 .

After histopathological examination, the cancer was classified according to Dukes’ stage (21) , and the differentiation state of the tumors was classified as high, moderate, or low.

Tissue Samples.
Paraffin-embedded, formalin-fixed specimens of resected tumors were analyzed immunohistochemically. Two sections (4 µm thick), taken from different parts of a primary tumor, and sections from lymph node metastases were analyzed for TS expression and p53 overexpression.

Expression of TS and p53.
The monoclonal antibody TS 106 (10) was used to detect TS and the monoclonal antibody DO-7 (Dakopatts, Glostrup, Denmark; Ref. 22 ) was used to detect p53. The standard avidin-biotin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Inc, Burlingame, CA; Ref. 23 ) technique was used. Tumor sections were deparaffinized in xylene and then hydrated in decreasing concentrations of ethanol. To quench the endogenous peroxidase activity, the sections were incubated in a solution of 3% hydrogen peroxide for 10 min. For reduction of nonspecific background staining, the sections were exposed to 20% horse serum for 30 min (sections to be examined for p53 expression had been pretreated in a microwave oven). Sections were incubated with TS 106 monoclonals at room temperature for 90 min for TS staining and and with DO7 overnight at 4°C for p53 staining. After rinsing, biotinylated horse antimouse secondary antibodies were applied for 30 min, followed by further washing, and then were incubated with avidin-biotin peroxidase complexes. Immunostaining was developed by immersion in 0.05% 3,3'-diaminobenzidine tetrahydrocloride and then counterstaining with a modified Harris hematoxylin.

Scoring of Immunohistochemical Staining.
Each time a set of tumor samples was stained, we included positive and negative reference slices from tumors that were previously classified as low- or high-intensity staining for TS expression and negative or positive for p53 expression. The intensity of TS-staining of the tumor cells was arbitrarily graded from 0 to 3. As described previously, 0 and 1 were defined as low intensity, and 2 and 3 were defined as high-intensity staining (1) . The highest staining intensity (low/high) found in a tumor was used for classification of the tumor, even if the area with high staining was small. Tumors were scored as positive for p53 overexpression if more than 5% of the tumor cells showed immunoreactivity. The agreement in scoring of TS intensity and p53 expression reached by two independent observers was more than 90%. In case of disagreement, intensity was determined by consensus.

Heterogeneity in TS expression was defined as a difference in TS expression (high/low) in the two sections taken from different parts of the primary tumor.

Statistical Analysis.
The Gehan-Wilcoxon univariate test (24) was used to examine the possible relationships between disease-free survival, overall survival, locoregional recurrence, or occurrence of distant metastasis and sex, Dukes’ stage, site of tumor, tumor differentiation, TS expression, and p53 expression. According to National Surgical Adjuvant Breast and Bowel Project criteria (25) , when disease-free survival was used as an end point, an event included recurrence of disease, death from cancer, death from noncancer causes, and the occurrence of a second noncolorectal primary. Multivariate analysis was performed using Cox regression (26) . Survival curves were constructed using the Kaplan-Meier method (27) . The Spearman correlation test was used to determine the correlations between TS expression in primary tumor and metastases and between TS expression and Dukes’ stage.

	RESULTS

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The characteristics of the patients in the present study (n = 243) did not differ from the overall study population (n = 1406) with respect to sex, age, stage of disease, differentiation of tumor, or percentage receiving radiation therapy treatment (15 , 16) .

Surgery was considered curative by both the surgeon and the pathologist in all 243 patients presented in this report. Preoperative irradiation was given to 117 patients, and 126 were treated with surgery only.

TS expression was homogenous (the same TS expression in the two different parts of a tumor) in 162 of 221 (73%) tumors.

By immunohistochemical analysis, 152 tumors (63%) had high intratumoral TS expression, and 91 (37%) had low TS expression.

TS Expression and Locoregional Recurrence.
During the follow-up period (median, 94 months; range, 43–202 months), 61 (25%) of the 243 patients who received curative surgery developed locoregional tumor recurrence. By univariate analysis, locoregional recurrence was significantly linked to Dukes’ stage, TS expression, and preoperative radiotherapy (Table 1) . Sex, tumor cell differentiation, and p53 expression did not correlate with the development of locoregional recurrence (Table 1) . By multivariate analysis, only Dukes’ stage (P < 0.001) and TS expression (P = 0.038) remained independent prognostic factors for locoregional recurrence (Table 1) . By multivariate analysis, preoperative radiation therapy showed a borderline (P = 0.051) correlation to locoregional recurrence (Table 1) .

