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© 2001 American Society for Clinical Oncology Polymorphisms of the Repeated Sequences in the Enhancer Region of the Thymidylate Synthase Gene Promoter May Predict Downstaging After Preoperative Chemoradiation in Rectal CancerFrom the Department of Oncology, Clínica Universitaria, University of Navarre, and Department of Oncology, Hospital of Navarre, Pamplona, Spain. Address reprint requests to Elena Villafranca, MD, Department of Oncology, Clínica Universitaria de Navarra, Avda. Pío XII, 36, Pamplona, 31008, Spain; email: evillafran{at}unav.es
PURPOSE: Thymidylate synthase (TS) is an important target enzyme for the fluoropyrimidines. TS gene promoter possesses regulatory tandemly repeated (TR) sequences that are polymorphic in humans, depending on ethnic factors. These polymorphisms have been reported to influence TS expression. TS expression levels affect tumor downstaging after preoperative fluoruracil (5-FU)–based chemoradiation. Tumor downstaging correlates with improved local control and disease-free survival. The aim of this study is to correlate TR polymorphisms with downstaging and disease-free survival. PATIENTS AND METHODS: Sixty-five patients with rectal cancer underwent tumor resection after preoperative 5-FU–based chemoradiation. Tumor downstaging was evaluated by comparing the pretreatment T stage with the pathologic stage observed in the surgical specimen. TS polymorphism genotype was determined by polymerase chain reaction amplification of the corresponding TS promoter region, and products of amplification were electrophoresed, obtaining products of 220 bp (2/2), 248 bp (3/3), or both (2/3). The TS polymorphism genotype results were subsequently compared with the downstaging observed and with disease-free survival. RESULTS: Patients who were homozygous for triple TR (3/3) had a lower probability of downstaging than patients who were homozygous with double TR or heterozygous patients (2/2 and 2/3): 22% versus 60% (P = .036; logistic regression). Furthermore, a trend toward improved 3-year disease-free survival was detected in the 2/2 and 2/3 groups, compared with that in the 3/3 group (81% v 41%; P = .17). CONCLUSION: This preliminary study suggests that TS repetitive-sequence polymorphisms are predictive for tumor downstaging. TR sequences in TS promoter may be useful as a novel means of predicting response to preoperative 5-FU–based chemoradiation.
CURRENTLY, FLUOROURACIL (5-FU) is the most important chemotherapy agent used in the treatment of rectal cancer. In addition, it is a good radiosensitizer1 and, when given concurrently with radiation, improves local control rates and overall survival, compared with results seen with radiation alone.2 Traditionally, 5-FU–based chemoradiation has been used postoperatively. However, there is a trend toward the preoperative use of 5-FU–based chemoradiation because it induces tumor shrinkage, facilitates complete resection, and maximizes the probability of performing a sphincter-saving procedure.3 Some patients treated with preoperative 5-FU–based chemoradiation experience a notable tumor downstaging effect, which has been correlated with an improvement in both local control and disease-free survival.4-7 Multiple factors may be predictive for tumor downstaging. Clinical factors include performance status, tumor stage, tumor mobility, and circumference of the rectal wall involved by tumor.4 Treatment factors include radiation dose8 and time elapsed from radiation to surgery.9 In addition, there are some biologic factors that may be involved in the tumor response process: thymidylate synthase (TS) expression, mutations of the p53 gene,10,11 and BAX protein expression levels.12 TS is an enzyme that catalyzes transformation of deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymydine-5'-monophosphate (dTMP), which is essential for DNA replication. TS is an important target enzyme for the fluoropyrimidines. An active metabolite of fluorouracil, 5'-fluorodeoxyuridylate (5FdUMP), prevents DNA synthesis by forming stable complexes with TS and folate cofactor, thus blocking the conversion of dUMP to dTMP. Information regarding TS levels before the initiation of the chemoradiation treatment may be important because patients with low TS levels have a better response to 5-FU than do those with higher TS levels.13 TS expression is a tightly controlled process regulated by the proliferative status of the cell. Special 28-base pair–sized, tandemly repeated (TR) sequences have been described in the region downstream of the cap site, which contribute to the efficiency of TS gene expression. No DNA-binding factor capable of coupling with TR has been identified; therefore, a role in posttranscriptional regulation has been proposed.14,15 Kaneda et al14 constructed mutant TScDNA clones with different TScDNA plasmids introduced into TS-negative mutants of murine FM3A cells. These plasmids are constructed with different restriction endonucleases to delete part of the TTR. They use a transient expression system: (3H)dUrd is incorporated into the cells and then converted to deoxyuridylate by