Thymidylate Synthase Genotype Research Articles

The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

Abstract 5805: Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy

Abstract The primary mechanism of action for the chemotherapeutic 5-fluorouracil (5-FU) is inhibition of thymidylate synthase (TS), which leads to nucleotide imbalance and subsequent cellular death. Expression of TS has been suggested to be determined by a variable number of tandem repeats located within the TS enhancer region (TSER). TSER genotype has been inconsistently linked to 5-FU-related adverse events, including severe grade ≥3 drug-related toxicity. Our recent studies demonstrated that the number of repeats within the TSER, as well as the presence or absence of a polymorphism within each repeat, was significantly associated with 5-FU toxicity in 629 patients. Our findings are consistent with a model in which the number of upstream stimulatory factor (USF1) transcription factor binding sites in the TSER, which is determined both by repeat status and genotype within each repeat, defines TYMS expression and contributions to 5-FU toxicity risk. We present an updated nomenclature that concisely and unambiguously identifies both the number of repeats and the number of USF1 binding sites. The difficulty in obtaining clear genotypes within this highly repetitive and variable region limits the ability to integrate this biomarker into predictive tests. We present methodologies for assigning TSER genotypes using conventional and high-throughput sequencing, including whole genome sequence data. Our results are expected to promote the improved study of TSER genotype in future clinical studies and the integration of TSER status into clinical predictive tests for severe 5-FU toxicity. Citation Format: Huixing Huang, Dominic Schaerer, Remington Schmidt, Ting Zhang, Tanja K Froehlich, Kelly Bouchonville, Robert B Diasio, Ursula Amstutz, Carlo Largiadèr, Steven M Offer. Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5805.

Thymidylate synthase locus LOH in combination with genotype has prognostic and predictive significance in colorectal cancer.

The aim of the current study was to investigate the prognostic and predictive significance of polymorphisms in the thymidylate synthase (TS) gene, alongside the loss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation to clinicopathological features, including the prognostic and predictive significance of TS genotype in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was obtained in 226 cases. No significant associations were observed between normal tissue TS genotype status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH were classified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status had poor outcomes when treated by surgery alone, but their survival was similar to patients with other genotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotype of the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should be considered when TS genotype is explored as a potential prognostic and predictive marker for 5-FU-based adjuvant chemotherapy in colorectal cancer.

Association of expression and genotypes of thymidylate synthase in non-small cell lung cancer patients with different clinicopathological characteristics

Abstract Objective To explore the expression and genotypes of thymidylate synthase (TS) in patients of non-small cell lung cancer (NSCLC) with different clinicopathological characteristics. Methods The expression profiles of TS were examined by immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 160 patients with NSCLC. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect TS-5′UTR tandem repeats, G/C nucleotide polymorphisms, and 3′UTR 6 bp deletion/insertion polymorphisms. The relationships between clinicopathological characteristics and TS expression or genotypes were investigated through χ 2 test. Kaplan–Meier survival analysis was used to analyze the association between TS expression and overall survival (OS) and disease-free survival (DFS) of NSCLC patients. Results The expression levels of TS protein and TS gene in NSCLC tissues were significantly higher than that in paracancerous tissues (P < 0.05). Furthermore, high expression of TS protein and 5′UTR polymorphism of TS gene showed significant correlation with differentiation, TNM stage, and lymph node metastases. The frequency of −6 bp/−6 bp genotypes in patients with NSCLC was 43.13% (69/160), which was higher than others. In addition, the rate of TS protein overexpression in NSCLC patients with 3R/3R was 79.79%, which was higher than others. Interestingly, high expression of TS protein predicted shorter DFS and OS and lower 3-year DFS rate and 3-year OS rate. Conclusions The expression levels of TS in NSCLC were significantly increased and may help to predict the prognosis of NSCLC, and high expression of TS protein and 5′UTR polymorphism of TS gene were significantly related to differentiation, TNM stage, and lymph node metastases.

Evaluation of effects of thymidylate synthase and excision repair cross-complementing 1 polymorphisms on chemotherapy outcome in patients with gastrointestinal tumors using peripheral venous blood.

