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Thymidylate Synthase Expression in Colorectal Cancer: A Prognostic and Predictive Marker of Benefit From Adjuvant Fluorouracil-Based Chemotherapy

From the Department of Surgery, South Hospital, Stockholm; Department of Oncology, Uppsala Akademiska Hospital, Uppsala; Department of Oncology Karolinska Hospital, Stockholm, Sweden; Department of Oncology, Vejle Hospital, Vejle, Denmark; and Department of Oncology, Queens University of Belfast, Belfast, Northern Ireland.

Address reprint requests to David Edler, MD, Department of Surgery, Karolinska Hospital, S-17176 Stockholm, Sweden; email: david.edler{at}swipnet.se

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: We studied the prognostic value of thymidylate synthase (TS) expression in primary colorectal cancer (CRC) and the role of TS expression as a predictor of chemotherapeutic benefit in patients treated with adjuvant chemotherapy.

PATIENTS AND METHODS: TS expression was immunohistochemically assessed on tumor sections from 862 patients with CRC Dukes’ stages B and C enrolled onto randomized trials evaluating fluorouracil (5-FU)-based adjuvant chemotherapy.

RESULTS: TS expression was an independent prognostic factor for disease-free (P = .05) and overall survival (P = .05). In the subgroup treated with surgery alone, TS was an independent prognostic factor for disease-free (P < .001) and overall survival (P = .001), whereas this was not the case in the subgroup of adjuvantly treated patients. Patients whose tumors expressed high TS levels had a tendency to improved outcome after adjuvant therapy (not significant). The group whose tumors expressed the highest TS grade, grade 3 (34% of the patients), had a significantly longer disease-free survival if they were treated with adjuvant therapy compared with surgery alone (multivariate analyses, P = .02), whereas patients whose tumors expressed low TS levels (28% of the patients) had an impaired outcome after adjuvant therapy (multivariate analyses: disease-free survival, P = .01; overall survival, P = .01).

CONCLUSION: TS expression predicts for survival independent of Dukes’ stage in patients with CRC treated with surgery alone. The study indicates that patients with high TS levels may benefit from adjuvant 5-FU–based chemotherapy. However, patients with low TS levels seem to have a worse outcome when treated with adjuvant chemotherapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
COLORECTAL CANCER (CRC) is one of the most common tumor types in the world, with approximately 400,000 deaths annually.¹ At present, only the stage of the disease is widely accepted as a prognostic factor.² However, in each stage, there are patients whose tumors are more aggressive and others that behave more indolently. Therefore, it is important to identify additional factors that may enhance the prognostic strength.

Intratumoral expression of the enzyme thymidylate synthase (TS) may be a factor that, independent of stage, can forecast prognosis in primary CRC^3-8 and in disseminated disease. The TS levels are not only of importance in predicting the natural course of the disease but may also predict the sensitivity of cancer cells to fluorouracil (5-FU),^9-16 which is widely used both in the adjuvant setting and in the treatment of disseminated CRC. TS is involved in the biosynthesis of DNA and is considered to be the main intracellular target of 5-FU.¹⁷

In the present investigation, we retrospectively examined the possible prognostic relevance of intratumoral levels of TS in 862 CRC patients who were included in Nordic adjuvant trials examining the clinical value of postoperative 5-FU–based chemotherapy in patients with Dukes’ B and C cancer. Since patients were randomized between surgery only and surgery followed by chemotherapy, we had the possibility to assess the relationships between TS expression and clinical events both in chemotherapy-treated and non–chemotherapy-treated patients.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Tumor specimens examined for TS expression were obtained from 862 patients with CRC Dukes’ stages B and C who were operated on during the time period September 1991 to December 1996. All patients were included in prospective randomized trials. In 1991, several Nordic groups initiated trials in which patients were randomized between surgery alone or surgery plus adjuvant therapy, this being either 5-FU plus levamisole according to the Moertel regimen,^18,19 or 5-FU plus leucovorin according to a modified Mayo schedule,²⁰ or according to the Nordic schedule,²¹ with or without levamisole. Each group chose their own adjuvant therapy. The addition of levamisole was based on randomization at participating hospitals. A total of 2,191 patients have been enrolled onto these trials during the time period September 1991 to December 1996. Our patient sample was obtained from 28 hospitals from two health care regions in Sweden and from two hospitals in Denmark. From the Stockholm region, 323 patients were examined with respect of TS expression, 457 patients from the Uppsala-Örebro region, and 82 patients from Århus-Åhlborg. Tumor material was available from 862 (93%) of 928 patients enrolled onto the trials at the 28 hospitals contributing patients to this study.

