High Thymidylate Synthase Expression Research Articles

1240P - Randomized phase 2 trial of gefitinib with and without pemetrexed as first-line therapy in East Asian patients with advanced, epidermal growth factor receptor (EGFR) mutation-positive (mt+), nonsquamous (NS) non-small cell lung cancer (NSCLC): Translational research and interim overall survival (OS)

The primary analysis of this open-label, randomized, multicenter, phase 2 study found significantly longer progression-free survival (PFS) for pemetrexed plus gefitinib (P + G) vs gefitinib alone (G) in 191 East Asian patients with EGFR mt+ NS NSCLC (adjusted hazard ratio [HR]: 0.68; 95% confidence interval [CI]: 0.48-0.96; P = 0.029). Translational research, interim OS data and updated safety results are reported. Treatment-naive patients with advanced, EGFR mt+ (local laboratory testing), NS NSCLC were randomized (2:1) to receive P + G (pemetrexed 500 mg/m2, Day 1 of 21-day cycles plus gefitinib 250 mg/day; n = 126) or G (gefitinib 250 mg/day; n = 65). Mandatory pretreatment tissue samples were assayed for EGFR mutations by central evaluation (CE) (n = 159) and thymidylate synthase (TS) by immunohistochemistry (n = 146). OS in the intention-to treat (ITT) population and correlation of biomarkers with clinical outcomes were analyzed by adjusted Cox regression. By CE, 149/159 patients (93.7%) were EGFR mt+. In the CE+ population, PFS was longer for P + G (n = 96) vs G (n = 53) (HR: 0.72; 95% CI: 0.48-1.06; P = 0.093), consistent with the primary analysis based on local testing. In patients with low (n = 67) and high (n = 79) TS expression, the PFS HR was 0.47 (95% CI: 0.26-0.87) and 0.83 (95% CI: 0.47-1.46), respectively; the interaction effect was not statistically significant (P = 0.177). At the interim OS analysis (80 events, 58% censoring), OS was not significantly different for P + G vs G (HR: 0.79; 95% CI: 0.49-1.28; P = 0.342). Postdiscontinuation systemic therapy was common (P + G: 62.7%; G: 72.3%). The incidence of grade 3/4 drug-related adverse events was significantly (P = 0.002) higher for P + G (42.9%) vs G (20.0%). The PFS HR for the CE+ population was consistent with the primary ITT analysis. No significant interaction effects were seen for TS. At this interim analysis, there was no statistical difference in OS for P + G vs G. Final OS analyses will be reported when 130 events have occurred.

Immunohistochemical analysis of thymidylate synthase expression in gastric carcinoma: correlation with clinicopathological parameters and survival.

The correlation of thymidylate synthase (TS) expression in gastric cancers with tumor histology and prognostic or predictive information remains unclear. Most studies have involved Asian populations, with few conducted in European cohorts. Moreover, all published studies analyze TS expression using semi-quantitative methods. This retrospective study evaluated the association of TS expression in tumor cells with gastric carcinoma histological type, with selected clinicopathological parameters, and with the prognosis of patients who underwent surgical treatment. TS expression was detected using immunochemistry and objectively assessed by computerized image analysis of tumor cells in 100 gastric cancers. We found that high TS expression was significantly more common in intestinal than in diffuse type of gastric cancer according to Lauren classification (P=0.0003); in type I carcinomas compared to type IV according to Goseki classification (P=0.002); and in gastric cancers in men than women (P=0.04). Low TS expression was found more often in carcinomas in the middle and lower third of the stomach than in cancers in the upper third of the stomach (P=0.009 and P=0.001, respectively). In the subgroup of 25 patients without lymph node metastases (stage I+II), high TS expression was associated with better DFS (83% for high TS expression versus 38,5% for low TS expression, P=0.03). The results (1) indicate significant correlation between the Lauren and Goseki histopathological classifications of gastric cancer and TS expression in tumor cells, (2) suggest that high TS expression may be a positive prognostic marker with regard to DFS in patients with gastric cancer without involvement of regional lymph nodes who underwent radical surgical treatment and were not treated with preoperative chemotherapy. Prognostic results need confirmation in larger cohorts.

Association of thymidylate synthase expression and clinical outcomes of gastric cancer patients treated with fluoropyrimidine-based chemotherapy: a meta-analysis.