The aim of the present study was to evaluate the effects of thymidylate synthase (TYMS) and excision repair cross-complementing 1 (ERCC1) polymorphisms on chemotherapeutic efficacy in patients with gastrointestinal tumors using peripheral venous blood. Preoperative peripheral venous blood and tumor tissue samples of 43 patients with gastric cancer and the peripheral venous blood samples of 76 patients with cancer who underwent chemotherapy were studied. The 3R/3R and 2R/2R or 2R/3R genotypes of TYMS were identified in 72.09 and 27.91%, respectively (P<0.01), of untreated patients, and the C/C and T/T or C/T genotypes of ERCC1 were present in 81.39 and 18.61%, respectively (P<0.01), of patients. The 3R/3R and 2R/2R or 2R/3R genotypes of TYMS were identified in 65.79 and 34.21%, respectively, of chemotherapy-treated patients. The overall response rates (ORRs) for the two aforementioned genotypes were 18.00 and 57.69%, respectively (P<0.01), and those for the C/C and T/T or C/T genotypes of ERCC1 were 63.16 and 36.84%, respectively. The ORRs were 47.91 and 3.57%, respectively (P<0.01). In conclusion, peripheral blood samples may be used to replace tumor tissue for detecting TYMS and ERCC1 polymorphisms, and may be used to evaluate the efficacy of 5-fluorouracil and platinum drugs.

Homozygosity for the 2R Tandem Repeat Polymorphism in the Thymidylate Synthase (TS) Promoter Is Associated with Increased Risk for Bony Morbidity Among Children Treated for Acute Lymphoblastic Leukemia on DFCI Protocol 05-001

Introduction: Both fractures and avascular necrosis (AVN) of bone frequently complicate therapy for childhood acute lymphoblastic leukemia (ALL), resulting in significant pain and loss of function. Risk of bony morbidity has been associated with age >10 years and exposure to crucial components of leukemia therapy including corticosteroids, asparaginase, and methotrexate. To further define risk factors, we tested whether 19 common genetic polymorphisms were associated with bony morbidity among 637 children treated for ALL on Dana Farber Cancer Institute ALL Consortium protocol 05-001.Methods: Genomic DNA was isolated from peripheral blood or bone marrow collected at the time of remission. Nineteen candidate polymorphisms were selected a priori, targeting common variants within genes related to glucocorticoid metabolism, oxidative damage, and folate physiology. Single nucleotide polymorphisms were detected using PCR-based allelic discrimination assays. The number of 28bp nucleotide repeats within the 5' untranslated region of the gene for thymidylate synthase (TS) was assessed by PCR-product length analysis. The incidence of AVN and fracture were estimated by the cumulative incidence method identifying death and relapse as competing risks. Because the incidence of bony morbidity differed significantly between children above and below the age of 10 years, relationships between polymorphisms and bony morbidity were explored within these two age subgroups separately. Comparisons at the 0.10 significance level were modeled univariately and were considered in multivariable modeling using competing risks regression. Multivariable models for bony morbidity included sex, WBC at diagnosis, race, final risk group, and asparaginase randomization. Blood was collected during treatment for analysis of serum and RBC folate by HPLC.Results: Of the 637 patients tested, 627 achieved a complete remission and received post-induction therapy, of which 61 (9.7%) experienced AVN and 138 (22.0%) had ≥1 fracture. Both AVN and fracture were more common among those ≥10 years of age (22.9% AVN, 30.7% fracture) than younger children (5.5% AVN, p<0.0001 and 19.2% fracture, p=0.002). Homozygosity for the 2R TS polymorphism was observed in 129 (20.6%) of the 626 patients tested. This 2R/2R genotype was associated with a greater 5-year estimated incidence of AVN (11.6%; 95% CI 6.1-18.9%) than among those with 2R/3R or 3R/3R genotypes (4.1%, 95% CI 2.4-6.5%; p=0.0037; Figure [A]) among children < age 10, but not among older children. The increased risk of AVN in younger children remained significant in the multivariable risk regression model (adjusted hazard ratio 2.92; 95% CI 1.36-6.28; p=0.0059). Among children over age 10, this polymorphism was associated with an increased risk of bony fracture (multivariable HR 2.13; 95% CI 1.13-3.99; p=0.019; Figure [B]). No significant association was observed between bony morbidity and the other tested polymorphisms, including PAI-1 (rs6092), ABCB1 (rs1045642), and the vitamin D receptor Fok1 restriction site (rs228570) which have been previously linked to risk of AVN. Samples were available for analysis of serum and RBC folate at diagnosis (n=367), remission (n=347), 15 months post-remission (n=251), and at the conclusion of therapy (n=247). No significant association was observed between TS genotype and serum or RBC folate at any time point.Conclusions: Homozygosity for the 2R polymorphism in TS was associated with increased risk of bony morbidity among children treated for ALL. Interestingly, the impact of the polymorphism differed between those above the age of 10 years (associated with risk of fracture) and younger children (risk for AVN), suggesting that the pathophysiology of bony morbidity differs between these two age groups. This is the first report identifying a genetic risk factor for AVN specifically among children younger than 10 years, a group where AVN is significantly less frequent. This result suggests young children with ALL and the 2R/2R genotype should be monitored more closely for the development of AVN during therapy for ALL. [Display omitted] DisclosuresSilverman:Seattle Genetics, Inc.: Research Funding.

