Thymidylate Synthase Expression In Tumors Research Articles

A Study of Thymidylate Synthase Expression as a Biomarker for Resectable Colon Cancer: Alliance (Cancer and Leukemia Group B) 9581 and 89803.

Tumor levels of thymidylate synthase (TS), a target of 5-fluorouracil (5-FU)-based chemotherapy for colorectal cancer, have been studied as a predictive or prognostic biomarker with mixed results. Tumor TS levels were prospectively evaluated in two adjuvant therapy trials for patients with resected stage II or III colon cancer. TS expression was determined by standard immunohistochemistry and by automated quantitative analysis. Tumor mismatch repair deficiency (MMR-D) and BRAF c.1799T > A (p.V600E) mutation status were also examined. Relationships between tumor TS, MMR-D, and BRAF mutation status, overall survival (OS), and disease-free survival (DFS) were investigated in the subset of stage III patients. Patients whose tumors demonstrated high TS expression experienced better treatment outcomes, with DFS hazard ratio (HR) = 0.67, 95% confidence interval (CI) = 0.53, 0.84; and OS HR = 0.68, 95% CI = 0.53, 0.88, for high versus low TS expression, respectively. No significant interaction between TS expression and stage was observed (DFS: interaction HR = 0.94; OS: interaction HR = 0.94). Tumors with high TS expression were more likely to demonstrate MMR-D (22.2% vs. 12.8%; p = .0003). Patients whose tumors demonstrated both high TS and MMR-D had a 7-year DFS of 77%, compared with 58% for those whose tumors had low TS and were non-MMR-D (log-rank p = .0006). Tumor TS expression did not predict benefit of a particular therapeutic regimen. This large prospective analysis showed that high tumor TS levels were associated with improved DFS and OS following adjuvant therapy for colon cancer, although tumor TS expression did not predict benefit of 5-FU-based chemotherapy. The Oncologist 2017;22:107-114Implications for Practice: This study finds that measurement of tumor levels of thymidylate synthase is not helpful in assigning specific adjuvant treatment for colorectal cancer. It also highlights the importance of using prospective analyses within treatment clinical trials as the optimal method of determining biomarker utility.

TS mRNA levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer

The purpose of the study is to analyze the relationship between tumor thymidylate synthase (TS) mRNA expression levels and raltitrexed/pemetrexed/5-FU sensitivity. We collected freshly removed colorectal tumor specimens from 50 patients. Chemosensitivities to anticancer drugs were evaluated by histoculture drug response assay. We adopted quantitative reverse transcription polymerase chain reaction for TS mRNA detection and immunohistochemical staining for assessing TS expression in tumor tissues. There is a significant relationship between TS mRNA expression levels and in vitro chemosensitivity of freshly removed colorectal tumor specimens to pemetrexed (P<0.001)/raltitrexed (P=0.004)/5-FU (P=0.007). TS mRNA expression levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.

Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer

Background:Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.Methods:We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.Results:The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.Conclusion:A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.

Association of thymidylate synthase (TS) expression with response to S-1 plus carboplatin (CBDCA) in non-small cell lung cancer (NSCLC) patients (pts).

e21025 Background: S-1, a novel oral fluoropyrimidine derivative, is active against NSCLC. Phase II studies of S-1 plus platinum compounds have shown activity for first-line treatment of advanced NSCLC. It is important to design an optimal therapeutic strategy according to tumor biology to improve the treatment of NSCLC. In the present study, we evaluated the correlations between immunohistochemical expression of TS, orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD), and response to S-1 plus CBDCA in advanced NSCLC pts. Methods: Chemotherapy-naïve pts with advanced stage (IIIB/ IV) NSCLC, ECOG PS of 0-1, adequate organ functions and archival tumor tissue were assigned to receive either CBDCA AUC 5 mg/mL/min on day 1 plus oral S-1 80 mg/m2/day (40 mg/m2 b.i.d.) on days 1 to 14 (22 pts) or CBDCA AUC 6 mg/mL/min plus paclitaxel (PTX) (200 mg/m2) on day 1 every 21 days (26 pts). Association between TS, OPRT and DPD protein expression levels in tumors and response to chemotherapy was evaluated. Results: There was no statistical difference in TS, OPRT and DPD expression level between S-1 plus CBDCA and PTX plus CBDCA. In the S-1 plus CBDCA groups, TS expression of responding tumors was significantly lower than that of nonresponding ones (p = 0.006), whereas no significant difference was observed in the PTX plus CBDCA groups. Furthermore, patients with low TS expression survived longer than those with high TS expression in patients receiving combination therapy with S-1 and CBDCA, but not in those treated with PTX plus CBDCA. Conclusions: Our results suggest that tumor TS expression levels is predictive markers for response to S-1 plus CBDCA chemotherapy in advanced NSCLC pts. No significant financial relationships to disclose.