PurposeAlthough several studies have suggested an association between thymidylate synthase (TS) expression and outcomes of gastric cancer (GC) patients treated with fluoropyrimidine-based chemotherapy (FUC), the predictive value of TS for response and survival in this setting is unclear. This meta-analysis aimed to estimate prognostic and predictive significance of TS more precisely.MethodsWe searched PubMed, Embase, Cochrane Library, and Web of Science databases for literature published up to June 2015. Primary outcomes included hazard ratios (HRs) for overall survival (OS), and event-free survival (EFS) and odds ratio (OR) for chemotherapy response. Fixed- or random-effects models were used to calculate pooled HR and OR according to heterogeneity.ResultsA total of 2,442 GC patients in 25 studies met our inclusion criteria. Response rates for FUC were significantly lower in patients with high TS expression than in those with low expression (OR: 0.43, 95% confidence interval [CI]: 0.22–0.84, P=0.013). High TS expression was significantly correlated with unfavorable OS (HR: 1.62, 95% CI: 1.28–2.05, P<0.001) and EFS (HR: 1.54, 95% CI: 1.22–1.93, P<0.001) in advanced disease. However, TS expression was not significantly related to OS (HR: 1.06, 95% CI: 0.74–1.50, P=0.760) or EFS (HR: 1.16, 95% CI: 0.84–1.61, P=0.374) in the adjuvant setting.ConclusionHigher TS expression might predict drug resistance and adverse prognosis in patients with advanced GC treated with FUC.

Prognostic implications of thymidylate synthase gene polymorphisms in patients with advanced small bowel adenocarcinoma treated with first-line fluoropyrimidine-based chemotherapy.

Thymidylate synthase (TS) gene polymorphisms such as tandem repeat (TR) polymorphisms and single-nucleotide polymorphisms (SNPs) affect transcriptional efficiency of the TS gene and may be prognostic markers for fluoropyrimidine-based therapy in various gastrointestinal cancers. However, data for TS polymorphisms on clinical outcomes in advanced small bowel adenocarcinoma (SBA) are limited. We retrospectively enrolled 58 locally advanced/metastatic SBA patients treated with first-line fluoropyrimidine-based chemotherapy and analyzed the relationship between TS genotypes and clinical outcomes in 30 patients who were available for tumor tissue. Based on TR polymorphisms and a G>C SNP in the promoter region of the TS gene, 74% of patients had high TS expression genotypes (2R/3RG, 3RG/3RC, 3RG/3RG); the remainder had low TS expression genotypes (2R/2R, 2R/3RC, 3RC/3RC). After a median follow-up of 48.8 months, median progression-free survival (PFS) and overall survival (OS) in all patients were 6.0 and 11.3 months, respectively. However, patients with low TS expression genotypes had better median PFS (12.8 vs. 4.3 months, P=0.027) and OS (28.8 vs. 8.9 months, P=0.025) than those with high TS expression genotypes. In multivariate analysis, poor Eastern Cooperative Oncology Group performance status [hazard ratio (HR), 2.85; 95% CI, 1.02-7.93] and high TS expression genotypes (HR, 3.49; 95% CI, 1.13-10.78) were independent prognostic factors for worse OS. Therefore, TS genotypes, based on a G>C SNP in the TR sequence of the TS gene, may be a useful biomarker for predicting outcomes for fluoropyrimidine-based chemotherapy in patients with locally advanced/metastatic SBA.

Expression Analysis of Genes Involved in DNA Repair or Synthesis in Mixed Neuroendocrine/Nonneuroendocrine Carcinomas

Background: Mixed neuroendocrine/nonneuroendocrine carcinomas are heterogeneous tumors with poorly defined diagnostic and clinical features and without pathological or molecular markers of prognosis or markers predicting their response to therapy. We aimed at analyzing the pathological features and the expression of genes involved in DNA repair or synthesis in a cohort of patients with mixed carcinomas from different sites as compared to the patients' outcome. Methods: Relative cDNA quantification of ribonucleotide reductase, large subunit 1, excision repair cross-complementation group 1, thymidylate synthase and topoisomerase IIa genes was tested using real-time PCR on microdissected neuroendocrine and nonneuroendocrine tumor components of 42 mixed cases (from the lung as well as the gastrointestinal and genitourinary tracts) and on 45 control cases of pure neuroendocrine and nonneuroendocrine carcinomas. Results: The expression levels of all genes were stable comparing nonneuroendocrine and neuroendocrine components of mixed cases (except for topoisomerase IIa in lung samples) but significantly different as compared to control nonneuroendocrine and neuroendocrine tumors. In the multivariate analysis including all clinical and pathological parameters and gene expression levels available, a predominant nonneuroendocrine component, the administration of additional therapy other than surgery and a high thymidylate synthase expression in nonneuroendocrine tumor tissue were significantly associated with a lower risk of a patient's death. Conclusions: Our data show that mixed neuroendocrine/nonneuroendocrine carcinomas are different at the molecular level from their pure neuroendocrine and nonneuroendocrine counterparts, and detailed analyses of their clinical, pathological and molecular features may improve the clinical strategies for the treatment of these rare and underestimated tumors.