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.

Thymidylate synthase polymorphisms in genomic DNA as clinical outcome predictors in a European population of advanced non-small cell lung cancer patients receiving pemetrexed.

BackgroundWe studied whether thymidylate synthase (TS) genotype has an independent prognostic/predictive impact on a European population of advanced non-small cell lung cancer (NSCLC) patients receiving pemetrexed.MethodsTwenty-five patients treated with pemetrexed-based regimens were included. Genomic DNA was isolated prior to treatment. The variable number of tandem repeat (VNTR) polymorphisms, the G > C single nucleotide polymorphisms (SNP) and the TS 6-bp insertion/deletion (6/6) in the 3′ untranslated region (UTR) polymorphisms were analyzed and correlated with overall response rate (ORR), progression-free survival (PFS), overall-survival (OS) and toxicity.ResultsThe genotype +6/+6 predicted a higher ORR among active/former smokers compared to +6/-6 genotype (100% vs. 50%; p = 0.085). Overall, the 3R/3R genotype predicted a higher ORR (100%) over the rest VNTR polymorphisms (p = 0.055). The presence of 3R/3R genotype significantly correlated with a superior ORR in patients without EGFR activating mutations (100%) compared to 2R/2R, 2R/3R and 3R/4R genotype (77.8%, 33.3% and 0% respectively; p = 0.017). After a median follow-up of 21 months, a trend towards a better PFS, although not significant, was found among subjects showing 3R/3R polymorphisms (p = 0.089). A significantly superior OS was found in patients showing 3R/3R genotype rather than other VNTR polymorphisms (p = 0.019). No significant correlation with the toxicity was observed.ConclusionIn our series, 3R/3R polymorphism correlated with a superior OS. Also, this polymorphism, when associated to wild type EGFR, was related to a higher ORR to pemetrexed. Toxicity was not significantly correlated with a specific TS genotype.

Association between polymorphisms in the thymidylate synthase gene and risk of breast cancer in a Mexican population.

Breast cancer (BC) is the leading cause of cancer-related deaths among women in Mexico. Two single-nucleotide polymorphisms (SNPs) in the thymidylate synthase (TS) gene, the 28-base pair (bp) tandem repeat in the TS 5'-untranslated enhanced region (TSER) and the 6-bp insertion/deletion in the TS 3'-untranslated region (TS 3'-UTR), increase the rate of misincorporation of uridylate into DNA and may lead to chromosomal damage. We examined the association between these polymorphisms and BC risk in Mexican women according to menopause status. Mexican patients with initial BC diagnosis (N = 230) and 145 individuals from a reference general population group (RGP) were included. For statistical analysis, the BC group was divided into pre- and post-menopause groups (PRE and POST groups, respectively). We analyzed both TS polymorphisms (TSER and TS 3'-UTR) using polymerase chain reaction. Finetti analysis was used to evaluate inter-and intra-group differences. The results showed a high frequency for the 3R and ins6 alleles in the BC, RGP, PRE, and POST groups. No significant differences were observed for the TS and TSER genotype and allele frequency distributions between groups. We found that the TSER and TS 3'-UTR SNPs are not associated with BC risk in Mexican patients.