A phase Ib/II study of PXD101 alone and in combination with 5-fluorouracil in patients with advanced solid tumors

3501 Background: PXD101 is a hydroxamate HDAC inhibitor that has broad anti-neoplastic activity in vivo and in vitro. Preclinical data showing PXD101 down-regulation of thymidylate synthase (TS), an in vivo target of 5-fluorouracil (5-FU) provide a rationale for combination of PXD101 with 5-FU. Methods: This is a phase Ib/II trial of PXD101 plus 5-FU in patients with advanced solid tumors, with dose escalation to establish the maximum tolerated dose (MTD) and an expansion at the MTD in colorectal cancer (CRC) patients. Patients (pts) must be = 18 yrs, have measurable disease, and had disease progression after standard chemotherapy. PXD101 was administered as a 30-min IV infusion on Days 1–5 of a 3-wk cycle. 5-FU was administered from cycle 2 on, as a continuous 96-hr IV infusion starting on Day 2 of the same 3-wk cycle. The primary endpoints were safety, and effect of belinostat on TS expression in patients’ tumors. Dose-limiting toxicities (DLTs) were PXD101-related = Gr 3 non-hematologic or Gr 4 hematologic toxicities. Expression of TS mRNA was measured by RTQ-PCR. Results: Twenty pts have been treated for a median of 2 cycles (range 1–8). Cohorts of 3–6 pts were tested at 5 dose levels of PXD101/5-FU (mg/m2/d): 300/250, 600/250, 1,000/250, 1,000/500, and 1,000/1,000. Two pts did not complete cycle 2, and one pt at the 600/250 dose level received 1,000 mg/m2/d 5-FU. There were no DLTs in the first 4 dose cohorts; the 5th cohort is ongoing. In the first 4 dose cohorts (17 pts), the most frequent AEs were fatigue, nausea/vomiting, dysgeusia, dehydration, constipation, edema, anorexia and anemia. There were 2 Gr 4 AEs (pulmonary embolism, bacteremia); Gr 3 AEs in = 2 pts were fatigue and dehydration. All Gr 3/4 events were assessed as not related to PXD101. One pt with stage IV CRC treated at 600/1,000 mg/m2/d PXD101/5-FU had stable disease (SD) for 8 cycles, and one pt with stage IV esophageal cancer treated at the 1,000/250 mg/m2/d dose had SD for 4 cycles. Conclusions: PXD101 in combination with 5-FU has been well tolerated up to 1,000/500 mg/m2/d PXD101/5-FU. The study is ongoing and data from the MTD expansion part, including effect of PXD101 on tumor TS expression, will be presented. No significant financial relationships to disclose.

Multifactorial prospective study of tumoral factors related to disease-free survival (DFS) in colorectal cancer patients

3604 Background: There is still a need to identify faithful biological prognostic factors in colorectal cancer, particularly in stage 2, so as to decide upon adjuvant treatment. We thus conducted a prospective multicentric multifactorial study to this end. Methods: Primary colorectal tumors (30 stage 1, 119 stage 2, 107 stage 3) were prospectively collected in 256 patients undergoing total tumor resection (152 men, 104 women ; mean age 69, extremes 29–90). Adjuvant 5FU-based chemotherapy was administered in 92 patients. Median follow-up was 54 months (53 patients developed metastasis or recurrence). Tumors were analyzed for thymidylate synthase (TS), thymidine phosphorylase and dihydropyrimidine dehydrogenase expression (RT-PCR), TS activity (radioenzymatic assay), EGFR level (ligand-binding assay), VEGF (Elisa), DNA content and cell cycle (flow cytometry), p53 mutations (exon 4 to 8), microsatellite instability (bat 25, bat 26), EGFR genotype (CA repeats in intron 1 and -216G&gt;T), TS genotype in 3’ (6 bp deletion) and 5’ (28 bp repeats including the G&gt;C mutation), and methylenetetrahydrofolate reductase genotype (677C&gt;T and 1298A&gt;C). Results: With the exception of tumor TS expression (p = 0.034, the higher the expression, the better the DFS), none of the analyzed parameters were linked to DFS. In multivariate Cox analysis, tumor staging (p = 0.001) and TS expression (p = 0.062) were the sole factors associated to DFS. Focus on tumoral EGFR revealed large inter-patient variability (from 1 to 510 fmol/mg prot) with a significant influence of tumor localisation (p = 0.009, higher in proximal colon) and differentiation status (p = 0.01, higher in poorly differentiated tumors). EGFR within the tumors was 30 % lower than in adjacent normal mucosa (p&lt;0.001). The longer the intron 1 CA repeats, the higher the tumor EGFR (p = 0.044). EGFR -216G&gt;T polymorphism was linked to intron 1 polymorphism, although -216G&gt;T did not influence EGFR levels. Conclusions: The present results underline the major impact of TS expression as a prognostic factor and provide new insights in the knowledge of EGFR in colorectal cancer. No significant financial relationships to disclose.