Predictive value of thymidylate synthase for the prognosis and survival of lung adenocarcinoma patients.

Chemotherapy represents an important treatment modality for lung adenocarcinoma. Thymidylate synthase (TS) is an essential enzyme in DNA synthesis, and its overexpression has been associated with reduced sensitivity to antifolate agents. The aim of the current study was to investigate the expression of TS and the effect on prognosis in lung adenocarcinoma patients. Adenocarcinoma and adjacent carcinoma tissues were resected from 100 patients with lung adenocarcinoma and the TS levels were detected by immunohistochemical analysis. The values for overall survival (OS) and disease-free survival (DFS) were determined using the Kaplan-Meier analysis. The results indicated that the TS protein was expressed predominantly in adenocarcinoma tissues, which exhibited higher TS expression compared with the adjacent tissues (P<0.001). The statistical analysis indicated that TS expression was associated with the clinical stage and history of smoking (P<0.05). The Kaplan-Meier analysis results indicated that the DFS and OS in patients with high TS expression levels were significantly shorter compared with those with low expression levels (P<0.05). In conclusion, the results from this study suggested that TS may serve as an independent predictive factor for survival rate, which may indicate the prognosis of lung adenocarcinoma patients.

Prognostic significance of thymidylate synthase expression in pancreatic adenocarcinoma: A meta-analysis.

The prognostic significance of thymidylate synthase (TS) overexpression in pancreatic adenocarcinoma has been extensively investigated; however, data on the survival of patients with pancreatic adenocarcinoma remain equivocal. We performed a meta-analysis of previous studies to assess the effects of TS overexpression on the overall survival (OS) of patients with pancreatic adenocarcinoma, using hazard ratio (HR) with its 95% confidence interval (CI). A total of 5 studies, including 425 patients, were subjected to the final analysis. The pooled HR was 0.72 (95% CI: 0.41-1.25; Z=1.18, P=0.238), indicating that TS expression exerted no significant survival effect on patients with pancreatic adenocarcinoma. The combined HR was 0.46 (95% CI: 0.31-0.68; Z=3.95, P<0.001), limiting the analysis to the studies assessing R0 resection patients, which indicated that a high expression of TS was significantly correlated with better OS in patients with pancreatic adenocarcinoma who underwent R0 resection. This meta-analysis identified TS as an independent factor predicting favourable outcome following R0 curative resection in patients with pancreatic adenocarcinoma.

Abstract LB-220: Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS)