phosphorylation. The subsequent reductive methylation to thymidylate is catalyzed by TS, and the thymidylate formed is incorporated into DNA. If (3H)dUrd is used as a substrate, the amount of radioactivity incorporated into the cells that have been transfected with the TScDNA must reflect the amount of transiently expressed TS activity. Although (3H)dUrd can be incorporated into RNA via uridylate, the amount incorporated was less than 0.5% of that incorporated into DNA. Those authors concluded that the first and the second upstream of the third TR elements have an inhibitory effect on the translation efficiency of TS mRNA and that the third element has an activator effect. The inhibitory effect of the first and the second elements on the translation efficiency of TS mRNA may result from the formation of a stem-loop structure by association of the inverted repeated sequence CGCCGCG within the TTR sequence. In 1995, Horie et al15 made a similar study. They examined the role of the TTR in the expression activity of TS gene. They constructed deletion mutants of this region and examined expression activity using another transient expression assay. They concluded that the mutant clones with the three sequences had a higher efficiency of expression than did the mutant clones with the first and second sequences. So the polymorphisms of the TTR contributed to the efficiency of expression of the TS gene. This is in agreement with other studies reporting that TS expression is primarily controlled on the posttranscriptional level.16 There are three predominant genotypes of TR: (a) homozygous with two TR (2/2), (b) homozygous with three TR (3/3), and (c) heterozygous with both alleles (2/3). Novel alleles with more TR have also been described, but these appear very infrequently.17 Direct correlation between TR genotype and the efficiency of TS expression has been reported in both in vitro and in vivo studies.17,18 Hence, the assessment of the TS genotype may be useful in selecting for 5-FU therapy only those patients who have a higher probability of response.19 Patients with a TS genotype that is predictive for a poor response may be directed to other treatment options from the beginning. The present study has been designed to determine whether the different TR genotypes may be predictive for tumor downstaging and disease-free survival (DFS) in patients with rectal cancer treated with preoperative 5-FU–based chemoradiation and surgery.
Between July 1996 and June 1999, 65 patients with rectal cancer at American Joint Committee on Cancer stages II and III were enrolled in this study. Patients were treated in the Hospital de Navarra, Pamplona, Spain and in the Clínica Universitaria, University of Navarre, Pamplona, Spain. Tumor and patient characteristics are described in Table 1.
Pretreatment evaluation included physical examination, pancolonoscopy, complete blood cell count, biochemical profile, carcinoembryonic antigen (CEA) and CA 19.9 tumor markers, abdominopelvic computed tomography (CT) scan, and chest x-ray or thoracic CT scan. Transrectal ultrasound was used in the last 25 patients. Fresh tissue samples were obtained before the initiation of the treatment program. All patients received preoperative radiation with 15- or 18-MV photons using 3 to 4 fields, except for three patients who were treated with a 60Co unit. The median dose prescribed was 45 Gy (range, 45 to 54 Gy). Chemotherapy was based on 5-FU, modulated by different drugs depending on the institutional policy of the center where the patient was treated: 5-FU (325 mg/m2 IV in 30 minutes) and folinic acid (20 mg/m2 IV in 30 minutes, 5 days per week, in the first and fifth weeks of radiotherapy) in nine patients; Tegafur (400 mg q12 hours PO) and levovorin (5 mg q12 hours PO, 5 days per week during radiotherapy) in 19 patients; 5-FU (800 mg/m2 [maximum 1,500 mg] 24-hour IV infusion) and carboplatin (55 mg/m2 in 1 hour IV, 5 days per week, in the first and the fifth week of radiotherapy) in 17 patients; 5-FU (800 mg/m2 [maximum 1,500 mg] 24-hour IV infusion), oxaliplatin (20 mg/m2 IV in 30 minutes in 20 patients, 5 days per week, in the first and the fifth week of radiotherapy). Surgery was performed 4 to 6 weeks after completion of chemoradiation. The surgical procedure included abdominoperineal resection in 17 patients (26%), low anterior resection in 36 patients (56%), Hartman procedure in 4 patients (6%), and exploratory laparotomy without resection in 8 patients (12%). Tumor downstaging was determined by comparison of pretreatment T stage (determined by radiographic or ultrasound staging) and the pathologic stage observed in the surgical specimen.20-22 Thirty patients (45%) with residual tumor or positive nodes in the surgical specimen received adjuvant chemotherapy with 5-FU (425 mg/m2 IV in 30 minutes) and folinic acid (20 mg/m2 IV in 30 minutes after the surgery, 5 days per week, every 4 weeks in nine patients); 5-FU (800 mg/m2 [maximum 1,500 mg] 24-hour IV infusion, 5 days), folinic acid (20 mg/m2 IV in 30 minutes, 5 days), and levamisole (50 mg per 8 hours PO, 3 days, with a 14-day schedule every 4 weeks) in 21 patients.
