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Clinical Cancer Research Vol. 12, 2738-2744, May 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Thymidylate Synthase Expression in Colon Carcinomas with Microsatellite Instability

Frank A. Sinicrope, Rafaela L. Rego, Kevin C. Halling, Nathan R. Foster, Daniel J. Sargent, Betsy La Plant, Amy J. French, Carmen J. Allegra, John A. Laurie, Richard M. Goldberg, Thomas E. Witzig and Stephen N. Thibodeau

Authors' Affiliation: Mayo Clinic and Mayo College of Medicine, Rochester, Minnesota

Requests for reprints: Frank A. Sinicrope, Gastroenterology/Hepatology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-286-8660; Fax: 507-284-9111; E-mail: sinicrope.frank{at}mayo.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Colon cancer cells with high-frequency microsatellite instability (MSI-H) display resistance to 5-fluorouracil (5-FU) that can be reversed by restoring DNA mismatch repair (MMR) proficiency. Given that thymidylate synthase (TS) is inhibited by 5-FU, we studied the relationship between MSI and TS expression, and the prognostic effect of these and other markers (i.e., p53 and 17p allelic imbalance).

Experimental Design: Dukes' stage B2 and C colon carcinomas (n = 320) from participants in 5-FU-based adjuvant therapy trials were analyzed for MSI and 17p allelic imbalance. Expression of MMR (hMLH1, hMSH2), TS, and p53 proteins were analyzed by immunohistochemistry. Correlations between markers and associations with overall survival were determined.

Results: Of 320 cancers studied, 60 (19%) were MSI-H. TS expression variables were similar in MSI-H and microsatellite stable/low-frequency MSI (MSS/MSI-L) cancers, and unrelated to MMR proteins. MSI-H tumors had lower stage (P = 0.0007), fewer metastatic lymph nodes (P = 0.004), and improved overall survival (P = 0.01). Loss of MMR proteins was also associated with better overall survival (P = 0.006). None of the TS variables were prognostic. Histologic grade (P = 0.0008) and nodal status (P = 0.0002) were associated with overall survival, in contrast to 17p allelic imbalance or p53. Only MSI status or loss of MMR proteins, histologic grade, and tumor stage were independent markers for overall survival.

Conclusions: MSI-H tumors show earlier stage at presentation and better stage-adjusted survival rates. MSI status and TS expression were unrelated and TS was not prognostic, suggesting that TS levels cannot explain therapeutic resistance to 5-FU reported in MSI-H colon cancers.

Colorectal cancer is the fourth most incident cancer in the United States and is the second leading cause of cancer-related mortality (1). The majority of sporadic colorectal cancers show chromosomal instability due to loss of heterozygosity and DNA aneuploidy (2, 3). Another pathway that accounts for up to 20% of colon cancers is characterized by microsatellite instability (MSI). MSI is caused by defective DNA mismatch repair (MMR), and in sporadic cases is nearly always due to hypermethylation of the hMLH1 gene promoter (4). Colon cancers with high-frequency MSI (MSI-H) are characterized by proximal location, frequent DNA diploidy, and pathologic features that commonly include poor differentiation, mucinous histology, and peritumoral lymphocytic infiltration (5–7). In colon cancer cell lines with defective DNA MMR, resistance to the cytotoxic effects of 5-FU have been observed in vitro (8). Specifically, the HCT-116 colon cancer cell line contains a mutated hMLH1 gene and displays resistance to 5-FU. Transfer of chromosome 3 containing the intact hMLH1 gene into these cells was shown to restore 5-FU sensitivity (9). Furthermore, a 5-FU-resistant cell line displaying MSI due to hypermethylation of the hMLH1 promoter was rendered sensitive after demethylation and reexpression of hMLH1 using 5-azadeoxycytidine (10). The potential clinical relevance of these findings for MSI-H were shown by Ribic et al. (11), wherein patients with resected Dukes' stage B2 and C colon cancers with MSI-H did not benefit from 5-FU-based adjuvant therapy, whereas microsatellite stable/low-frequency MSI (MSS/MSI-L) patients showed a survival advantage. These retrospective data, although not definitive, suggest that molecular features associated with the MSI-H phenotype may confer resistance to 5-FU.