Abstract Background: Tumor resistance to chemotherapy is a major challenge in cancer therapeutics today. 5-Fluorouracil (5-FU) is one of the most commonly used cancer therapeutic agents. The Forkhead box transcription factor M1 (FOXM1) has been shown to play vital roles in cell cycle progression, apoptosis, and cell survival. FOXM1 is over expressed in many types of cancers, including colorectal carcinoma and has been implicated in drug resistance in breast cancer. Thymidylate synthase (TS) enzyme is an important precursor in DNA synthesis. The mechanism of action of 5-FU has been associated with TS. High TS expression levels in tumors are generally related to 5-FU resistance. In this study we investigated the role of FOXM1 in 5-FU resistance and the therapeutic potential of the FOXM1 inhibitor thiostrepton (a thiazole antibiotic) in combination with 5-FU in colorectal cancer. We further analyzed the relation between TS and FOXM1 gene expressions in colorectal cancer. Methods and Results: HCT116, DLD1, and HT29 cells were treated with 5-FU to detect the protein, mRNA expression of FOXM1 and TS. This resulted in a decrease in TS and FOXM1 expression in p53 wild type HCT116 cells but not in p53 mutant DLD1 or HT29. We also observed that FOXM1 expression was significantly correlated with TS expression by thiostrepton treatment. The IC50 of 5-FU was determined by SRB assay and was found to be 0.92ug/mL, 0.45ug/mL and 2.8ug/mL, while thiostrepton IC50 was found to be 0.60uM, 1.07uM, and 3.54uM, in HCT116, DLD1, and HT29 cells respectively. Combination of 5-FU and thiostrepton showed synergistic effect in all 3 colorectal cancer cells. Chromatin immunoprecipitation (ChIP) was performed in HCT116 and DLD1 cells to determine if FOXM1 binds to the TS promoter region (0 - 2000bp upstream of the TS transcription start site). Three negative primers were designed further to 2000bp upstream region. ChIP data revealed enrichment for FOXM1 at the TS promoter (0 - 1500 bp upstream) and no enrichment observed in the negative control. Inhibition of FOXM1 by thiostrepton led to a decrease in enrichment in the TS promoter region. These results confirmed the role of FOXM1 in TS regulation. Chip Sequencing (ChIP-seq) was then performed to study further FOXM1 targets and binding sites of FOXM1 to other well-known 5-FU targets such as thymidine phosphorylase (TP) and thymidine kinase 1 (TK1). The FOXM1 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. ChIP-seq confirmed that FOXM1 significantly binds to both these factors. FOXM1 binding to cell cycle regulatory genes E2F1, E2F2 and E2F3 was also shown in both cell lines. Conclusion: FOXM1 plays a critical role in 5-FU drug resistance by regulating 5-FU target genes such as TS, TK1. Inhibition of FOXM1 acts synergistically with 5-FU in colorectal cancer and warrants further investigation as a new treatment strategy. Citation Format: Vidhya Varghese, Luca Magnani, Narumi Harada, Francesco A. Mauri, Eric W. Lam, Laura M. Kenny. Forkhead box transciption factor M1 (FOXM1) plays a critical role in colorectal cancer resistance by regulating thymidylate synthase (TS). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2014-LB-220

CES2, ABCG2, TS and Topo-I primary and synchronous metastasis expression and clinical outcome in metastatic colorectal cancer patients treated with first-line FOLFIRI regimen.

Enzymatic activation of irinotecan (CPT-11) is due to carboxylesterase (CES), and its pharmacological behavior is influenced by drug resistance-related proteins. We previously reported that the clinical response and prognosis of metastatic colorectal cancer (mCRC) patients did not differ in tumors with different thymidylate synthase (TS) or topoisomerase-I (Topo-I) expression. Using immunohistochemistry (IHC), we evaluated the biological role of CES2 and the expression of breast cancer resistance protein (BCRP/ABCG2) in 58 consecutive mCRC patients, who had undergone a first-line CPT-11/5-FU/leucovirin (FOLFIRI) regimen. The expression of these proteins was also examined in a group of synchronous lymph nodes and liver metastases. Furthermore, all samples were revaluated for TS and Topo-I expression. High expression of CES2, ABCG2, TS and Topo-I was observed in 55%, 56%, 38% and 49% of patients, respectively. There was a significant association between high TS and high ABCG2 expression (p = 0.049). Univariate analysis showed that only TS expression significantly impacted on time to progression (p = 0.005). Moreover, Cox’ multivariate analysis revealed that TS expression was significantly associated with overall survival (p = 0.01). No significant correlation was found between investigated markers expression and clinical response. Topo-I expression resulted in being significantly higher in liver metastases with respect to the corresponding primary tumors (p < 0.0001), emphasizing the role of Topo-I expression in metastatic cancer biology. In primary tumor tissues, CES2 expression tended to be higher than that observed in liver metastasis tissues (p = 0.05). These preliminary data may suggest CES2 over-expression as a potential marker of malignant phenotype. In light of these findings, we suggest that Topo-I expression together with TS expression could be associated with metastatic progression of CRC. Further studies are warranted with the aim of evaluating the potential predictive and prognostic role of CES2 and ABCG2 in larger series of patients.