DNA Extraction and Polymerase Chain Reaction for TS TR Polymorphisms Polymerase chain reaction (PCR) analysis was performed in a total volume of 50 µL: 1 µL (250 ng) of DNA, 1.5 µL (40 pmol) of each specific sense and antisense primer, 0.5 µL (2.5 U) of AmpliTaq Gold DNA Polymerase, 5 µL of 10x Gold PCR Buffer, 3 µL of 25 mmol/L MgCl2, 4 µL of 10 mmol/L mix of dNTP, 5 µL of dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany), and water to 50 µL on a GeneAmp PCR System 9700 (Perkin Elmer, Foster City, Norwalk, CT). All PCR reagents were obtained from PE-Biosystem (Madrid, Spain). Primers (5'-GTG GCT CCT GCG TTT CCC CC-3' [sense] 5'-CCA AGC TTC GCT CCG AGC CGG CCA CAG GCA TGG CGC GG-3' [antisense])14 were synthesized on the Oligo 1000M DNA Synthesizer (Beckman Instruments Inc, Fullerton CA). After 30 cycles of amplification (denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 2 minutes), amplification products were electrophoresed in 3% agarose gel. Products of 220 bp (2/2), 248 bp (3/3), or both of these products (2/3), depending on the TS TR genotype, were obtained ( Fig 1).
Statistical Analysis Different variables that might influence downstaging were analyzed by using the  2 or Fisher’s exact two-tailed test. Logistic regression analysis was used to assess the relative influence of different prognostic factors on downstaging after preoperative chemoradiation.23 Because of the limited number of events, only a limited model could be run; however, this model included all of the variables that were predictive for downstaging after preoperative chemoradiation by univariate analysis. Overall survival (OS) and DFS were calculated from the start of chemoradiation until death, progression, or last follow-up visit. OS and DFS were calculated using the Kaplan-Meier method.24 Univariate analyses were performed to determine the statistical value of the studied factors, and differences in survival were calculated using the log-rank test.
Tumor Downstaging Pathologic evidence of tumor downstaging after preoperative chemoradiation was observed in 29 patients (44%); in 30 patients (47%), the disease remained stable, and only 6 patients (9%) had evidence of progression. The pathological stages for the entire series were as follows: pT0, 9 patients (14%); pT1, 3 patients (5%); pT2, 21 patients (32%); pT3, 23 patients (35%); pT4 1, patient (2%); and unresectable tumor in 8 patients (12%).
TS TR Polymorphisms: Distribution in the Sample
Correlation Between TS TR Polymorphisms and Downstaging The TS TR genotype was the most important predictor of downstaging. Patients with the 3/3 genotype had a lower probability (22%) of downstaging after preoperative chemoradiation than did those with 2/3 and 2/2 genotypes (60%); ( 2 = 9.37, P = .002; Fig 3).
Among all patients who had a complete pathological response after preoperative chemoradiation (pT0), 11% had the 3/3 genotype, and 89% had the 2/3 and 2/2 genotypes (Fisher’s exact two-tailed test, P = .06).