Thymidylate Synthase Gene Polymorphism and Survival of Colorectal Cancer Patients Receiving Adjuvant 5-Fluorouracil

Limited studies indicate a possible association of 5'-UTR thymidylate synthase enhancer region polymorphism and treatment outcome in patients medicated with 5-fluorouracil (5-FU). The study was designed to verify the relationship in patients with colorectal cancer (CRC), a Polish population that received 5-FU-based adjuvant chemotherapy. The study analyzed 145 Astler-Coller B2 and C CRC patients. Genotyping for a variable number of tandem repeats and G to C single-nucleotide polymorphism in the 5'-UTR of the thymidylate synthase (TS) gene was carried out. TS genotypes were classified into high expression (high TS) and low expression types (low TS). High TS was found in 22.8% of patients. The right-side tumors were more frequently associated with high TS than the left-side tumors (p=0.024). High TS was only found in 9.3% of rectal tumors, but in 29.7% of colon cancers (p=0.0042). Disease-free survival after 20 months (DFS 20) was longer in subjects with low TS than in high TS (p=0.043). Patients who underwent chemotherapy had longer DFS 20 in the low TS than in the high TS subgroup (p=0.051). The low TS was found to be an independent good prognostic factor for DFS 20 in the whole group as well as in the subgroup treated with chemotherapy (p=0.024 and p=0.034, respectively). Patients with low TS did not show any differences in DFS 20 whether they were treated with adjuvant chemotherapy or not. Proximal CRC tumors are characterized by higher TS expression genotypes than distal tumors, and are at significantly greater risk of early recurrence during the first 20 months after surgery.

Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients

PurposeTo assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS).MethodsThree-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method.ResultsHALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53–3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/106 cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): ‘A+A’; OR for ‘A+B’ vs. ref.: 1.39 [0.69–2.80]; OR for ‘B+A’ vs. ref.: 2.16 [1.22–3.83]; OR for ‘B+B’ vs. ref.: 3.13, 95%CI: 1.54–6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56–4.14, P = 0.001), (c) use of EFV (1.10 [1.00–1.21], P = 0.008, per year of use).ConclusionHALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine.

Thymidylate synthase (TS) polymorphisms (Pol) in buffy coat-derived genomic DNA as a clinical outcome predictor in non-Asian stage III/IV non-small cell lung cancer (NSCLC) patients (pts) receiving pemetrexed (Pem).