584P - Low Level of Thymidylate Synthase Gene Expression in Tumor Tissues is Associated with Response to Preoperative Chemoradiotherapy Including S-1 or Uft in Patients with Rectal Cancer

ABSTRACT Aim: Preoperative chemoradiotherapy (CRT) including 5-fluorouracil-based oral drug, S-1 or uracil/tegafur (UFT), significantly decreases local recurrence in locally advanced rectal cancer. However, reliable biomarkers to predict the response remain to be established. In the present study, we examined the association of the response to CRT with the expression levels of 18 drug-related genes in tumor tissues. Methods: Eighty two patients with locally advanced rectal cancer who received preoperative CRT with UFT or S-1 (± bevacizumab) for 5 weeks were analyzed. We assessed pathological tumor response according to the Japanese classification of colorectal carcinoma criteria. A patient with Grade 2 or 3 was defined as a responder. The mRNA expressions of pyrimidine-related enzymes, reduced folate-related enzymes and radiation-related enzymes (18 genes) in tumor biopsy specimens before CRT were quantitatively evaluated using a RT-PCR assay. The relationships between tumor response and gene expression levels were analyzed. The patients were divided into low and high TYMS groups using the cut-off value determined by the receiver operating characteristic curve. Results: Pathological response was observed in 72% (59/82) of the patients. There was no significant difference in response rates among clinical parameters. The gene expression level of thymidylate synthase (TYMS) was significantly higher in nonresponders than in responders (p = 0.028). There were no significant associations of tumor response with the expressions of other genes such as folylpolyglutamate synthase (FPGS) and hypoxia inducible factor 1 alpha subunit (HIF1A). In high TYMS group (response rate; 48% (10/21)), the gene expression level of FPGS and HIF1A were higher in nonresponders than in responders (p = 0.035 and p = 0.036, respectively). Conclusions: The TYMS gene expression levels in tumor tissues may be useful for predicting the efficacy of preoperative CRT including S-1 or UFT in patients with rectal cancer. In the patients with rectal cancer having high expression of TYMS, combination chemotherapy with leucovorin may be required in order to potentiate TYMS inhibition of 5-FU-based drug. Disclosure: H. Nagase: Mr. H. Nagase is a emplloyee of Taiho Pharmaceutical Co.,Ltd. All other authors have declared no conflicts of interest.

The pemetrexed-containing treatments in the non-small cell lung cancer, is -/low thymidylate synthase expression better than +/high thymidylate synthase expression: a meta-analysis

BackgroundThe predictive value of thymidylate synthase (TS) for clinical sensitivity to pemetrexed-containing chemotherapy in patients with non-small cell lung cancer (NSCLC) remains controversial. This meta-analysis is performed to provide an assessment of whether expression variations of TS are associated with objective response in patients with NSCLC treated with pemetrexed-containing chemotherapy.MethodsAn electronic search was conducted using the databases MEDLINE, EMBASE and CNKI, from inception to June 10th, 2013. A systemic review of the studies on the association between TS expression in NSCLC and objective response of pemetrexed-containing regimen was performed. Pooled odds ratios (OR) for the response rate were calculated using the software Revman 5.0.ResultsThere were a total of 526 patients in the eight studies that met our criteria for evaluation. +/high expression of TS was found in 269 patients (51.1%), and -/low expression for this gene was found in 257 (48.9%) patients. The objective response rate for pemetrexed-containing chemotherapy was significantly higher in patients with -/low expression TS expression (OR = 0.45; 95% CI, 0.29–0.70; p = 0.0004). Although patients with -/low expression of TS have a longer median overall survival time and progression free survival time than those with +/high expression of TS, the difference was not statistically significant.Conclusions−/low expression of TS was associated with higher objective response in NSCLC patients treated with pemetrexed-containing chemotherapy. TS may be a suitable marker of sensitivity to pemetrexed-based chemotherapy in patients with NSCLC.

The Pemetrexed-Containing Treatments in the Non-small Cell Lung Cancer, Is -/Low Thymidylate Synthase Expression Better than +/High Thymidylate Synthase Expression? A Meta-analysis

SESSION TITLE: Lung Cancer I

Thymidylate synthase expression and p21WAF1/p53 phenotype of colon cancers identify patients who may benefit from 5-fluorouracil based therapy