5-Fluorouracil (5-FU)-based treatment remains the standard of care for the adjuvant treatment of resected colorectal cancer where it is combined with oxaliplatin (12–14) and for metastatic disease where it is coadministered with either oxaliplatin or irinotecan (15). Fluoropyrimidines irreversibly inhibit the thymidylate synthase (TS) enzyme, leading to DNA damage and blockade of DNA replication and repair (16). TS catalyzes conversion of dUMP to dTMP, the latter being necessary for DNA synthesis. Overexpression of TS protein or mRNA levels in primary colorectal cancers, as determined by immunohistochemistry or reverse transcription-PCR, have been associated with reduced responsiveness to 5-FU and adverse outcome in many (17–21), but not all, studies in patients with metastatic disease (22, 23). In the adjuvant setting, the relationship between TS protein levels and patient survival rates after 5-FU-based treatment is less well established (21, 24–26). Differences in the molecular profile of MSI-H versus MSS/MSI-L colorectal cancers may contribute to potential differences in their responsiveness to the cytotoxic effects of 5-FU. However, whether such differences include TS expression is largely unknown and remains a highly relevant clinical issue. p53 has been shown to transcriptionally regulate TS (27) and alterations in TS levels may contribute to the effect of p53 functional status upon 5-FU sensitivity. p53 is a known negative regulator of the cell cycle via its downstream effector protein p21WAF1/CIP1, a cyclin-dependent kinase (28). p21WAF1/CIP1 inhibits the cell cycle after DNA damage (29) and was found to be overexpressed in a higher proportion of MSI-H compared with MSS/MSI-L colorectal cancers (30–32). Metastatic colorectal cancers with wild-type p53 were found to have significantly lower TS levels compared with tumors with mutated p53 (18). Furthermore, MSI-H colorectal cancers have reduced rates of p53 mutation (33–35) and nuclear p53 protein expression compared with MSS/MSI-L tumors (36, 37). The potential relevance of these findings are underscored by the observation that wild-type p53 was associated with an improved response and prolonged survival in colon cancers after 5-FU-based adjuvant therapy (11, 38, 39), as were tumors that retained heterozygosity at either 17p or 18q (40).

In this study, we analyzed the relationship between the level of TS protein expression and MSI status or MMR proteins with clinical outcome in Dukes' B2 and C colon cancer patients treated in 5-FU-based adjuvant therapy trials. We also examined TS levels in relation to p53 expression and chromosome 17p allelic imbalance.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Primary colon carcinomas were analyzed from patients who underwent surgical resection and who participated in one of five 5-FU-based adjuvant therapy trials conducted by the North Central Cancer Treatment Group. Paraffin-embedded tumor tissue was available from a nonrandom subset of study participants and included Dukes' stage B2 (n = 54) and C (n = 266) colon cancers (total n = 320). Tumor histologic grade was defined by the American Joint Committee on Cancer Prognostic Factors Consensus Conference (41) as follows: grade 1, well differentiated; grade 2, moderately differentiated; grade 3, poorly differentiated; grade 4, undifferentiated. All patients were censored at 5 years after randomization for disease-free survival. Patients were followed for a minimum of 8 years after study randomization for overall survival data.

Details of the five completed, randomized 5-FU-based adjuvant chemotherapy trials have been previously reported (25). The current analysis was in accordance with the original informed consent document. Of 320 patients, 233 were randomized to treatment arms and 87 received observation alone. Given that 21 patients received treatment that was later determined to be ineffective (i.e., portal venous 5-FU), there were a total of 212 patients who received effective treatment that included 5-FU. Accordingly and for purposes of the analyses, treatment was categorized as none or ineffective [n = 108; observation only (n = 87) + portal venous 5-FU (n = 21)] versus effective (i.e., consisting of modulated 5-FU; n = 212).

MSI testing and 17p allelic imbalance. Tumors (n = 320) had been analyzed for MSI and allelic imbalance using 11 dinucleotide microsatellite markers as previously described (7, 42). Chromosome 17p allelic loss was determined using markers that included D17S261 and TP53 on 17p at or near the p53 locus. The median number of markers that gave a PCR product for both normal DNA and tumor DNA was nine (range, 5-11). Tumors were classified into three groups: (a) MSS with no MSI at any of the loci examined; (b) low instability (MSI-L, <30% of the loci demonstrating MSI); or (c) high instability (MSI-H, ≥30% of the loci demonstrating MSI; refs. 42, 43). Because extensive data indicate that tumors with MSI-L are not biologically distinct from those exhibiting MSS, these two molecular phenotypes were grouped together in all analyses (42–44). The presence of MSI at a microsatellite marker makes interpretation for allelic imbalance potentially inaccurate. Therefore, loci with MSI and those without obvious instability were considered noninformative for allelic imbalance in MSI-H tumors (42).