The age of the patients ranged between 23 and 75 years (mean, 62 years; median, 65 years). Other characteristics such as sex, tumor localization, stage of the disease according to Dukes’ classification, and TS-expression of the primary tumors are listed in Table 1.

1. 5-FU 450 mg/m² bolus injection on days 1 to 5, and levamisole 50 mg 3 times a day (oral) on days 1 to 3 and 15 to 17. From day 29, 5-FU was given as a bolus injection once a week during 48 weeks and levamisole 50 mg 3 times a day (oral) on days 1 to 3 every second week during the rest of the treatment period of 48 weeks (Moertel regimen^18,19). This treatment was given in some hospitals in Uppsala-Örebro and in Denmark (n = 69).

2. 5-FU 425 mg/m² bolus injection, and after 30 to 40 minutes leucovorin 20 mg/m² intravenously on days 1 to 5 (modified Mayo regimen²⁰). A new course of treatment was started on day 29. A total of four courses were given. This treatment was given in Stockholm (n = 95).

3. The same modified Mayo regimen as in regimen 2 together with levamisole, 50 mg for 3 days (oral) on days 1 to 3 and 15 to 17. This treatment was given in Stockholm (n = 86).

4. 5-FU 500 mg/m² bolus injection, and after 30 to 40 minutes leucovorin 60 mg/m² bolus intravenously on days 1 and 2 (Nordic regimen²¹). A new course of treatment was given on day 15. The patients were scheduled for 10 courses. This treatment was given in Uppsala-Örebro (n = 118).

5. The Nordic regimen plus levamisole 50 mg for 3 days (oral) on days 1 to 3. This treatment was given in Uppsala-Örebro (n = 52).

Clinical data, such as the development of local recurrence, distant metastases, and death, were obtained from the registries at the oncologic centers in Stockholm, Uppsala, and Denmark.

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

TS Staining Technique
The standard avidin-biotin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Inc, Burlingame, CA)²² 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 minutes. For reduction of nonspecific background staining, the sections were exposed to 20% horse serum for 30 minutes. Sections were incubated with the TS 106 monoclonal antibody²³ at room temperature for 90 minutes. They were then rinsed and incubated with biotinylated horse antimouse secondary antibodies for 30 minutes followed by further washing, and then incubated with avidin-biotin-peroxidase complexes. Immunostaining was developed by immersion in 0.05% 3,3'-diaminobenzidine tetrahydrochloride and then counterstained with a modified Harris hematoxylin.

Scoring of Immunohistochemical Staining
All samples were read blindly (ie, the observers did not know which patient they were examining). The intensity of TS staining of the tumors was arbitrarily graded from 0 to 3. Each time a set of tumor samples was stained, reference slices were included from tumors, which had previously been classified as TS staining intensity 0 or 1 and 2 or 3. As described previously, 0 and 1 was defined as low intensity, and 2 and 3 as high.⁴ The highest staining intensity (low/high) found in a tumor was used for its classification. Thus, even if the high-staining area only constituted a small part of the whole tumor area, the tumor was classified as high. The agreement of TS intensity scoring reached by two independent observers was greater than 90%. In case of disagreement, intensity was determined by consensus.

Statistical Analyses
The Gehan-Wilcoxon univariate test²⁴ was used to examine the possible relationships between disease-free survival (DFS), overall survival (OS), and disease-specific survival (dead in CRC) and Dukes’ stage, site of tumor, treatment, and TS expression. When DFS was used as an end point, an event included recurrence of disease, death from cancer, and death from noncancer causes. Multivariate analyses were performed using Cox regression.²⁵ Survival curves were constructed using the Kaplan-Meier method.²⁶ Differences in distribution between groups were compared with the ² test and differences in mean with Student’s t test.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patient Demographics
The TS study population comprised 862 patients. The age of the study population ranged between 23 and 75 years (mean, 62 years). Two hundred sixty-nine patients (31%) developed tumor recurrence. During the follow-up period, 308 patients (36%) died, 244 (79%) of them because of CRC.