TS Expression and Distant Metastasis.
Distant metastases occurred in 64 (26%) of the 243 patients (Table 2) . Twenty-seven of the 243 patients (11%) had both locoregional recurrence and distant metastatic spread. The occurrence of distant metastases was significantly linked to Dukes’ stage, tumor differentiation, and TS expression. However, by multivariate analysis, only Dukes’ stage (P < 0.001) and TS expression (P = 0.011) remained as independent prognostic factors (Table 2) .

The relationships between disease-free survival and the other variables are presented in Table 3 . Univariate analysis demonstrated that Dukes’ stage (P < 0.001) and TS expression (P < 0.001) correlated significantly, whereas the other variables tested did not. Fig. 1a illustrates the disease-free survival of patients with rectal cancer graded as high or low TS. The independent prognostic significance of Dukes’ stage (P < 0.001) and TS expression (P = 0.014) are shown in Table 3 .

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Kaplan-Meier life table analysis of disease-free survival (a) and overall survival (b) in 243 curatively operated patients with rectal cancer Dukes’ stage A–C according to their TS expression (low or high) in primary tumor. Patients with low intratumoral TS expression had a significantly better outcome than those with high TS expression.

TS Expression and Relative Hazards for Recurrence, Occurrence of a New Noncolorectal Primary Tumor, or Death in Different Dukes’ Stages.
Model-based fitted values from the Cox regression analysis is shown in Fig. 2 . High TS expression was associated with a worse clinical outcome.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Relative hazard for recurrence, occurrence of a new noncolorectal primary tumor, or death during follow-up time in 243 curatively operated patients with rectal cancer Dukes’ stage A–C with respect to TS expression. Shown are the model-based fitted values from the Cox regression.

TS Expression in Primary Tumor and Its Lymph Node Metastases.
We observed a significant correlation between TS expression in metastatic lymph nodes and the primary tumor (n = 56; P < 0.001; Spearman r_s = 0.36).

p53 Overexpression.
p53 overexpression was analyzed in tumors from 114 patients that were obtained from the first trial (15) . No correlation was noted between p53 overexpression and disease-free survival, overall survival, local recurrence, distant metastasis, or TS expression.

	DISCUSSION

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Rectal adenocarcinoma is a common tumor type among both men and women in the Western world (12) . It represents a severe health problem due to the high risk of locoregional recurrence and development of distant metastasis, and almost half of all patients will die from their disease. However, recent progress in surgical management (19, 20) , adjuvant local radiation therapy (15, 16, 17, 18) and systemic chemotherapy (25 , 28 , 29) has resulted in an improvement in the overall outcome for these patients.

The stage of disease usually determines whether a patient should be offered adjuvant pre- or postoperative radiation or chemotherapy. However, because patients with a relatively favorable stage may also die from this disease, it is important to identify factors other than stage that may help form an accurate prognosis.

In the present study, we have examined the possible prognostic value of TS expression, as measured immunohistochemically on paraffin-embedded primary tumors from patients undergoing surgery for rectal cancer during the years 1980–1993. Local radiation therapy, which was given to approximately half of the patients shortly before surgery, did not seem to have any impact on TS expression in viable tumor tissue, because its distribution in the two treatment groups was similar, as was the distribution of sex, age, Dukes’ stage, and differentiation of the tumor (data not shown).

This study has demonstrated that stage of the disease, according to Dukes’ classification (21) , remains the most powerful prognostic factor in rectal cancer. However, we have also shown that the expression of TS is an independent prognostic factor for locoregional recurrence, distant metastasis, overall survival, and disease-free survival. The multivariate analysis (Table 3) has shown that the relative risk of recurrence, occurrence of a second noncolorectal primary, or death during the follow-up time is 1.5 times higher in the group with high TS staining tumors than in the group with the low TS staining tumors.

In the present article, we have made the assumption that high TS staining of a tumor governs the prognosis. Therefor, even if only a small part of a tumor had a high-intensity staining, the entire tumor has been classified as high.

We are currently examining whether there is any relationship between the proportion of high TS intensity staining in a tumor and prognosis. We are also studying the relationship between TS expression and proliferation and between TS expression in primary tumor versus distant metastases, which may give us more information about why high TS expression is correlated with poor prognosis.