e19112 Background: TS is the main biological target of the antifolate Pem. Some TS Pol may confer a short survival and a poor response to antifolate-treated colo-rectal cancer pts. Whether TS genotype has an independent prognostic/predictive impact on non-Asian advanced NSCLC pts treated with Pem is unknown. Methods: Twenty-five non-Asian advanced NSCLC pts treated with Pem-based regimens (1st, 2nd or 3rd line) were included. Genomic DNA was isolated from periferal blood prior to treatment. The variable number of tandem repeat (VNTR) Pol, the G&gt;C single nucleotide Pol (SNP) and the TS 6-bp insertion/deletion (6/6) in the 3' untranslated region (UTR) Pol were analyzed and correlated with response rate (RR), progression-free survival (PFS), overall-survival (OS) and toxicity (Tx). Results: Regarding the VNTR Pol, most of the pts showed 2R/2R or 3R/2R Pol (17 pts; 68%), and 8 pts (32%) showed 3R/3R or 3R/4R Pol. A SNP (G&gt;C) in VNTR was observed in 18 pts (72%). The Pol found in the 3´UTR region were +6/+6 in 12 pts (48%), +6/-6 in 11 pts (44%) and -6/-6 in 2 pts (8%). In the subgroup of T3-T4 pts, there was a higher RR among those showing 3R/3R Pol than those with 2R/2R Pol (100% vs 83.8%; p=0.029). The genotype +6/+6 predicted a higher RR among active/former smokers (A/FS) compared to +6/-6 (100% vs 37.5%; p=0.026). A 3R/3R Pol followed by 2R/2R and 2R/3R Pol predicted a superior RR in pts with EGFR mutations (p=0.018). Overall, a trend towards a better PFS in pts showing 2R/2R Pol was found (p=0.076). The cohort’s median overall survival (OS) was not reached during follow-up. The most frequent Tx was grade (G)1 anemia (28%) and nausea (20%) and G2 leucopenia (40%). G3-4 anemia (4%), leucopenia (16%), neutropenia (4%), astenia (8%) and dyspnea (4%) were uncommon but more frequent in pts with 2R/2R Pol (p=0.545). Conclusions: In this cohort of NSCLC pts 3R/3R Pol among T3-T4 pts and EGFR mutant pts and +6/-6 bp among A/FS significantly predicted a higher RR to Pem and may aid in treatment selection. Tx was not significantly correlated with a specific TS genotype. These interesting preliminary data warrant further validation in larger series.

Abstract 2369: Pharmacogenomic assessment of outcomes of pemetrexed-treated patients with adenocarcinoma of the lung.

Abstract Purpose: The main objective of this study was to evaluate the association between polymorphisms of the target genes of pemetrexed and clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with pemetrexed. Materials and Methods: We assessed polymorphisms at 8 sites in 4 genes (thymidylate synthase (TS), dihydrofolate reductase (DHFR) [1610, 680, 317, intron 1], methylenetetrahydrofolate reductase (MTHFR) [677, 1298], glycinamide ribonucleotide formyl transferase (GARFT) [2255]) associated with pemetrexed metabolism using polymerase chain reaction, gene scanning, and restriction fragment length polymorphism analysis in 90 patients with adenocarcinoma of the lung. Results: Survival was significantly longer with pemetrexed in patients with TS 3RGCC/3RGCC or 3RGGC/3RGGC compared with the other groups (PFS; 5.2 months vs. 3.7 months, p=0.03: OS; 31.8 months vs. 18.5 months, p=0.001). Patients with DHFR 680CC experienced fatigue more frequently (50% vs. 8.6%, p=0.008). Polymorphisms of MTHFR and GARFT were not significantly associated with clinical outcomes with of pemetrexed. Conclusions: The TS genotype was associated with survival and one DHFR polymorphism was associated with fatigue in NSCLC patients treated with pemetrexed. Further large prospective studies are required to identify other biomarkers that affect patients being treated with pemetrexed for adenocarcinoma of the lung. Citation Format: Minkyu Jung, Chul Ho Lee, Hyung Soon Park, Ji Hyun Lee, Dae Joon Kim, Sun Min Lim, Joo Hang Kim, Byoung Chul Cho. Pharmacogenomic assessment of outcomes of pemetrexed-treated patients with adenocarcinoma of the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2013-2369 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Prognostic significance of thymidylate synthase polymorphisms in rectal cancer patients treated with neoadjuvant chemoradiotherapy

There is a lack of prognostic factors of preoperative chemoradiation for locally advanced rectal cancer. Thymidylate synthase (TS) is the most important target of 5-fluorouracil; three main genetic polymorphisms of TS have been described. We analysed the prognostic value of these in patients with locally advanced rectal cancer treated with fluoropyrimidine-based chemoradiation. Ninety-nine patients treated between November 2001 and March 2009 were included. All were treated by radiotherapy (5040 cGy) and concomitant fluoropyrimidine-based chemotherapy. Three polymorphisms were analysed: (i) a double (2R) or triple (3R) repeat of a 28 base pair (bp) tandem sequence upstream of the ATG codon initiation site in the 5'-terminal regulatory region, (ii) a functional G > C single nucleotide polymorphism present in the second repeat of the 3R alleles and (iii) a 6 bp deletion at nucleotide 1494 in the 3'-untranslated region. DNA was extracted from paraffin-embedded core biopsies taken from the tumour and the genotype was analysed using polymerase chain reaction restriction fragment length polymorphism. The 6 bp polymorphism was significantly associated with disease-free survival (+ 6 bp/+ 6 bp vs-6 bp/-6 bp, P = 0.032 logistic regression). No differences were found in disease-free survival according to the other polymorphisms studied. No relationship was observed between the different TS genotypes and pathological regression. The study suggests that the TS 6 bp polymorphism may be a predictor of disease-free survival in patients with locally advanced rectal cancer treated with fluoropyrimidine-based chemoradiation.

