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Molecular Oncology, Markers, Clinical Correlates |
1 Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California; 2 Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Tokyo, Japan; and 3 Response Genetics, Inc., Los Angeles, California
ABSTRACT
Thymidylate synthase (TS), the target enzyme of the fluoropyrimidine class of drugs, has a 28-bp repeat polymorphism in the promoter region that has been associated with response of tumors to 5-fluorouracil-based therapy. Patients homozygous for the double repeat (2R/2R) in the TS gene have an overall better outcome from treatment than patients homozygous for the triple repeat (3R/3R). However, due to loss of heterozygosity at the TS locus on chromosome 18 in cancer cells, heterozygous 2R/3R individuals can acquire the 2R/loss or the 3R/loss genotype in their tumors. The purpose of this study was to determine whether the response of colorectal cancer to fluoropyrimidine therapy is associated with the resulting tumor TS genotype when loss of heterozygosity occurs in tumor DNA. A total of 30 colorectal cancer patients treated with the fluoropyrimidine-based combination S-1, all of whom had stage IV disease, were studied. The response rate to S-1 in this group of patients was 13 of 30 (43%). The heterozygous 2R/3R genotype was found in 22 of 30 normal tissues, whereas 10 (45%) of the matched cancer tissues showed only the 2R-sequence band (2R/loss), and 7 cancer tissues (32%) showed only the 3R-sequence band (3R/loss). The response rate of the 2R/loss tumor genotype patients was 80% (8 of 10) compared with 14% (1 of 7) in the 3R/loss genotype group (P = 0.029). Patients with tumor 3R/loss genotypes had significantly lower survival than 2R/loss genotypes. Heterozygous patients with a 2R/loss tumor genotype had the same survival as 2R/2R patients, whereas patients with a 2R/3R tumor genotype had a short survival similar to homozygous 3R/3R genotypes. These results show that: (a) response to 5-fluorouracil-based therapy is determined by tumor genotype; and (b) the 3R repeat is a direct negative determinant of outcome.
Introduction
Thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridylate by 5,10-methylenetetrahydrofolate to form thymidylate and dihydrofolate. Because TS is the only de novo source of the thymine base and its reaction is one of the rate-limiting steps in DNA synthesis, inhibition of TS has been a fruitful approach in cancer chemotherapy (1)
. TS is the target enzyme for 5-fluorouracil (5-FU), which for 
50 years has been one of the mainstay drugs for treatment of many cancers. 5-FU exerts its cytotoxic effect through TS inhibition by forming a stable ternary complex among 5,10-methylenetetrahydrofolate, TS, and 5-fluoro-2'-deoxyuridylate, the active metabolite of 5-FU. Since the appearance of 5-FU, other fluoropyrimidine-based therapies such as 5-fluoro-2'-deoxyuridine, UFT, S-1, and capecitabine, as well as folate-based TS inhibitors such as ratitrexed, pemetrexed, and nolatrexed have been developed. In vitro studies have shown that cells become resistant to TS inhibitors by up-regulating TS expression and raising intracellular TS levels (2)
, leading to the expectation that the amount of TS in tumors might be a predictor of response to TS-targeted therapy. Indeed, recent studies have shown that the TS expression does vary considerably among tumors and that sensitivity of various tumors to 5-FU-based chemotherapy is correlated with the intratumoral level of TS (3, 4, 5, 6, 7)
. Moreover, high TS levels in tumors have also been shown to be associated with worse prognosis (8
, 9)
.