Pathologic Correlations
The TS staining was predominantly a granular cytoplasmatic staining pattern noted in the cribriform glands of the colorectal cancer. TS staining was also noted in lymphocytes and macrophages adjacent to tumors, and some minor cross-reactivity was observed with smooth muscle cells. Twenty-eight percent of tumors were classified as having low TS expression, and 72% of tumors were classified as having high TS expression. No significant difference in TS expression was noted in tumors from patients with Dukes’ stage B or C. There was a significantly higher frequency of tumors with low TS expression in right-sided tumors (34%) compared with the left-sided and rectal tumors (26%) (P = .02).

Clinical Correlations
Tumor site distribution, age, and sex were similar per stage. Site of tumor, stage, age, and sex were similarly distributed in the group treated with surgery alone, compared with the group treated with surgery and adjuvant therapy. In our study group of 862 patients, which represents a subgroup of the 2,191 patients included in the trials, no adjuvant treatment-related benefit was found (Fig 1).

View larger version (15K): [in this window] [in a new window]   Fig 1. Overall survival of the study population (n = 862), which represents a subgroup of 2,191 patients included in the adjuvant trials. The patients were treated with surgery alone, or surgery followed by adjuvant 5-FU–based chemotherapy.

View larger version (15K): [in this window] [in a new window]   Fig 2. Overall survival for the entire study population (n = 862) with respect to low versus high TS expression.

View larger version (17K): [in this window] [in a new window]   Fig 3. Overall survival in patients treated with surgery alone with respect to low versus high TS expression. (A) Patients with Dukes’ B and C tumors; (B) patients with Dukes’ B tumors; and (C) patients with Dukes’ C tumors.

View larger version (22K): [in this window] [in a new window]   Fig 4. Disease-free survival (A) and overall survival (B) in patients with high TS expression Dukes’ B and C (n = 617) with respect to treatment with surgery alone versus surgery followed by adjuvant 5-FU–based chemotherapy.

View larger version (15K): [in this window] [in a new window]   Fig 5. Disease-free survival in patients with tumors expressing the highest TS grade, grade 3 (34% of the patients), with respect to treatment with surgery and adjuvant therapy versus surgery alone.

View larger version (23K): [in this window] [in a new window]   Fig 6. Disease-free survival (A) and overall survival (B) in patients with low-TS-expressing tumors (n = 245) with respect to treatment with surgery and adjuvant therapy versus surgery alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

The results of this study confirm that the level of TS expression in primary CRC correlates with survival. Patients whose tumors contain low TS levels survive significantly longer than patients with high TS levels. Moreover, the ability of TS to predict survival is independent of Dukes’ stage. Dukes’ staging still represents the most powerful prognostic factor in CRC.² Yet, within each of the Dukes’ stages examined (Dukes’ B and C), patients with low TS expression treated with surgery alone had a better outcome than those with high TS expression.

This study also examined the impact of TS and its correlation with clinical outcome after adjuvant 5-FU–based chemotherapy. Patients whose tumors expressed high TS, constituting almost 70% of the tumors, and who received adjuvant treatment had a slightly improved clinical outcome, although this did not reach statistical significance. This was in contrast to the results in patients with low TS expression. Patients with low-TS-expressing tumors who received adjuvant therapy had a worse outcome than those who did not, suggesting that 5-FU–based therapy had a deleterious effect on survival in a low-TS population.

Several previous reports have suggested that patients whose primary tumors had high TS expression may benefit from 5-FU–based adjuvant treatment and that patients whose tumors had low TS expression do not.^4-6 In the study by Johnston et al,⁴ the monoclonal antibody TS 106 and immunohistochemical technique were used to study tumors from 194 patients with rectal cancer Dukes’ stage B and C who were entered onto the National Surgical Adjuvant Breast and Bowel Project protocol R-01 from 1977 to 1986.²⁸ The treatment arms were surgery with or without radiation or surgery plus lomustine, 5-FU, and vincristine chemotherapy. Adjuvant chemotherapy demonstrated significant improvements in DFS and OS for patients with high TS levels and no benefit in patients whose tumors expressed low TS levels. In the study presented here, 254 of 862 tumors were rectal cancers. Among them, the high TS expressers possibly benefited somewhat, with respect to DFS, after adjuvant therapy (P = .2). In the group of patients with rectal cancer expressing the highest TS grade, grade 3 (n = 89), there was a significant improvement of DFS of patients who were treated with surgery and adjuvant therapy compared with surgery alone (P = .04). There was no deleterious effect of adjuvant therapy in the group of patients with rectal cancer expressing low levels of TS. In the report presented by Johnston et al,⁴ most rectal cancer patients were operated on before the development of the total mesorectal excision technique,^29,30 which reduces the rate of local recurrences, whereas in our study material, many of the rectal cancer patients were operated on using the total mesorectal excision technique.