BackgroundStudies on the expression of thymidylate synthase (TS) in colorectal cancers (CRCs) have failed to provide unequivocal prognostic or predictive information. Here, we assessed the prognostic significance of TS expression in Astler-Coller stage B2 and C CRCs defined by a p21WAF1/p53 immunophenotype in patients subjected to 5-fluorouracil (5FU)-based adjuvant therapy.MethodsA cohort of 189 CRCs was asssessed for TS, p21WAF1 and p53 expression on tissue microarrays using immunohistochemistry, and associations with disease-free survival (DFS) and overall survival (OS) of the patients were assessed using univariate and multivariate analyses.ResultsTS expression led to the stratification of patients with colon cancer, but not rectal cancer, with immunophenotypes other than p21WAF1+/p53- (referred to as P&P) into subgroups characterized by a worse (P&P TS+) and a better (P&P TS-) DFS and OS, in univariate (P = 0.006 and P = 0.005, respectively) and multivariate (P = 0.0004 and P = 0.002, respectively) analyses. The p21WAF1+/p53- immunophenotype was associated with a favorable prognosis, irrespective of TS expression.ConclusionsThe strong association observed between the P&P TS+ immunophenotype and a worse DFS and OS suggests a predictive significance of TS expression for 5FU-based adjuvant therapy in patients with colon cancers exhibiting the P&P immunophenotype. In addition, our findings suggest that the appropriate target for assessment of TS expression as a prognostic/predictive marker is a subgroup of colon cancers with an immunophenotype other than p21WAF1+/p53-, and that only in this subgroup high TS expression is associated with an unfavorable DFS and OS. Therefore, we suggest that assessing TS expression in conjunction with p21WAF1/p53 immunophenotyping of colon cancers may improve the selection of patients suitable for 5FU-based adjuvant chemotherapy.

Role of topoisomerase I and thymidylate synthase expression in sporadic colorectal cancer: Associations with clinicopathological and molecular features

Topoisomerase I (Topo I) and thymidylate synthase (TS) are essential enzymes for the replication, transcription and repair of DNA, and are potential biomarkers in colorectal cancer (CRC). The aim of the study was to correlate the tissue expression of Topo I and TS in sporadic CRCs with relevant pathological and molecular features and patients’ outcome.Topo I and TS expression was assessed by immunostaining in 112 consecutive primary CRCs. Increased expression of Topo I was found in 36% of tumors, preferentially rectal (50%) and with not otherwise specified (NOS) histology (44%). Topo I expression was associated with 18q allelic loss (LOH), (p=0.013), microsatellite stable phenotype (p=0.002) and normal expression of mismatch proteins hMLH1 and hMSH2 (p=0.0012 and p=0.02, respectively). High TS expression was found in 60% of tumors, more frequently in distal sites (62%) and with NOS histology (66%); no association with microsatellite instability was observed.Topo I seems to be involved in the chromosomal instability pathway of sporadic CRCs. Conversely, high TS expression is unlikely to affect the clinical behavior of microsatellite unstable CRCs.

Thymidylate synthase (TS) expression as a prognostic molecular marker in stage II/III colon cancer.

3577 Background: Studies on the association between colorectal cancer (CC) outcome and thymidylate synthase expression have provided inconsistent results. In this study we attempted to resolve the issue by assessing the associations between TS expression and outcome in a population of primary CC patients (pts), who after resection were randomized to 5-FU/FA vs. FOLFIRI adjuvant therapy. Methods: Immunohistochemical staining for TS protein was successfully performed for 1211 pts in the PETACC3 trial. TS immunoreactivity was scored as high expression (≥75% positive) and low expression (&lt;75% positive). Gene expression respectively copy number data were available for 853 respectively 306 of these samples. Twelve single nucleotide polymorphisms (SNPs) close to the TYMS gene were assessed in 923 pts. Association of variables with relapse-free (RFS) and overall survival (OS) was assessed using Cox regression models. Results: High TS expression and RNA level were strongly associated (log fold change 0.65, p&lt;0.001). Both were significantly higher in proximal CC. As expected, both were associated with other characteristics of proximal CC: MSI, BRAF mutation, high tumor grade. RNA was significantly correlated with gene copy number, distal CC showing more frequent allelic loss. Three SNPs were associated with gene expression which was validated in data from the 1000 genomes project, but none with survival. High TS expression was more strongly associated with better OS in pts receiving FOLFIRI (HR 0.4, 95% CI 0.3–0.6, p&lt;0.001), than 5-FU/FA (HR 0.8, 95% CI 0.5–1.1, p=0.13), with a significant interaction (p=0.05). Similar results were observed for RFS (HR 0.5, p&lt;0.001 vs. HR 0.7, p=0.07; interaction p=0.11). TS expression is still highly prognostic in multivariate models adjusting for factors associated with risk or proximal tumors in FOLFIRI treated pts (OS: HR 0.5, p=0.008; RFS: HR 0.6, p=0.02), but not in F-FU/FA treated pts (OS and RFS: HR=1, p=1). Conclusions: TS expression is lower in distal CC, partly due to deletion of the TYMS locus. Pts with high TS expression have longer RFS and OS, notably when treated with FOLFIRI. For these pts addition of irinotecan to 5-FU/FA adjuvant chemotherapy might be considered. Clinical trial information: NCT00026273.