TS TR polymorphism was also a predictor of nodal pathological stage. In the 11 patients with positive pathological nodes, 82% had the 3/3 genotype, and 12% had the 2/3 and 2/2 genotypes (
Predictive value of other variables over downstaging was also analyzed. In univariate analysis, the pretreatment CEA level was the only variable significantly associated with downstaging. Fifty-four percent (27/50 patients) of the patients with CEA of less than 6 ng/mL experienced downstaging. If the CEA level was more than 6 ng/mL, only 13% of the patients (2/15 patients) responded to the treatment (
Logistic regression analysis of these prognostic factors indicated that only the TS TR polymorphisms genotype was an independent predictor of downstaging after preoperative chemoradiation. The relative probability of achieving downstaging among patients homozygous for double TR and heterozygous patients (2/2 and 2/3) was 3.7-fold higher than for the patients homozygous for triple TR (3/3). TS TR genotype was a major predictor of downstaging, independent of pretreatment CEA level, stage, and the type of chemotherapy ( Table 3).
Correlation Between TS TR Polymorphisms and DFS and OS After a median follow-up of 23 months (range, 7 to 48 months), the 3-year DFS was 58% for the entire series. The 3-year local control was 82% (87% with completion of the protocol, including surgery). Local failures included eight patients (12%) with unresectable disease after preoperative chemoradiation and three local relapses (4%) after surgery. Eight patients (12%) presented distant metastases. Patients with downstaging had a longer 3-year DFS (88%) than those without downstaging (30%) (P = .017; log-rank test; Fig 4). In patients with pT0, the 3-year DFS was 100%, compared with 79% in patients with residual disease (P = .09).
Patients with the 3/3 genotype had a 41% 3-year DFS, compared with 81% in patients with 2/3 and 2/2 genotypes (P = .17; log-rank test; Fig 5). This difference does not reach statistical significance, probably because of the limited follow-up period.
A significant difference in DFS has also been observed in univariate analysis with regards to the pretratment CEA (P = .009) and the pretreatment stage: T2-3 versus T4-other (P < .001).The patients who received adjuvant chemotherapy had a worse prognosis, with a 36% 3-year DFS compared with 85% in patients who did not receive adjuvant chemotherapy. After a median follow-up of 23 months, seven patients (11%) died of disease progression, and another two patients (3%) died of unrelated causes. The 3-year OS and cause-specific survival were 74% and 76%, respectively. In statistical analysis, none of the factors studied were predictive for overall survival.
In the present study, tumor downstaging has been correlated significantly with the TS TR genotype in a logistic regression analysis (P = .036). The downstaging effect observed in patients homozygous for double TR and heterozygous patients (2/2 and 2/3) was 3.7 times higher than in the patients homozygous for triple TR (3/3). Twenty-two percent of the patients in the 3/3 group experienced a downstaging effect, compared with 60% in the 2/3 and 2/2 groups combined. In addition, the genotype 3/3 was a predictor of a lower pT0 rates and an indicator of nodal-positive pathological stage (P = .005). The presence of positive nodes after preoperative chemoradiation has been correlated with an increase in the incidence of distant metastases and a poorer overall survival rate.25 A direct correlation between TR genotype and the efficiency of the TS expression has been reported both in vitro and in vivo.26 In vitro, the presence of a triple TR showed a 2.6-fold higher TS expression than did double ones.18 It was reported that TS levels were significantly higher in patients with gastrointestinal cancer and the 3/3 genotype than in with those with 2/2 and 2/3 genotypes.17 High levels of TS determine a lower response to 5-FU19,27,28 and a decrease in DFS and OS. In the analysis of the TS expression level in 294 patients of the 801 patients enrolled in the NSABP R-01 protocol, the 5-year OS was 60% in patients with low TS compared with 40% in patients with high TS levels (P < .01).29 So it is possible that patients with the TS TR 3/3 genotype have higher expression levels of TS. In the present study, the 3/3 genotype correlated with a lower downstaging effect after preoperative chemoradiation (22% v 60%, P = .002) and a decrease in DFS (41% v 81%, P = .17), compared with those seen in the 2/2 and 2/3 groups. Different methods to measure TS expression and activity exist. The classical assay for TS-activity determination (high-performance liquid chromatography with output monitored by the radioactive flow detector) is labor