Allelic imbalance was assessed as previously described (42). Tumors were classified as positive for allelic imbalance when the PCR assay for the control normal tissue showed heterozygosity of the microsatellite marker and the relative intensity of the two alleles in the tumor DNA differed from the relative intensity in the normal DNA by a factor of at least 1.5 (45).

Immunostaining for TS, p53, and MMR proteins. Immunohistochemistry for TS and p53 proteins were done in paraffin-embedded tissue sections as previously reported (25). Each slide contained a unique number that enabled blinding with respect to patient identity and clinical characteristics. Tissues were incubated for 50 minutes with TS106 primary antibody at a 1:500 dilution (Zymed Laboratories, San Francisco, CA), as previously described (25). For p53 staining, antigen retrieval was done by microwaving the slides in a 10 mmol/L citric acid buffer for 3 x 3 minutes. Slides were subsequently incubated with the p53-D07 primary antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, United Kingdom) at a 1:50 dilution for 1 hour at room temperature, as previously described (25). In a subset of patients (n =220), expression of MMR proteins (hMLH1, hMSH2) were analyzed using anti-hMLH-1 (G168-782 clone; PharMingen, San Diego, CA) and anti-hMSH-2 antibodies (FE11 clone; Oncogene, Cambridge, MA) as previously described (37).

Immunohistochemical scoring. For TS staining, each slide was assigned a score for intensity (0-3) and extent was determined and dichotomized at leg50% immunopositive tumor cells at light microscopy, as previously described (25). Staining intensity was categorized as follows: 0, no staining; 1, trace staining; 2, definite staining of light to moderate intensity; 3, bright and/or dark intensity. The categories were grouped into weak (0, 1) and strong (2, 3). TS extent was determined in tissue sections where at least 1% of tumor cells stained positive for TS and otherwise, regarded as inevaluable for expression level. A weighted score was also calculated as the product of staining intensity and the extent. All specimens had been analyzed independently by two investigators who were blinded to all clinical information. Discrepant scores (~15% of cases) were resolved by consensus. Nuclear p53 staining was regarded as positive when ≥10% of the malignant nuclei showed staining; all others were regarded as p53 negative (25). Slides were scored as either positive or negative based on the presence (+) or absence (–) of staining for hMLH-1 or hMSH-2 (37).

Statistical analysis. {chi}2 tests were used to test for an association between prognostic markers for categorical variables. Wilcoxon rank-sum tests were used to test for associations of continuous variables (i.e., age) with two-level categorical data. Patients were observed routinely for 5 to 8 years after randomization for disease recurrence on each of the five treatment trials. The disease-free interval (i.e., disease-free survival) is calculated as the number of days from randomization to the date of recurrence or death. Overall survival was calculated as the number of days from randomization to death or date of last contact in those patients lost to follow-up. Overall survival for patients was censored at 8 years. The distributions of overall survival and 5-year disease-free survival were estimated using Kaplan-Meier methodology. Cox proportional hazards models were used to explore the association of clinical and laboratory variables with overall survival and disease-free survival. All these models were stratified according to the patient's original treatment study and adjusted for histologic grade, stage and treatment for overall survival, and disease-free survival. The likelihood ratio test was used to test the significance of covariates in all models tested. Graphical methods were used to examine whether underlying model assumptions were satisfied (e.g., proportional hazards; ref. 46). Analyses were done using SAS (SAS Institute, Cary, NC). All P values reported are two-sided, and values <0.05 are considered statistically significant. No adjustment for multiple comparison was attempted. Interaction test P values were calculated using a likelihood ratio test comparing a model with and without the interaction term included.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinicopathologic features. We analyzed surgically resected colon adenocarcinoma specimens from patients enrolled in 5-FU-based adjuvant chemotherapy trials. The study population (n = 320) included 54 (17%) Dukes' stage B2 and 266 (83%) Dukes' stage C colon cancers. Clinicopathologic features of the study cases are shown in Table 1 . The median patient age was 65.5 years (range 30-86). The median duration of follow-up for patients who remain alive was 8.0 years.