Preoperative radiation correlated with locoregional recurrence, but in this relatively small number of patients, it showed a borderline value of independence. We failed to demonstrate any prognostic significance of preoperative irradiation to overall survival in curatively operated patients, as has been reported for one of the two clinical trials from which our patient sample group was obtained (15 , 16) . This discrepancy is likely due to the fact that our patient material only represented 243 of 1406 randomized patients in the overall study population.

Recent studies examining the level of TS expression in clinical tumor samples suggest that TS expression predicts for overall clinical outcome and response to cytotoxic therapy. Using the present immunohistochemical technique on paraffin-embedded cancer sections, Johnston et al. (1) have shown that TS protein expression predicts for disease-free survival and overall survival in patients with rectal cancer independently of Dukes’ stage. Disease-free survival and overall survival was significantly better in patients whose tumors stained less intensely for TS. Similarly, Pestalozzi et al. (30) found that lower TS protein expression in patients with node-positive primary breast cancer correlates with improved event-free and overall survival independently of other established prognostic factors for this disease. In both of these studies, adjuvant chemotherapy had the greatest impact on survival in tumors expressing higher TS levels.

Other studies have suggested that the level of TS staining in primary tumor sections may not necessarily correlate with chemosensitivity of subsequent metastases, suggesting that changes in TS expression may occur with tumor evolution. In a study of colorectal tumors, TS staining in sections of primary tumors failed to predict for the response of subsequent advanced disease to palliative chemotherapy (31) .

The predictive value of TS overexpression may be further enhanced if combined with other molecular characteristics. Kitchens and Berger (32) found higher TS expression in colon cancer cell lines that also expressed a mismatch repair deficient (RER+) phenotype.

Lenz et al. (4) reported that increased pretreatment TS expression together with expression of mutant p53 identified a subgroup of primary stage II colon cancers with a poorer prognosis.

However, in the present study, we did not observe any prognostic significance of p53 expression in the group of 114 patients with rectal cancer of Dukes’ stage A–C who were analyzed for p53 overexpression. A large number of studies on the association between p53 immunohistochemistry or mutational analysis and survival have been published (33) . There are several studies reporting a poor survival in tumors overexpressing p53, whereas others show no association between p53 expression and clinical outcome. p53 gene mutation studies have been conflicting, with the majority showing no association with patient survival. The conflicting results regarding the prognostic value of 53 suggests the need for several large prospective studies with long follow-up times.

In the setting of advanced metastatic disease, both high TS mRNA, quantified by RT-PCR, and high TS protein expression have been shown to predict for poor response to fluoropyrimidine-based therapy in colorectal (5 , 34, 35, 36) , gastric (37, 38, 39, 40, 41) , and head and neck (42) cancer. Considerable overlap between responders and nonresponders was often present in the low TS category, but patients with TS levels above the median tended not to respond. In the metastatic disease setting, the presence of mutant p53 has also been associated with higher TS expression and also predicts for failure of therapy (43) .

The results of the present investigation demonstrate that the measurement of TS expression in primary rectal cancer adds important prognostic information about locoregional recurrence, distant metastases, disease-free survival, and overall survival in a manner independent of that provided by Dukes’ stage. This would suggest that TS expression in rectal cancer may consistently add prognostic information independent of that provided by Dukes’ stage. No other factor has as yet emerged as consistently adding prognostic information.

Immunohistochemical assessment of TS expression in primary rectal cancer may also be of value when deciding the agressiveness of the treatment, as it may be possible to select high-risk patients in Dukes’ stage A and B groups and offer them more frequent follow-up.

	ACKNOWLEDGMENTS

 
We thank associate professor Björn Cedermark for helpful discussion and Bo Nilsson for assistance with statistical analysis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Dagmar Ferb Memorial Foundation, the Gustaf V Jubilee Foundation, the Swedish Cancer Society, and the Ihre Foundation of the Swedish Society of Medicine.

² To whom requests for reprints should be addressed, at Radiumhemmets Forskningslab, CCK R8-03, Karolinska Hospital, S-171 76 Stockholm, Sweden. Fax: 46-8-339031; E-mail: david.edler{at}kirurg.sos.sll.se

³ The abbreviations used are: TS, thymidylate synthase; 5-FU, 5-fluorouracil.

Received 8/17/99; revised 12/14/99; accepted 12/21/99.

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