intensive and expensive. Quantitative reverse transcriptase PCR allows quantification of basal and chemotherapy-induced TS mRNA expression levels in peripheral mononuclear cells and tumor biopsies. Although this method has shown a good correlation between TS mRNA expression and TS protein levels,30 its accuracy is disputable. Thus, a genotyping approach is proposed. PCR amplification of the TS TR polymorphic regions as an indirect method for determination of the TS expression levels seems to be a good alternative to the above mentioned methods. The strong correlation observed in the present study between the TS TR genotype and the downstaging effect after preoperative 5-FU–based chemoradiation might be obscured by the fact that patients were treated with four different 5-FU–based combinations. However, multivariate analysis ruled out the type of 5-FU–based combination as a predictor of downstaging (P = NS). Subset analysis by type of 5-FU combination shows that the rate of downstaging is dependent on the number of patients with favorable TS TR genotype included in each group rather on than the specific 5-FU combination used. In fact, the 14% pT0 rate observed in this study is similar to the 15% to 25% pT0 (or minimal microscopically residual disease) rate reported in the literature for preoperative 5-FU–based chemoradiation.3,31-34
The presence of downstaging may be an important clinical predictor for both local control and disease-free survival. In our series, the 3-year DFS was 30% in the patients without downstaging, compared with 88% in the patients with downstaging. In the M. D. Anderson Cancer Center series,4 the 3-year DFS was 61% for patients without downstaging, compared with 87% for patients with downstaging (P < .001). In addition, the presence of downstaging may facilitate the performance of a sphincter-saving procedure. In the present study, sphincter-saving procedures for tumors located less than 6 cm from the anal verge were performed in 50% of the patients with downstaging but only in 18% of the nonresponding patients ( The presence of downstaging was a strong predictor of DFS but did not significantly influence overall survival in the present study. This may be due to the shortness of the follow-up period, with a median of only 23 months. In other studies with a longer follow-up, the presence of downstaging was an important prognostic factor for cancer-specific survival. Kaminsky et al7 reported a 5-year cancer-specific survival of 100% in patients with downstaging, compared with only 45% in patients without downstaging. The data presented in this manuscript need to be confirmed in a larger prospective study with longer follow-up. However, the results suggest that TS TR polymorphisms might serve as a novel prognostic factor for predicting the probability of downstaging after preoperative 5-FU–based chemoradiation in rectal cancer patients. In addition, the pretreatment determination of the TS TR genotype might be useful to select the chemotherapy combination that the best probability of response for each particular patient. In patients with the 5-FU resistant 3/3 subgroup, which has a low probability of response, other TS-targeting chemotherapy agents may be an alternative. Ethnic variations of the TS TR genotype have been reported in the literature. The frequency for homozygous triple TR (3/3) in the Caucasian population has been reported to be in the 30% to 37% range.18 In the Latino population, TS TR genotype distribution has been reported to be 16% in the 2/2 group, 42% in the 2/3 group, and 42% in the 3/3 group.35 In the present study with a Mediterranean population, the 42.5% frequency of the 3/3 genotype is slightly higher than that in reports of Caucasian patients. These data differ from those reported for Japanese patients, in whom the allelic frequency of double repeats was 19% and that of triple repeats was 81%.14 Thus, the geographic variation of the TS TR genotype may need to be taken into account when comparing the results of 5-FU series that include patients belonging to different ethnic groups. Finally, if prospective, large-scale trials confirm that the TS TR genotype is a strong predictor of response to 5-FU therapy, it then could be evaluated in other patients with malignant tumors that are commonly treated with 5-FU–based chemotherapy.
We thank María G. Guzman, laboratory technologist, for her technical assistance in the Oncology Laboratory of the Clínica Universitaria de Navarra. We also thank the Departments of Digestive Medicine and Surgery of the Clínica Universitaria de Navarra, Hospital de Navarra, and of Hospital Virgen del Camino for the collection of the samples.
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ABSTRACT
INTRODUCTION