The mechanisms by which TS expression is regulated in vivo have not yet been clearly defined. However, it has been suggested that polymorphisms occurring in the TS promoter might be one regulatory factor. The TS gene is known to have a unique 28-bp tandemly repeated sequence in the 5'-untranslated region and is polymorphic in the numbers of this repeat (10) . Most individuals have a double tandem repeat (2R/2R), a triple repeat (3R/3R), or a heterozygous (2R/3R) genotype, although higher order repeats are found in a few cases (11) . It has been reported that the 3R/3R genotype is associated with higher levels of TS gene and/or protein expression than the 2R/2R genotype, suggesting that this thymidylate synthase enhancer region (TSER) polymorphism can regulate TS transcription or translation (10 , 12, 13, 14) . This observation, taken together with the role of TS expression in 5-FU-based chemotherapy, suggested that the TS genotype with respect to the number of tandem repeats might be at least a partial predictor for 5-FU-based chemotherapy. Indeed, several recent clinical correlative studies have obtained preliminary evidence that the genotype of this TSER is associated with response and survival of colorectal cancer (CRC) patients treated with 5-FU-based therapies (14, 15, 16, 17, 18) .
The discovery of TS polymorphisms that might be tumor response determinants was of considerable interest because of the possibility that prediction of tumor response could be done by analysis of readily available normal tissue (e.g., peripheral blood cells). This expectation is based on the assumption that the genotype in the normal tissue would be identical to that in cancer tissue. However, recently obtained evidence shows that this assumption is not always true in the case of TS genotype. A high incidence of loss of heterozygosity (LOH) has been observed at the TS locus in cancer tissues, which leads to modification of TS genotype in the tumor when it is heterozygous in normal tissue (19 , 20) . That is, the occurrence of LOH in individuals who have a heterozygous 2R/3R genotype in their normal tissue would give rise to a tumor with either a 2R/loss or the 3R/loss TSER genotype. Thus, it is possible that patients who are heterozygous and who have LOH at the TS locus in their tumor tissue might experience considerably different outcomes from chemotherapy depending on which allele became deleted during the LOH event.
To address this question, we characterized the TSER tandem repeat genotype in normal and tumor tissue of CRC patients who had been treated with the fluoropyrimidine-based combination S-1 and examined the associations with clinical outcomes. We found that the response to S-1 therapy and survival of heterozygous individuals whose tumors had LOH at the TS locus varied in accord with the TS genotype of the tumor tissue. Thus, in heterozygotic individuals, it is necessary to analyze the tumor tissue for TS genotype to obtain a meaningful prediction of response.
Materials and Methods
Patient Population.
Eligible patients had: (a) a diagnosis of disseminated or recurrent colorectal cancer after surgical operation; (b) a Eastern Cooperative Oncology Group performance status of 0–2 with adequate hematological, hepatic, and renal function; (c) no treatment during the preceding 4 weeks; and (d) a lesion that was measurable by radiological examination.
Treatment.
Patients were treated with S-1 twice daily for 28 days, followed by a 2-week period rest. The S-1 was given orally after breakfast and dinner. As in previous Phase II studies (21
, 22)
, body surface area was used to determine the dose of S-1 administered, as follows: body surface area <1.25 m2, 40 mg; 1.25–1.5 m2, 50 mg; >=1.5 m2, 60 mg. These treatments were repeated until disease progression as determined by the treating physician or at the discretion of the physician.
The protocol was reviewed and approved by an institutional review board and an ethics committee before study activation, and informed consent was obtained from every patient according to the institutional regulations.
Evaluation.
After two cycles of treatment, measurable disease was reassessed. Response criteria were the standard definitions used for national cooperative group trials (23)
. Response was assessed by computed tomography in liver, lymph node, and lung metastases, as well as in primary lesions. To be classified as a responder, a tumor had to have a 50% reduction in the sum of the products of the perpendicular diameters of the indicator lesion without growth of other disease or the appearance of new lesions (23)
.
Microdissection.
A representative formalin-fixed, paraffin-embedded pre-S1 treatment tumor specimen was selected by a pathologist after examination of the H&E stained slides. Ten µm-thick sections were stained with neutral fast red to enable visualization of histology for laser capture microdissection (P.A.L.M. Microlaser Technologies AG, Munich, Germany), which was performed to ensure that only tumor cells were studied. Tissue collected from specimens having no cancer invasion by histopathology was considered to be normal tissue.
RNA Isolation and cDNA Synthesis.
