Low Thymidylate Synthase Research Articles

Evaluating the Prognostic Value of ERCC1 and Thymidylate Synthase Expression and the Epidermal Growth Factor Receptor Mutation Status in Adenocarcinoma Non-Small-Cell Lung Cancer.

The present study evaluated the prognostic value of the epidermal growth factor receptor (EGFR) mutation status, and excision repair cross-complementation group 1 (ERCC1) and thymidylate synthase (TS) expression following intercalated tyrosine kinase inhibitor (TKI) therapy and platinum- and pemetrexed-based chemotherapies (subsequent second-line treatment) for patients with adenocarcinoma non-small-cell lung cancer (AC-NSCLC). In total, 131 patients with AC-NSCLC were enrolled. The EGFR mutation status and ERCC1 and TS expression were evaluated through direct DNA sequencing and immunohistochemical analyses, respectively. The EGFR mutation status and ERCC1 and TS expression were the significant predictors of clinical outcomes. The EGFR mutation status was the main outcome predictor for overall survival (OS) benefits in the overall population. Further exploratory ERCC1 and TS expression analyses were conducted to provide additional insights. Low TS expression was predictive of improved OS of patients with negative EGFR-mutated advanced AC-NSCLC, whereas high ERCC1 expression resulted in poor OS in patients with positive EGFR-mutated advanced AC-NSCLC. TS and ERCC1 expression levels were effective prognostic factors for negative and positive EGFR-mutated AC-NSCLC, respectively. In conclusion, the present results indicate that the EGFR mutation status and TS and ERCC1 expression can be used as the predictors of OS after subsequent second-line treatments for AC-NSCLC.

The role of thymidylate synthase in non-small cell lung cancer treated with pemetrexed continuation maintenance therapy

Pemetrexed continuation maintenance therapy has been proven to be beneficial for patients with advanced non-squamous non-small cell lung cancer (NSCLC). However, the eligibility criteria for maintenance treatment are too simple. This study sought to evaluate thymidylate synthase (TS) as a predicting biomarker for pemetrexed continuation maintenance treatment in NSCLC. Specimens were collected from 87 patients treated with pemetrexed continuation maintenance therapy before and after four-cycle induction chemotherapy. Real-time quantitative PCR was used to detect TS expression in tissues. The TS expression level was correlated with characteristic clinical data, radiographic response, progression-free time (PFS) and overall survival (OS). Low total TS expression (<8.47) was associated with improved PFS (median: 4.7 months vs. 3.5 months, p = 0.034) and improved OS (time from random assignment: 16.4 months vs. 11.7 months, p = 0.026; time from induction: 19.7 months vs. 14.8 months, p = 0.022). Our results indicate that in NSCLC patients treated with pemetrexed continuation maintenance therapy, low TS expression is associated with improved PFS and OS.

Efficacy of crizotinib and pemetrexed-based chemotherapy in Chinese NSCLC patients with ROS1 rearrangement.

BackgroundROS1 rearrangement is a novel molecular subgroup of non-small-cell lung cancer (NSCLC). This study aimed to investigate the efficacy of crizotinib and pemetrexed-based chemotherapy in Chinese NSCLC patients with ROS1 rearrangement.ResultsA total of 2309 patients received ROS1 fusion detection and 51(2.2%) patients had ROS1 rearrangement. There was no significant difference between ROS1 fusion-positive and fusion-negative cohorts in demographic data. For the ROS1 fusion-positive patients, crizotinb-treated group had a higher overall response rate (ORR, 80.0%), disease control rate (DCR, 90.0%) and longer progression-free survival (PFS, 294 days) compared with the rates in pemetrexed-treated group (ORR, 40.8%; DCR, 71.4%; PFS, 179 days) and non-pemetrexed-treated group (ORR, 25.0%; DCR, 47.7%; PFS, 110 days). Besides, ORR, DCR and PFS were similar in three major ROS1 fusion partners. For the first-line treatment, patients received pemetrexed had a significant longer PFS than those received non-pemetrexed chemotherapy (209 vs. 146 days, P = 0.0107). In pemetrexed-treated cohorts, ROS1-positive patients with low TS expression had a statistically significant longer PFS than those with high TS expression (184 vs. 110 days, P = 0.0105).Materials and methodsWe retrospectively identified patients with NSCLC who were screened for ROS1 fusion using multiplex reverse transcription-polymerase chain reaction (RT-PCR) from October 2013 to February 2016. The thymidylate synthase (TS) mRNA levels were tested using quantitative real-time RT-PCR.ConclusionsCrizotinib was also highly active at treating Chinese NSCLC patients with ROS1 rearrangement. TS expression could predict the efficacy of pemetrexed-based therapy in ROS1 fusion-positive patients.

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.