Polymorphisms in the Thymidylate Synthase Gene Predict Response to 5-Fluorouracil Therapy in Colorectal Cancer

Introduction Advances in the field of pharmacogenomics have enhanced our knowledge of the molecular basis of cancer and provided a link between detection of genetic polymorphisms and response to treatment. Different variations of genetic polymorphisms, such as tandem repeats, single nucleotide polymorphisms, or nucleotide deletions, can lead to alterations in the transcription, translation, or stability of certain gene products that could be critical for the clinical activity and/or toxicity of chemotherapeutic drugs. One such target is thymidylate synthase (TS), an enzyme that catalyzes the reductive methylation of deoxyuridine-5'monophosphate (dUMP) to deoxythymidine-5'-monophosphate (dTMP) in the cell and generates the sole intracellular de novo source of thymidylate, a key precursor needed for DNA biosynthesis. This folate-dependent enzyme is an important target for chemotherapy drugs such as 5fluorouracil (5-FU), fluorodeoxyuridine (FUDR), oral 5-FU prodrugs (eg, uracil/tegafur, S-1, and capecitabine), and other novel folate-based drugs (eg, raltitrexed and pemetrexed). These agents directly inhibit TS enzymatic function, thereby resulting in thymidylate depletion with subsequent DNA damage and cell death. In the treatment of colorectal cancer (CRC), 5-FU–based chemotherapeutic regimens have yielded overall response rates ranging from 10% to 30% for advanced disease1 and provide approximately 15% improvement in 5year survival as adjuvant therapy for stage III disease.2 One key question that remains, however, is how to best predict which patients will benefit from such a therapeutic treatment strategy. Several studies have observed that high levels of TS expression at the protein or messenger RNA (mRNA) level, in the primary tumor or at the metastatic sites, have correlated with a lack of response to 5-FU–based chemotherapy. In contrast, low expression levels of TS have been associated with a positive clinical response. In order for these drugs to be effective in killing a tumor cell, active enzyme levels have to be reduced for a sufficient period for cell cycle arrest to be initiated. If TS expression is increased, then standard drug doses would be ineffective in attenuating enzyme activity. This reasoning has been supported in a retrospective analysis of TS levels, as measured by immunohistochemistry, in tumor specimens of patients with advanced CRC. Among 48 patients who received 5-FU–based therapy, the overall response rate was lower (24% vs. 67%; P = 0.003), and median time to progression (6.2 months vs. 9.6 months; P = 0.005) and median overall survival (OS) times (15.4 months vs. 18.4 months; P = 0.02) were shorter in patients with high tumor TS expression compared with patients with low TS expression.3 Discrepancies have been reported in TS expression levels detected in the primary colorectal tumor versus hepatic metastases. In 14 patients with CRC and hepatic metastases matched samples, TS mRNA levels, as measured by semiquantitative reverse-transcriptase polymerase chain reaction, were higher in the primary tumor (mean, 1.14 ± 0.34) compared with the liver metastases (mean, 0.76 ± 0.37; Wilcoxon test; P < 0.05).4

Thymidylate synthase expression levels: A prognostic and predictive role in advanced colorectal cancer

Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides, required for DNA synthesis, and is also a critical target for fluoropyrimidines, which are widely used in the treatment of gastrointestinal tumours. We examined TS expression in tumours from 86 patients with advanced colorectal cancer who received one of two chemotherapy regimes (either irinotecan alone or irinotecan and 5-flurouracil with folinic acid). TS expression was determined immunohistochemically in 86 paraffin-embedded primary tumour sections and assessed using image analysis software. TS was significantly associated with survival, with lower levels of TS expression associated with longer patient survival (p=0.02). Patients with an objective response to treatment had a longer median survival than those who did not show an objective response to treatment (p=0.001). A significant association was found between tumour TS expression and response to treatment with 5-FU plus FA with irinotecan (p=0.05). Sixty-four percent of patients with TS expression levels below the median showed an objective (complete or partial) response to treatment while, 38% with TS expression levels above the median responded. Immunohistochemical TS expression levels might be used both as a prognostic marker and to help identify patients who would benefit from 5-FU based regime.