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  		Table 1. Tumor markers and overall survival

 
Thymidylate synthase and other cellular or molecular markers. We observed strong TS intensity (n = 236, 74%), a high TS extent [>50% tumor cell positivity; n = 123 (38%)], and increased dichotomized TS score (n = 188, 59%) in Dukes' B2 and C colon cancers (Table 1; see Materials and Methods). Of the 320 cases, 60 (19%), were MSI-H and 260 (81%) were MSS/MSI-L. Of 44 MSH-H cases available for testing, 41 showed loss of hMLH1 (n = 39) or hMSH2 (n = 2) proteins. For MSS/MSI-L cases, 175 of 176 showed intact hMLH1 and hMSH2 protein expression. Allelic imbalance at chromosome 17p was found in 80 (25%) cases and nuclear p53 staining was considered positive in 184 (57%) tumors. No significant correlations were found between TS expression variables and MSI status, MMR proteins, p53 expression, 17p status, gender, or patient age. Patient tumors that were positive for p53 were more likely to show 17p allelic imbalance. In this regard, 55 (76%) p53-positive tumors showed 17p allelic imbalance in contrast to 25 of 44 (57%) p53-negative tumors (P = 0.0271).

Tumor characteristics stratified by MSI status. We compared clinicopathologic features and cellular and molecular marker data in MSI-H versus MSS/MSI-L colon cancers. No differences were found in TS expression variables in relation to MSI status (Table 3) nor with DNA MMR protein expression (Table 2 ). MSI-H tumors were more frequent in women [42 of 60 (70%); P = 0.0003], and were more likely to show high-grade histology [32 of 60 (53%) versus 67 of 260 (26%) MSS/MSI-L; P < 0.0001] and to be negative for p53 [41 of 60 (68%) versus 94 of 259 (36%) MSS/MSI-L; P < 0.0001; Table 3 ]. Patients with MSI-H tumors were older than those with MSS/MSI-L tumors (P = 0.0063; Table 3). Because loci with MSI were considered noninformative for allelic imbalance in MSI-H cases, allelic imbalance at 17p was determined only in MMS/MSI-L tumors.


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  		Table 3. Tumor markers stratified by MSI-H status

 

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  		Table 2. Tumor markers stratified by MMR proteins

 
We found that MSI-H tumors are more likely to be Dukes' stage B2 than MSS/MSI-L tumors (32% versus 13%; P = 0.0007; Table 3). In addition, MSI-H colon cancers had fewer metastatic lymph nodes than did MSS/MSI-L tumors (P = 0.0037; Table 3).

Tumor characteristics and patient survival rates. Five-year overall survival rates were 78.3% for patients with MSI-H tumors versus 65% for MSS/MSI-L cases (hazard ratio, 0.53; 95% confidence interval, 0.31-0.90, P = 0.0115; Table 1; Fig. 1 ). Patient tumors with loss of MMR proteins had better overall survival rates than those with intact MMR (83.3% versus 64.6%; P = 0.0062; Fig. 2 ). Tumor stage, number of metastatic lymph nodes, and histologic grade were all highly significant prognostic variables in both disease-free survival and overall survival (Table 1). Neither TS expression variables, allelic imbalance at 17p, nor p53 expression were prognostic (Table 1). Specifically, 5-year overall survival rates were 66% for patients whose tumors had a high TS score versus 68% for those with low TS scores (Fig. 3 ). Data were analyzed after adjustment for grade, stage, and treatment, and stratified by adjuvant study.


Figure 1
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  		Fig. 1. Overall survival in patients with Dukes' stage B2 and C colon cancer by MSI status.

 

Figure 2
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  		Fig. 2. Overall survival in patients with Dukes' stage B2 and C colon cancer by defective DNA MMR (dMMR) indicated by loss of MMR proteins.

 

Figure 3
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  		Fig. 3. Overall survival in patients with Duke's B2 and C colon cancer by TS score (see Materials and Methods).

 
A multivariate analysis was conducted and is shown for disease-free survival and overall survival (Tables 4 and 5 ). Variables included in the models were MSI-H or loss of MMR proteins, TS weighted score, treatment, tumor stage, and histologic grade. Only MSI status or MMR proteins, tumor stage, and histologic grade were independent markers for overall survival. No significant interaction was found between TS variables and MSI-H or MMR proteins.