Association of Thymidylate Synthase Polymorphisms with Acute Pancreatitis and/or Peripheral Neuropathy in HIV-Infected Patients on Stavudine-Based Therapy

BackgroundLow expression thymidylate synthase (TS) polymorphism has been associated with increased stavudine triphosphate intracellular (d4T-TP) levels and the lipodystrophy syndrome. The use of d4T has been associated with acute pancreatitis and peripheral neuropathy. However, no relationship has ever been proved between TS polymorphisms and pancreatitis and/or peripheral neuropathy.MethodsWe performed a case-control study to assess the relationship of TS and methylene-tetrahydrofolate reductase (MTHFR) gene polymorphisms with acute pancreatitis and/or peripheral neuropathy in patients exposed to d4T. Student’s t test, Pearson’s correlations, one-way ANOVA with Bonferroni correction and stepwise logistic regression analyses were done.ResultsForty-three cases and 129 controls were studied. Eight patients (18.6%) had acute pancreatitis, and 35 (81.4%) had peripheral neuropathy. Prior AIDS was more frequent in cases than in controls (OR = 2.36; 95%CI 1.10–5.07, P = 0.0247). L7ow expression TS and MTHFR genotype associated with increased activity were more frequent in patients with acute pancreatitis and/or peripheral neuropathy than in controls (72.1% vs. 46.5%, OR = 2.97; 95%CI: 1.33–6.90, P = 0.0062, and 79.1% vs. 56.6%, OR = 2.90, 95%CI: 1.23–7.41, P = 0.0142, respectively). Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; ORA+B = 0.34 [95%CI: 0.08 to 1.44], ORB+A = 3.38 [95%CI: 1.33 to 8.57], ORB+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm3 (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018).ConclusionsLow expression TS plus a MTHFR genotype associated with increased activity is associated with the development of peripheral neuropathy in d4T-exposed patients.

Pharmacogenomic Assessment of Outcomes of Pemetrexed-Treated Patients with Adenocarcinoma of the Lung

PurposeThe main objective of this study was to evaluate the association between polymorphisms of the target genes of pemetrexed and clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with pemetrexed.Materials and MethodsWe assessed polymorphisms at 8 sites in 4 genes [thymidylate synthase (TS), dihydrofolate reductase (DHFR; 1610, 680, 317, intron 1), methylenetetrahydrofolate reductase (MTHFR; 677, 1298), glycinamide ribonucleotide formyl transferase (GARFT; 2255)] associated with pemetrexed metabolism using polymerase chain reaction, gene scanning, and restriction fragment length polymorphism analysis in 90 patients with adenocarcinoma of the lung.ResultsSurvival was significantly longer with pemetrexed in patients with TS 3RGCC/3RGCC or 3RGGC/3RGGC compared with the other groups (PFS; 5.2 months vs. 3.7 months, p=0.03: OS; 31.8 months vs. 18.5 months, p=0.001). Patients with DHFR 680CC experienced fatigue more frequently (50% vs. 8.6%, p=0.008). Polymorphisms of MTHFR and GARFT were not significantly associated with clinical outcomes of pemetrexed.ConclusionThe TS genotype was associated with survival and one DHFR polymorphism was associated with fatigue in NSCLC patients treated with pemetrexed. Further large prospective studies are required to identify other biomarkers that affect patients being treated with pemetrexed for adenocarcinoma of the lung.