Relationship Between Thymidylate Synthase (TS) Genotype and TS Expression: A Tissue Microarray Analysis of Colorectal Cancers

Response of colorectal cancers to 5-fluorouracil appears to be influenced by differences in thymidylate synthase (TS) expression. To explore the relationship between TS 5' genotype and expression, we analyzed paired tumor and normal tissue from 87 colorectal cancers by tissue microarray. A trend to an association between TS genotype and expression was observed, but the correlation was weak. Although the 2R homozygote was preferentially associated with TS expression (p<0.03), no relationship was observed for the 3R homozygote (p=1.0). The relationship between 5' TS genotype and TS expression is not simple. For clinical trials incorporating TS status, detection of TS expression in tumors by immunohistochemistry must still remain the benchmark over genotype.

Thymidylate synthase polymorphism in the development of Barrett's associated adenocarcinoma

9528 Background: Thymidylate synthase (TS), one of the rate-limiting enzymes in the de novo synthesis of DNA, has a unique 28bp tandemly repeated sequence in the promoter region. Patients who are homozygous for the double repeat (2R/2R) of this sequence have been reported to have lower TS expression and better outcome from chemotherapy than patients who are homozygous for the triple repeat (3R/3R). However, loss of heterozygosity (LOH) at the TS locus during tumor development can cause the heterozygous (2R/3R) patients to acquire either the 2R/loss or the loss/3R genotype in their cancer cells, which could affect tumor TS expression as well as clinical outcome. The aim of this study was 1) to verify the relationship between TS mRNA expression and TS genotype in Barrett's associated adenocarcinoma (BA), and 2) to analyze the frequency and timing of LOH at the TS locus in BA and its precursory lesion such as intestinal metaplasia (IM) and high grade dysplasia (HGD). Methods: Sixty-six patients (including 21 with IM, 6 with HGD and 23 with BA) were included in this study. Biopsies were obtained during endoscopy from: (1) normal squamous epithelium at 20cm (HNE); (2) normal squamous epithelium 3cm above the gastroesophageal junction (LNE); (3) IM, HGD or BA. All samples were analyzed for the TS genotype and TS mRNA expression using a quantitative real-time RT-PCR method after laser-capture microdissection. Results: TS mRNA expression from HNE tissue of 3R/3R genotype patients (n=8) was significantly higher than that of 2R/2R genotype patients (1.8-fold; n=14; p=0.0024) and that of 2R/3R genotype patients (1.4-fold; n=44; p=0.0282). Out of 44 patients with heterozygous (2R/3R) genotype in the HNE, no patient lost an allele in the LNE. However, 10 of 18 patients with IM had LOH (55.6%), as had 4/6 patients with HGD (66.7%) and 9/17 patients with BA (52.9%). Conclusions: Our results demonstrate 1) an association between TS genotype and TS expression in normal squamous epithelium and 2) that LOH at the TS locus is a frequent and early event in the IM-BA sequence, and thus tumor and normal genotypes will not be the same in these heterozygous patients. No significant financial relationships to disclose.

Association of gastric and intestinal phenotypic marker expression of gastric carcinomas with tumor thymidylate synthase expression and response to postoperative chemotherapy with 5-fluorouracil.