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  		Table 4. Multivariate models for MSI-H and TS score

 

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  		Table 5. Multivariate models for MMR proteins and TS score

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, 19% of colon cancers were found to be MSI-H, which is consistent with other studies where the study population is enriched with MSI-H tumors (6, 47, 48). Our data show and confirm that MSI-H colon cancers have distinct clinicopathologic differences compared with MSS/MSI-L tumors (5, 6, 36, 42, 43, 47). In this regard, MSI-H tumors were significantly more likely to have a higher histologic grade and to be negative for p53 expression (versus MSS/MSI-L tumors). Patients with MSI-H tumors were more frequently women (P = 0.0003) and older than those with MSS/MSI-L tumors (P = 0.0063), consistent with studies in sporadic colon cancers with defective DNA MMR due to hypermethylation of hMLH1 gene (49). MSI-H tumors had significantly fewer lymph node metastases than did MSS/MSI-L cases and, accordingly, were more frequently Dukes' stage B2 than C (P = 0.0007). In addition, patients with MSI-H tumors showed improved 5-year overall survival rates among Dukes' stage B2 and C patients compared with MSS/MSI-L tumors (hazard ratio, 0.53; 95% confidence interval, 0.31-0.90; P = 0.0115), after adjustment for grade, stage and treatment, and stratifying by study. Furthermore, patient tumors with loss of MMR proteins also had better overall survival rates (P = 0.0062). Immunostaining for hMLH1 and hMSH2 proteins was shown to accurately predict the molecular finding of MSI-H versus MSS/MSI-L status in a large number of colorectal cancers (50). Our results in MSI-H colon cancers confirm previous reports of a survival benefit for patients with these tumors (5, 6, 42, 47). Of note, prior studies demonstrating the prognostic effect of MSI were limited to young patients (47) and often included rectal cancers (5, 6, 42, 47).

We determined whether TS expression variables differ between MSI-H versus MSS/MSI-L colon cancers and whether such changes may explain the reported differential sensitivity to fluoropyrimidines in MSI-H tumors (11, 38, 51). We failed to detect significant differences in TS expression variables in relation to MSI status or p53 expression. In support of these data, analysis of DNA MMR proteins showed a similar lack of association with TS levels and concordant results as for MSI. In contrast to our results, a recent study reported that MSI-H sporadic colorectal cancers (n = 24) had significantly increased TS expression compared with MSS/MSI-L patients (32). In that study, however, only 14.8% MSS/MSI-L tumors had a high TS score determined by immunohistochemistry, which is inconsistent with our finding that 61% MSS/MSI-L tumors had a high TS score. In a systematic review and meta-analysis of TS expression in colorectal cancers, the median proportion of cases expressing a high level of TS in adjuvant studies was 50% [range 19-77%, median sample size 108 (range 16-617); ref. 21], which is similar to our findings. As in our study, the majority of these studies used anti-TS monoclonal antibody, TS106, and TS staining intensity has been the most frequently used semiquantitative method for analyzing TS expression (21). Whereas MSI-H tumor cell lines have shown resistance to DNA damage (9, 52) and such tumors may not benefit from 5-FU-based adjuvant therapy (11), our finding of similar TS expression in MSI-H versus MSS/MSI-L tumors indicates that TS alone cannot explain fluoropyrimidine resistance in MSI-H colon cancers.

TS expression variables were not prognostic. These data are consistent with aggregated studies of TS protein expression in patients receiving surgery and adjuvant 5-FU where the pooled hazard ratio for overall survival was 0.93 (95% confidence interval, 0.69-1.90; refs. 21, 25, 26). The association of TS expression and poor overall survival in colon cancers has been mostly limited to patients treated by surgery alone, where those treated with both surgery and adjuvant 5-FU have not generally shown an association between TS expression and clinical outcome (21). Importantly, TS expression measured in primary tumors may not reflect TS levels in lymph node or other metastases (53). Furthermore, TS levels were found to differ depending on organ site of metastasis (54). These data may explain, in part, why a weaker and inconsistent relationship between TS and clinical outcome have been found in 5-FU-based adjuvant studies. Additionally, the relationship of TS expression and poor overall survival has generally been stronger in studies using reverse transcription-PCR in fresh tissue compared with immunohistochemistry (17, 18, 55, 56). However, these data must be interpreted cautiously given the relatively small number of contributing studies.

In conclusion, MSI-H colon cancers are associated with better stage-adjusted 5-year overall survival rates, lower tumor stage, and fewer lymph node metastases compared with MSS/MSI-L tumors. Patient tumors with loss of MMR proteins also showed better prognosis compared with those with intact expression. Histologic grade was also found to be a significant prognostic variable. TS expression variables were unrelated to MSI status, MMR proteins, p53, or 17p. These findings indicate that TS cannot explain the observed lack of benefit of MSI-H colorectal cancers from 5-FU-based adjuvant chemotherapy.


   Acknowledgments
 
The authors thank Luanne Wussow for her very capable secretarial assistance.


   Footnotes
 
Grant support: National Cancer Institute grant CA104683 (F.A. Sinicrope).

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.

Received 1/24/06; revised 2/17/06; accepted 2/20/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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