Gene polymorphisms of folate metabolizing enzymes and susceptibility to lung adenocarcinoma in Serbia.

e12020 Background: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are important enzymes of the folate metabolism and are suggested as new prognostic factors for lung cancer. Cytosine to thymine transition at nucleotide 677 (C677T) leads to reduced MTHFR activity. The TS promoter has a tandem repeat polymorphism (2R, 3R) and a guanine to cytosine transition in the 3R allele related to TS protein expression. The aim of this study was to analyze the association of MTHFR and TS polymorphisms with lung adenocarcinoma in Serbia. Methods: A case-control study including 55 late-stage lung adenocarcinoma patients and 53 healthy subjects was performed. Restriction fragment length polymorphism analysis was used for MTHFR and TS genotyping. Depending on the presence of high (3RG) or low (2R, 3RC) expression alleles, TS functional groups were subclassified into HH (3RG/3RG), HL (2R/3RG, 3RG/3RC) and LL (2R/2R, 2R/3RC, 3RC/3RC) groups. Descriptive analyses included genotype and allelic frequencies; the odds ratio (OR) and 95 % confidence interval (CI) were calculated as an estimate of relative risk. Significance was considered for p &lt; 0.05. Results: The distribution of the MTHFR variants in patients vs. controls was 61.8 % vs. 24.5 % for CC, 32.7 % vs. 62.3 % for CT and 5.5 % vs. 13.2 % for TT. A significant difference in CC vs. TT+CT MTHFR genotype distribution was observed between patients and controls (χ2 = 13.79; OR = 4.98; 95 % CI, 2.14 – 11.61). There was no significant association between the TS polymorphisms and the risk of lung adenocarcinoma occurrence, but in the CT+TT MTHFR subgroup, a nonsignificant difference in LL vs. HL+HH TS genotype distribution was observed between patients and controls (χ2 = 0.04; OR = 1.07; 95% CI, 0.31 – 3.67). Conclusions: A significant corelation between the CC MTHFR genotype and lung adenocarcinoma occurence in Serbia was found. Also, there was no significant correlation with lung adenocarcinoma occurence in the CT+TT MTHFR subgroup for carriers of the LL TS genotype. As this study was performed on a relatively small sample size further large case-control studies including analysis of gene-gene interactions with genes coding for other folate metabolism enzymes is needed.

Thymidylate synthase genotype specific dosing of capecitabine: Proof of concept phase I study.

2551 Background: Thymidylate synthase (TYMS) is the target of fluoropyrimidines (FP). TYMS 3R3R is the predominant genotype in East Asian (EA) patients and is associated with increased TYMS expression, reduced FP related toxicity and relative FP resistance. Dose finding studies for FP were developed in Caucasians, a population that typically harbors TYMS 2R2R and 2R3R genotypes. We hypothesize the recommended phase II dose (RP2D) of capecitabine is higher in 3R3R and similar for 2R allele carriers compared to the FDA approved dose. Objectives 1) To determine the maximal tolerated dose (MTD) and RP2D of capecitabine in EA patients with advanced stage malignancy 2) To determine the safety and toxicity of this regimen and 3) To perform plasma pharmacokinetics (PK) of capecitabine and its metabolites. Methods: EA patients with advanced stage cancer were prospectively allocated into two cohorts: Group A (3R3R) and Group B (2R3R, 2R2R). In each cohort, dose escalation followed a standard phase I 3+3 design. Additional patients were treated at the R2P2 dose level (DL). Initial dose was 1250/m2 bd for 14 days q3w (DL 1) with 125 mg/m2 bd increments subsequently. Pharmacokinetics (PK) of capecitabine and its metabolites were performed using a LC-MS/MS method. Results: 23 patients (Group A=18; group B=5) received 94 cycles (median 2.5, range 1-8). Median age was 58 (range 34-74) years. Median turnaround time for TYMS genotyping was 1 day. In group A, grade 3-4 DLTs were seen in 3 patients: diarhoea, neutropenic fever, hand-foot syndrome, and mucositis. MTD was at DL 4 (1625mg/m2 bd) and the RP2D was 1500mg/m2 bd (DL 3). DL 3 was expanded to n=9. At DL 3, day 1 mean (± SD) capecitabine and 5FU Cmax was 12.3 ± 9.9 and 0.91 ± 0.73 µg/mL, respectively and AUC0 – t was 9.84 ± 5.53 and 1.21 ± 0.54 h*µg/mL, respectively. Compared with DL1, Cmax for capecitabine and 5FU was 2 and 2.02 fold higher and AUC 0-t was 1.49 and 1.59 fold higher at DL3. Accrual in group B was ceased at DL2 due to lack of patient enrolment; no DLT was seen. By ROC analysis, day 1 5FU AUC0 – t of 1.74 h*µg/mL predicted for DLT (p=0.022, sensitivity 100%, specificity 90%). Conclusions: In EA patients with TSER 3R3R, the RP2D was 1500 mg/m2 bd. The D1 5FU AUC0 – t at RP2D was 59% more than the FDA approved dose (DL1).