Impact of thymidylate synthase protein expression on efficacy of chemotherapy in advanced lung cancer patients

Advanced lung cancer is primarily treated with platinum combination chemotherapy, however, its prognosis remains poor. The aim of this study was to examine the correlation between the expression of thymidylate synthase (TS) in cancer tissues and the efficacy of chemotherapy in patients with advanced lung cancer in order to clarify the role of TS in the overall response. In total, 120 patients diagnosed with lung cancer between June 2004 and December 2010 at Nihon University Hospital, Tokyo, Japan, were included in this study. Cancer tissue specimens were obtained from the included patients by surgery or bronchofiberscopy prior to treatment. The expression of TS protein was evaluated using specimens immunostained with anti-TS antibody and H-scoring. TS protein expression tended to be higher in smokers compared with non-smokers. Overall survival (OS) (median value) was significantly prolonged in the low TS expression group compared with the high TS expression group. More favorable therapeutic effects were observed in the high TS expression group compared with the low TS expression group, when carboplatin + paclitaxel combined chemotherapy (CbPac therapy) was used. When the therapeutic effects were compared between CbPac therapy and carboplatin + pemetrexed combined chemotherapy (CbPem therapy) in the high TS expression group, prolongation of OS (median value) was observed with CbPac therapy. The present study suggests that TS protein expression is a critical factor in determining the efficacy of CbPac therapy in lung cancer. CbPac therapy is more effective when TS protein is highly expressed in lung cancer tissue.

Prognostic significance of 5-fluorouracil metabolism-relating enzymes and enhanced chemosensitivity to 5-fluorouracil by 5-chloro 2,4-dihydroxy-pyridine in urothelial carcinoma

BackgroundRecently, S-1, a novel 5-fluorouracil (5-FU)-based agent containing the strong dihydropyrimidine dehydrogenase (DPD) inhibitor, 5-chloro-2,4-dihydropyrimidine (CDHP) has been clinically used to treat various non-urothelial carcinomas (UC). High levels of thymidylate synthase (TS), the target enzyme of 5-FU and DPD which degrades the majority of 5-FU, are associated with poor prognosis in some cancers. However, only a few reports have dealt with this in UC. The aim of this study was to investigate the clinical significance of TS and DPD in upper tract urothelial carcinoma (UTUC) and evaluate the role of TS and DPD on the sensitivity of 5-FU in UC cell lines and the anti-tumor effect of S-1 in UC xenograft model.MethodsFirstly, we evaluated the immunohistochemical expression of TS and DPD in 176 patients with UTUC to determine their prognostic significance. Secondly, the levels of TS and DPD in UC cell lines were measured by ELISA and real-time PCR. Furthermore, the association between their levels and the sensitivity to 5-FU was examined using the small interfering RNA (siRNA) specific for TS and DPD. Thirdly, the anti-tumor effect of S-1 was evaluated in UC xenograft model.ResultsImmunohistochemical evaluation of TS and DPD in UTUC human samples revealed that TS expression was significantly associated with stage, grade, and lymphovascular invasion and DPD expression was significantly associated with grade. Multivariate analysis revealed that high TS expression was an independent predictor of disease-specific survival in them. In in vitro study using UC cell lines, high levels of TS and DPD were associated with low response to 5-FU and these associations were confirmed with siRNA specific for TS and DPD. In in vivo study using UC xenograft model, S-1 treatment dramatically inhibited tumor growth compared to controls, tegafur, or UFT in UC tumor with a high level of DPD.ConclusionsTS plays an important role in the prognosis of UTUC and S-1 may be a key agent for UC tumor, especially with a high level of DPD.