RNA isolation from paraffin-embedded specimens was done according to a proprietary procedure of Response Genetics, Inc. (Los Angeles, CA; United States patent number 6,248,535). After RNA isolation, cDNA was prepared from each sample as described previously (24)
.
Reverse Transcription-PCR.
Relative cDNA quantitation for TS and an internal reference gene (ß-actin) was done using a fluorescence based real-time detection method [ABI PRISM 7900 Sequence Detection System (TaqMan); Applied Biosystems, Foster City, CA], as described previously (25
, 26)
. The primers and probe sequences used are given in Table 1
. The PCR reaction mixture consisted of 600 nM of each primer, 200 nM probe, 2.5 units AmpliTaq Gold Polymerase, 200 µM each dATP, dCTP, dGTP, 400 µM dUTP, 5.5 mM MgCl2, and 1 x Taqman Buffer A containing a reference dye, to a final volume of 25 µl (all reagents Applied Biosystems). Cycling conditions were 50°C for 10 s, 95°C for 10 min, followed by 46 cycles at 95°C for 15 s and 60°C for 1 min. Colon, liver, and lung RNAs (Stratagene, La Jolla, CA) were used as control calibrators on each plate.
|
Statistical Analysis.
The gene expression values are expressed as ratios between two absolute measurements (gene of interest:internal reference gene). The gene expression values in each TS genotype group were analyzed using the Mann-Whitney U test. 
2 for independence test was used to assess the association between TS genotype and response to chemotherapy. The log-rank test was used to measure the association between TS genotype and survival.
Results
A total of 30 primary pre-S1 treatment colorectal cancer specimens from 30 patients, all of whom had stage IV disease, were studied. Thirteen of the patients (43.3%) were classified as responders to S-1, and 17 patients (56.7%) were nonresponders. TS genotype and TS expressions values were obtained for all 30 of the patients. Fourteen of the 30 patients (46.6%) included in the study were male, and the median age was 65 (range, 39–79). The median overall survival time for all 30 patients was 215.5 days (range, 98–627). The median overall survival for patients with S-1 responding tumors was 303 days (range, 139–627) and for nonresponders was 190 days (range, 98–435).
TS Polymorphism and LOH in the Number of TSERRepeat Sequences in Colorectal Normal Tissue and Cancer Tissue.
We obtained PCR fragments with estimated length of 107 and 135 bp (Fig. 1)
, as described previously (14)
. The 107- and 135-bp fragments represent the two- and three-repeat (2R and 3R) sequences, respectively. The TS genotypes were classified into 2R-homozygote (2R/2R), 3R-homozygote (3R/3R), and 2R/3R-heterozygote. The frequency of each genotype in the 30 colorectal normal tissues is shown in Table 2
. In the 22 normal tissues with the 2R/3R genotype the 10 cancer tissues showed only 2R-sequence band (2R/loss), and the 7 cancer tissues showed only 3R-sequence band (3R/loss; Table 3
; Fig. 1
). The rate of incidence of loss of heterozygosity in the TS locus is 45% (10 of 22) of 2R/loss genotype and 31.8% (7 of 22) of 3R/loss genotype (Table 3)
.
|
|
|
. Regarding relapse category, 13 of 30 patients (43%) had liver metastasis. The majority of tumors (29 of 30) were well or moderately differentiated adenocarcinoma histologically.
|
).
|
; Fig. 2
). The median overall survival periods were 308 days (95% confidence interval, 233–418 days) for colorectal patients with 2R/2R or 2R/loss genotype in the cancer tissue. This is significantly longer than that of patients with other genotypes in the cancer tissue (P = 0.002; Table 5
; Fig. 3
).
|
|
).
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The presence of variable numbers of a 28-bp tandem repeat sequence in the TSER has drawn considerable interest recently because of evidence that this is the only genomic lesion besides p53 mutation that may predict to some extent the clinical outcome of patients treated with 5-FU-based therapy. A potential advantage of using a genetic polymorphism as a tumor response marker is because, in contrast to tumor-specific gene expressions and lesions such as p53 mutations, a germ-line alteration in principle could be assayed in normal tissues without the necessity for delving into the tumor to capture tumor tissue samples. Possible genetic instability at the TS locus, however, adds a complicating factor to the use of genotypic analysis of TS polymorphisms in noncancerous tissue for prediction of tumor response.