In trials by Yamachika et al,⁵ including 86 colon cancer patients with stages I to III tumors, and by Takenoue et al,⁶ with 142 stages I to III colon tumors that were operated on and six stage IV tumors that were curatively operated on, a benefit of 5-FU–based adjuvant chemotherapy was found only in patients whose tumors expressed a high level of TS. In these studies, the percentage of low TS expressors was 81% and 72%, respectively, compared with 31% in the study presented by Johnston et al⁴ and 28% in this study. We analyzed TS expression in two different parts of each tumor, which increases the probability of classifying a tumor as a high expressor.³¹ This may be one reason why we recorded a lower rate of low-TS-expressing tumors than other authors.

Thus, four studies including the present one show a differential effect of 5-FU–based adjuvant therapy according to TS level. This study, however, is the first to suggest that patients who have low-TS-expressing primary tumors actually show a deleterious effect resulting from adjuvant treatment. The possible benefit of chemotherapy in patients with high TS levels and its deleterious effect in those with low TS levels would not have been predicted on the basis of preclinical models and suggests that the importance of TS expression in the primary tumors is related more to the biology of the disease than to chemoresistance.

There was no imbalance with respect to age, sex, site of tumor, stage, and TS expression between the adjuvantly treated group compared with the group treated with surgery only. The group with low-TS-expressing tumors treated with surgery alone showed a distribution of age, sex, site of tumor, and stage similar to the group treated with postoperative adjuvant chemotherapy. The reduced survival of the adjuvantly treated patients with tumors expressing low TS could possibly be because of an imbalance in the distribution of unknown prognostic factors between the treatment arms. It could also be because of chance, although the likelihood for this is minor in light of the high statistical significance (P = .01).

The methodology itself with the assumption that any area of high TS staining confers high-TS prognosis could be a reason for unexpected findings. We have already started to calculate the percentage of high-TS-staining area in every tumor and to correlate the degree of high staining to the clinical outcome.

At present, we prefer not to give any hypothesis concerning biologic explanation for these unexpected results. We have started to examine a new patient population to see whether the present results can be reproduced.

It is not possible to draw any conclusions about the effects of adjuvant chemotherapy in the Nordic trials on the basis of the presented analyses. This is a retrospective analysis of a subsample which, although comparatively large (about 40% of total), was neither prospectively designed nor powered to identify a 10% absolute gain in 5-year survival.

In conclusion, we have shown that the level of TS expression predicts for DFS and OS independent of Dukes’ stage in patients with CRC treated with surgery alone. The study, together with three previous studies,^4-6 also indicates that patients with high TS levels may benefit from adjuvant 5-FU–based chemotherapy. However, patients with low TS levels seem to have a worse outcome when treated with adjuvant 5-FU–based chemotherapy. Further prospective studies measuring TS levels are now necessary to understand the role of TS as a prognosticator of survival and DFS, and as a predictor of chemotherapeutic benefit in patients with CRC.

	ACKNOWLEDGMENTS

 
Supported by grants from the Dagmar Ferb Memorial Foundation, Cancerföreningen in Stockholm, Gustav V Jubilee Foundation, Bengt Ihre Foundation of the Swedish Society of Medicine, Minerva Foundation, Ruth and Rickard Juhlin Foundation, Robert Lundberg Foundation, Swedish Society for Medical Research, and Swedish Cancer Society.

We thank Bo Nilsson for assistance with statistical analysis, Göran Lundell for computer assistance, Inger Öst and Toom Singnomklao for data management, and Lena Kanter-Lewensohn for discussions about immunohistochemical analyses.

	REFERENCES

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Submitted December 13, 2000; accepted December 5, 2001.

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