1242P - Thymidylate Synthase Expression can Guide Treatment Selection for Advanced Non-Small Cell Lung Cancer Patients

ABSTRACT Background Combination cisplatin plus pemetrexed (PMT) and carboplatin plus paclitaxel with bevacizumab are standard first-line chemotherapies for advanced non-small cell lung cancer (NSCLC). However, it is unclear whether a PMT-based regimen or taxane-based regimen should be selected for patients with advanced NSCLC. Thymidylate synthase (TS) is an important enzyme in DNA synthesis and influences sensitivity to several anti-cancer drugs. The purpose of this study is to determine whether TS expression affects the therapeutic efficacy of PMT or taxane. Methods Participants were 94 patients with advanced NSCLC treated with PMT or taxane. Samples were obtained by tumor biopsy prior to treatment. Cancer cells were isolated from formalin-fixed paraffin-embedded tissue using laser microdissection, and mRNA levels were analyzed using real-time reverse transcription polymerase chain reaction. Protein expression was evaluated using immunohistochemistry. The association between TS expression and therapeutic efficacy was evaluated. Results Fifty patients received PMT, 51 patients received taxane, and 7 patients received both regimens. TS expression was significantly lower in PMT responders compared with PMT non-responders (p = 0.0142), but significantly higher in taxane responders compared with taxane non-responders (p = 0.0486). In patients with low expression of TS, progression-free survival (PFS) was prolonged for the PMT-based regimen compared with the taxane-based regimen (21.9 versus 18.7 weeks, p = 0.5661). Conversely, in patients with high expression of TS, PFS for the taxane-based regimen was superior to that for the PMT-based regimen (19.9 versus 13.3 weeks, p = 0.2433). Conclusion The PMT-based regimen afforded better outcome in patients with low expression of TS, whereas the taxane-based regimen was better in patients with high expression of TS. Thus, TS expression could be a useful biomarker for treatment selection in NSCLC patients receiving PMT- or taxane-based chemotherapy. Disclosure All authors have declared no conflicts of interest.

Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Although cisplatin and pemetrexed are key drugs in the treatment of malignant pleural mesothelioma, their drug-drug interactions, cross-resistance and resistance mechanisms in malignant pleural mesothelioma are not well understood. In the present study, the interaction of these 2 agents was determined by clonogenic assays followed by isobologram analysis of 4 human malignant pleural mesothelioma cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase mRNA expression was evaluated in the drug-resistant sublines. As a consequence, cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar pemetrexed sensitivity in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High thymidylate synthase expression did not correlate with natural pemetrexed resistance. Elevated thymidylate synthase expression correlated with acquired pemetrexed resistance in 2 sublines. In conclusion, cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 malignant pleural mesothelioma cell lines, suggesting the clinical relevance of their combination in chemotherapy. Thymidylate synthase expression did not necessarily correlate with pemetrexed resistance. The information together with the experimental model presented here would be useful for further investigating therapeutic targets of malignant mesothelioma.

Abstract 3821: Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Abstract Background: Malignant pleural mesothelioma (MPM) is one of the most lethal cancers with a rapidly increasing incidence worldwide. Although cisplatin and pemetrexed are recognized as key drugs, and their combination chemotherapy is a standard therapy for MPM, their drug-to-drug interactions, cross-resistance, and resistance mechanisms are not well understood. Methods: The interaction between cisplatin and pemetrexed was evaluated by colonogenic assays followed by isobologram analysis in 4 human MPM cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase (TS) mRNA expression was evaluated by RT-PCR, and gene expression profiles were assessed by cDNA microarray analysis and gene ontology analysis using Ingenuity Pathway Analisis (IPA) in the drug-resistant sublines. Results: Cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar sensitivity to pemetrexed in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High TS expression did not correlate with natural pemetrexed resistance and its elevatedexpression correlated with acquired pemetrexed resistance in 2 sublines. Microarray analysis disclosed that the genes which related to “cellular development”,” small molecule biochemistry,” “cellular movement,” “cell death, and “cellular growth and proliferation” affected drug-resistant mechanisms deeply, and that this tendency was common to cisplatin-resistant sublines and pemetrexed-resistant sublines. Conclusion: Cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 MPM cell lines, suggesting the clinical relevance of their combination chemotherapy. TS expression did not necessarily correlate with pemetrexed resistance. Multiple mechanisms of molecular and cellular functions may underlie acquired resistance to cisplatin and pemetrexed in MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3821. doi:1538-7445.AM2012-3821