It is well known that both gastric and intestinal phenotypic markers are expressed in gastric carcinomas, irrespective of their histological type. In the present study, the associations among phenotypic marker expression of gastric carcinomas, tumor thymidylate synthase (TS) expression, and the chemotherapeutic response to 5-fluorouracil (5-FU) were examined. The gastric and intestinal phenotypic marker expression of the tumor was determined by the combination of the expression of human gastric mucin (HGM), MUC6, MUC2, and CD10, and was evaluated in comparison with tumor TS expression in 137 advanced gastric carcinomas in 137 patients (75 with postoperative chemotherapy with 5-FU and 62 without postoperative chemotherapy). Tumors were classified into the gastric- (G-), gastric and intestinal mixed- (GI-), intestinal- (I-), or unclassified- (UC-) phenotype according to the immunopositivity of HGM, MUC6, MUC2, and CD10 stainings. The associations among the gastric and intestinal phenotypic marker expression of the tumor, tumor TS expression, effect of postoperative chemotherapy with 5-FU, and the patient's prognosis were examined. Of the 137 gastric carcinomas, 48 (35.0%), 58 (42.3%), 23 (16.8%), and 8 (5.8%)were classified as the G-, GI-, I- and UC-phenotype, respectively. The high TS expression of more than 25% tumor cell positivity was found in 25 (52.1%) of the 48 G-phenotype tumors, 39 (67.2%) of the 58 GI-phenotype tumors, 18 (78.3%) of the 23 I-phenotype tumors, and 4 (50.0%) of the 8 UC-phenotype tumors. The I-phenotype tumors were significantly correlated with the higher rate of the high TS expression as compared with the G-phenotype tumors (P<0.05). Among 48 patients with the G-phenotype tumor, the 5-year survival rate in patients with and without postoperative chemotherapy was 39.7 and 27.8%, respectively. The patients with postoperative chemotherapy had a significantly better prognosis than those without postoperative chemotherapy (P<0.05). Conversely, there were no significant correlations between the presence of postoperative chemotherapy and the patient's prognosis among patients with GI-, I-, and UC-phenotype tumors. These results indicate that postoperative chemotherapy with 5-FU could be effective for patients with the G-phenotype tumor, since the incidence of intratumoral expression of TS, the target enzyme of 5-FU, is significantly low in G-phenotype tumors.

Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer.

Thymidylate synthase (TS), an essential enzyme in DNA synthesis, is a target for the fluoropyrimidines, an important group of antineoplastic agents used widely in the treatment of head and neck cancer. We evaluated relationships between the level and/or pattern of tumor TS expression and response to fluorouracil (5-FU)-based neoadjuvant chemotherapy in patients with advanced head and neck cancer. Tumor specimens from 86 patients were available for this retrospective analysis. The patients were enrolled in four consecutive phase II studies that tested combinations of 5-FU, leucovorin, and cisplatin with or without added methotrexate plus piritrexim or interferon alfa-2b (IFN alpha-2b). TS protein expression in the tumors was assessed by use of the TS 106 monoclonal antibody and standard immunohistochemical staining techniques. TS immunostaining was classified according to its level of intensity (TS 0-1 = low, TS 2 = intermediate, and TS 3 = high) and according to its extent (focal pattern = less than 25% of tumor cells positive; diffuse pattern = greater than or equal to 25% of tumor cells positive). Data from 79 patients were available for an analysis of tumor TS expression and patient/tumor characteristics; 70 patients were assessable for their response to neoadjuvant chemotherapy. There was a statistically significant association between the level of tumor TS expression and the degree of tumor differentiation; a higher proportion of patients whose tumors exhibited TS 0-1 immunostaining had undifferentiated or poorly differentiated tumors than patients whose tumors exhibited TS 2 or TS 3 immunostaining (P = .04, Jonckheere-Terpstra trend test). Among the 70 patients who were assessable for response to neoadjuvant chemotherapy, TS 3 tumor immunostaining was associated with a lower rate of complete response (i.e., complete disappearance of clinically detectable disease for a minimum of 4 weeks from time of initial determination) than was TS 2 or TS 0-1 immunostaining, but this association was not statistically significant (P = .09, exact trend test); among the 39 patients who were treated with regimens that included 5-FU, leucovorin, cisplatin, and IFN alpha-2b, this inverse association between TS immunostaining intensity and response was statistically significant (P = .02, exact trend test). Tumor TS immunostaining intensity and overall survival were not found to be associated. Patients with tumors exhibiting a focal pattern of TS immunostaining have experienced significantly longer survival than patients with tumors exhibiting a diffuse pattern; for the 53 patients with diffuse tumor TS immunostaining, the median survival was 24.7 months, whereas the median survival has not yet been reached for the 22 patients with focal tumor TS immunostaining (P = .04, two-tailed logrank test). However, the survival advantage for the focal versus diffuse TS immunostaining pattern was limited to patients whose tumors also exhibited a TS 3 level of immunostaining intensity. Characterization of tumor TS expression may be of value in identifying patients with advanced head and neck cancer who would benefit from fluoropyrimidine-based neoadjuvant chemotherapy.