The TS gene has been localized to the telomeric region of the short arm of chromosome 18 at chromosome band 18p11.32 (27)
. Chromosome 18 is known to be a site of frequent deletions in a high percentage of colorectal cancers (28)
. TS itself is not likely to be the target of deletions because it is an essential gene, but if TS is in proximity to the real target gene(s) of chromosome 18 deletions, it may in some cases be contained within the deleted DNA segment. The idea of LOH at the TS locus was suggested by Zinzindohoue et al. (19)
and was confirmed in our laboratory when analysis of the TS genotype from 2R/3R individuals by gel electrophoresis showed a different ratio between 2R and the 3R bands in tumor tissue samples than in normal tissues (i.e., an allelic imbalance; Ref. 20
). In this previous study (20)
, the observed frequency of LOH at the TS locus was 62% (31 of 50), almost identical to that reported by Zinzindohoue et al. (Ref. 19
; 19 of 30; 63%) in their group of 30 CRC patients. The observed LOH frequency in the set of patients of the present study was even higher (77%), which might be ascribed to the use of laser-capture microdissection of all of the specimens to isolate the areas of tumor cells as free as possible of any stromal tissue. With this technique for purifying tumor tissue, it is possible to observe quite unambiguously, as demonstrated in Fig. 1
, the loss of one or the other TS alleles and, thus, to show definitively the presence or absence of LOH at the TS locus.
If LOH occurs at the TS locus, the tumor genotype of homozygous patients will be the same as that in normal tissue, but in the case of heterozygous 2R/3R individuals, the tumor will have either a 2R/loss or 3R/loss genotype. Because the presence of 3R (either 3R/3R or 3R/2R genotype) has consistently been associated with less favorable clinical outcomes than if 3R is not present (15, 16, 17, 18
, 29)
, the question is raised whether patients with 3R/loss tumors will therefore fare worse than those with 2R/loss tumor genotypes. The salient finding of this study is that the tumor genotype is in fact associated with the outcome of the therapy: 2R/3R patients with a 2R/loss genotype in their tumors had strikingly better results from the treatment than patients with the 3R/loss genotype, both in terms of response rate (80% versus 14%) and survival (333 days versus 203 days). These results strongly support the idea that TSER polymorphism status in the tumor tissue is a determinant of response to 5-FU-based therapy. The data additionally illustrate that the 3R negatively influences tumor response (as opposed to 2R having a positive effect). As shown in Table 5
and Fig. 2
, the survival times of patients who have the heterozygous 2R/3R tumor genotype are short, similar to those of the 3R-only patients, but loss of the 3R allele during LOH results in a 2R/loss tumor genotype with high response rate and long survival.