Abstract 3689: Association of thymidylate synthase gene polymorphisms with efficacy and toxicity of oral fluoropyrimidine based chemotherapy in advanced gastric cancer

Abstract Purpose: Oral fluoropyrimidines (S-1 or capecitabine) are metabolized by different enzymes. We evaluated the association between polymorphisms of cytochrome P450 2A6 (CYP2A6), carboxylesterase 2(CES 2), thymidylate synthase (TS), and excision repair cross complementation group 1 (ERCC1) and toxicity of advanced gastric cancer patients treated with oral fluoropyrimidine based chemotherapy. Methods: A total of 123 patients received S-1 80mg/m2 or capecitabine 2000 mg/m2 plus oxaliplatin 130 mg/m2 for advanced gastric cancer. We analyzed polymorphisms of CYP2A6, CES 2, TS and ERCC1 by using genomic DNA extracted from prospectively collected peripheral blood sample. Associations between polymorphisms and toxicity of these patients treated with oral fluoropyrimidines based chemotherapy were evaluated individually. Results: Patients who were 2R/3R for the double repeat in the TS promoter region had more toxicity of oral fluoropyrimidines. Patients with a 2R/3R and 2R/2R or 3R/3R genotype had a grade 2 or more gastrointestinal toxicity rate of 50 and 22%, respectively (P = .049). Response rate was higher in patients with a 2R/3R than those with a 2R/2R or 3R/3R (50% vs. 32%, P = .082). Polymorphisms of CYP2A6, CES 2, and ERCC1 were not associated with efficacy and toxicity of oral fluoropyrimidine based chemotherapy for advanced gastric cancer. Conclusion. This study demonstrated that TS genotyping could be of help in predicting toxicity to oral fluoropyrimidine based chemotherapy in advanced gastric cancer patients. 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 3689. doi:1538-7445.AM2012-3689

Thymidylate synthase inhibitors for non-small cell lung cancer

Introduction:The folate-dependent enzyme thymidylate synthase (TS) plays a pivotal role in DNA replication/repair and cancer cell proliferation, and represents a valid target for the treatment of several tumor types, including NSCLC. NSCLC is the leading cause of cancer-related mortality, and several TS inhibitors have gone into preclinical and clinical testing, with pemetrexed emerging for its approval and widespread use as first-/second-line and maintenance therapy for this disease.Areas covered:This review summarizes the therapeutic options in NSCLC, as well as the background and rationale for targeting TS. The authors also review recent pharmacogenetic studies and data from clinical trials evaluating novel TS inhibitors, hoping that the reader will gain a comprehensive overview of the field of TS inhibition, specifically relating to drugs used or being developed for lung cancer patients.Expert opinion:TS is a validated target in NSCLC. However, benefits from conventional chemotherapy in NSCLC have plateaued, and more cost-effective results should be obtained with individualized treatment. Accordingly, the clinical success for TS inhibitors may depend on our ability to correctly administer these agents following biomarker-driven patient selection, including TS genotype and expression, and using the right combination therapy.