The mechanism by which the TSER polymorphic repeats affect tumor response is still unclear. The observation of Horie et al. (12)
that the expression activity of a reporter gene linked to the TSER containing a double repeat was lower than that of the gene with the triple repeat suggested the possibility that TSER polymorphisms affect tumor sensitivity to 5-FU by regulating TS gene expression. This hypothesis seemed to be confirmed by the results of Pullarkat et al. (17)
, who found a significantly higher (3.6-fold) TS gene expression and a lower response and survival rate among 3R/3R CRC patients compared with those with 2R/2R genotypes. However, when other available data are considered, the association between the number of TSER polymorphisms and TS gene expression does not appear to be straightforward. We analyzed previously 130 CRC specimens and found no significant difference in TS gene expression between 2R/2R and 3R/3R genotypes. Instead, the 3R/3R tumors had a significantly higher TS protein level, which would also account for lower response rates among 3R/3R patients (14)
. This finding suggested that translational activity producing TS protein from mRNA is affected by TSER polymorphism, as is indeed supported by in vitro experiments showing that the 3R promoter caused an increase in translation activity (14)
. Etienne et al. (29)
analyzed 103 CRC tumors treated with 5-FU/folinic acid and found, interestingly, that 2R/3R tumors had higher TS enzyme activity than either 2R/2R or the 3R/3R tumors, as well as the shortest survival. In the present study, tumors with only 3R genotypes (3R/3R +3R/loss) showed higher mean TS gene expression than those with only 2R genotypes (1.5-fold). The finding that 3R/loss genotypes had higher expression than the 2R/loss genotypes (Table 6)
by a similar amount suggests that the 3R does exert a direct albeit modest up-regulation of TS gene expression. However, the small difference in mean TS gene expression between tumors bearing only 2R or 3R genotypes seems insufficient to account for the rather striking difference in response to S-1 treatment, suggesting that mechanisms other than (or in addition to) TS regulation may be involved. For example, several recent studies suggest that TSER repeats have a role in regulating the levels of folate metabolites, which could affect drug toxicity to patients and antitumor activity of drugs that interact with folate-using enzymes. Significantly lower levels of plasma folates and homocysteine were found in 3R/3R genotype individuals (30)
. This observation might account for the lower toxicity to 5-FU treatment experienced by 3R/3R patients (17)
because elevated levels of plasma homocysteine have been found to be associated with higher risk of drug toxicity (31)
. TSER repeat status has been linked to response of acute lymphocytic leukemia to methotrexate, a dihydrofolate reductase inhibitor, which may also be influenced by folate levels (32)
. Associations have been noted among TSER polymorphisms, folate intake, and risk of colorectal cancer (33)
, as well as with overall risk of developing risk of adult acute lymphocytic leukemia (34)
.
In summary, we have shown that a high percentage of cancer patients undergo LOH at the TS locus in the tumor, and those who have a 2R/3R normal tissue genotype segregate into two groups with different tumor TS genotypes, one of whom can anticipate considerably better clinical outcome from fluoropyrimidine treatment than the other. As far as we know, there is an equal probability of losing either the short or the long allele during LOH, and whether a particular individual will acquire a 2R/loss or a 3R/loss tumor is not predictable. Thus, if these TS gene polymorphisms are to be used as tools for prediction of response and selection of therapy, analysis only of normal tissue is insufficient but tumor tissue must also be analyzed to establish the TS polymorphism status in the tumor. Determination of TS genotype in tumor tissue, in conjunction with TS gene expression measurements, may identify patients who are good candidates for chemotherapy with TS-directed regimens and those who, due to their lower probability of response, should be considered for another type of treatment. Larger prospective clinical trials need to be done to establish more firmly the predictive importance of the TS promoter polymorphisms. Additional work should also be addressed for a better understanding of the mechanism(s) by which these TS gene polymorphisms affect tumor response. In this regard, it would be of interest to determine whether the number of TS repeats predicts response only to TS-directed drugs or also to other, mechanistically unrelated drugs.
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.
Requests for reprints: Peter V. Danenberg, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Room 5318, Los Angeles, CA 90033. Phone: (323) 865-0518; Fax: (323) 865-3478; E-mail: pdanenbe{at}usc.edu
Received 2/10/03; accepted 5/24/03.
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. 
with arrows in it indicates the thymidylate synthase enhancer region (TSER). Arrows represent tandemly repeated sequences and a complementary reverse sequence. The numbers indicate nucleotide position when the first nucleotide of the initiation codon is defined to be +1. The PCR primers are designed to flank the region of the tandem repeats, so that the presence of 3 repeats will give a longer PCR product than a 2-repeat TSER. These products can be separated by gel electrophoresis (below). The upper and lower bands represent PCR products from amplification of the TSER segment containing 3R and 2R, respectively. The numbers refer to different patient cases. Each patient has a heterozygous 2R/3R genotype in normal tissue, as indicated by the presence of both bands. Case 1: loss of heterozygosity (LOH) gives rise to a tumor with a 2R/loss genotype. Case 2: LOH does not happen. Case 3: LOH gives rise to a tumor with a 3R/loss genotype.