1283P - The role of thymidylate synthase in non-small-cell lung cancer treated with pemetrexed continuation maintenance therapy

Pemetrexed continuation maintenance therapy has been proven to be beneficial for patients with advanced non-squamous non-small cell lung cancer (NSCLC). However, the eligibility criteria for maintenance treatment are too simple. This study sought to evaluate thymidylate synthase (TS) as a predicting biomarker for pemetrexed continuation maintenance treatment in NSCLC. Specimens were collected from 87 patients treated with pemetrexed continuation maintenance therapy before and after four-cycle induction chemotherapy. Real-time quantitative PCR was used to detect TS expression in tissues. The TS expression level was correlated with characteristic clinical data, radiographic response, progression-free time (PFS) and overall survival (OS). Low total TS expression (<8.47) was associated with improved PFS (median: 4.7 months vs. 3.5 months, p = 0.034) and improved OS (time from random assignment: 16.4 months vs. 11.7 months, p = 0.026; time from induction: 19.7 months vs. 14.8 months, p = 0.022). Our results in...

Abstract 2944: Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer

Abstract Purpose: Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, is elevated in multiple cancers. Thymidylate synthase (TS) is an important enzyme in purine synthesis and DNA replication. In this study, we investigated the AEG-1 expression characteristics in non-small cell lung cancer (NSCLC) to determine whether its expression is correlated with TS regulation of NSCLC. Materials and Methods: A549, H157, CL1-0, CL1-5, PC9, H1975, H520 and H292 human lung cancer cell-lines were cultured in medium with RPMI1640 + 10% FBS. Immunohistochemical stain and Western blot of AEG-1 and TS were studied in all cell-lines. The primary antibodies used were anti-AEG-1 (1:1,000; chicken polyclonal), anti-actin (1:1,000; rabbit polyclonal; Santa Cruz), and anti-TS (1:1,000; mouse monoclonal; Abcam). Total RNA was extracted using a Qiagen mRNA easy mini kit (Qiagen). Real-time PCR was performed using ABI 7900 fast real-time PCR system to detect expression of AEG-1 and TS. Cell viability was determined by standard MTS assays to recognize IC50 of Pemetrexed in lung cancer cell-lines. Transfection of siRNA for AEG-1 was carried out to knock down AEG-1. AEG-1 pcDNA (Life technologies, CA, USA) were used to quantitatively examine AEG-1 activity. Expressive change of AEG-1associated with TS and signaling pathways were analyzed by Western blot and PCR. Results: TS and AEG-1 in H157, Cl1-0, CL1-5, H520 and H292 were higher detected by IHC stain, Western blot and RT-PCR mRNA level. The expression of TS was positive correlated with AEG-1 in lung cancer cell-lines. The IC50 values of Pemetrexed for cells were lower in the lung cancer cell-lines with lower TS gene expression. After knockdown of AEG-1 by siRNA in Pemetrexed-resistance PC-9 and A549 cell-lines, the expression of TS decreased associated with decreasing change IC50 of Pemetrexed. In contrast, transfection with AEG-1 pcDNA in PC-9 and A549 cell-lines overexpressed AEG-1 gene, TS level also enhanced associated with increasing change IC50 of Pemetrexed. AEG-1 expression can also activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that augments binding of c-Myc thereby regulating AEG-1 transcription. Conclusions: Our data indicated a significant correlation between TS and AEG-1 gene expression in NSCLC cell-lines. TS expression might be regulated by AEG-1 through activation of PI3K/AKT and c-Myc signaling pathways associated with development of chemoresistance to pemetrexed in NSCLC treatment Citation Format: Chung-Yu Chen, Ying-Yin Chen, Kuan-Yu Chen, Jin-Yuan Shih, Yih-Leong Change, Chong-Jen Yu, Pan-Chyr Yang. Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2944.

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.

Pemetrexed/Carboplatin/Bevacizumab followed by Maintenance Pemetrexed/Bevacizumab in Hispanic Patients with Non-Squamous Non-Small Cell Lung Cancer: Outcomes according to Thymidylate Synthase Expression.

ObjectiveTo evaluate the efficacy and safety of pemetrexed, carboplatin and bevacizumab (PCB) followed by maintenance therapy with pemetrexed and bevacizumab (PB) in chemotherapy-naïve patients with stage IV non-squamous non-small cell lung cancer (NSCLC) through the influence of thymidylate synthase (TS) protein and mRNA expression on several outcomes. The primary endpoints were the overall response rate (ORR), progression-free survival (PFS) and overall survival (OS).MethodsA cohort of 144 patients were administered pemetrexed (500 mg/m2), carboplatin (AUC, 5.0 mg/ml/min) and bevacizumab (7.5 mg/kg) intravenously every three weeks for up to four cycles. Maintenance PB was administered until disease progression or unacceptable toxicity.ResultsOne hundred forty-four Colombian patients with a median follow-up of 13.8 months and a median number of 6 maintenance cycles (range, 1–32) were assessed. The ORR among the patients was 66% (95% CI, 47% to 79%). The median PFS and (OS) rates were 7.9 months (95% CI, 5.9–10.0 months) and 21.4 months (95% CI, 18.3 to 24.4 months), respectively. We documented grade 3/4 hematologic toxicities, including anemia (14%), neutropenia (8%), and thrombocytopenia (16%). The identified grade 3/4 non-hematologic toxicities were proteinuria (2%), venous thrombosis (4%), fatigue (11%), infection (6%), nephrotoxicity (2%), and sensory neuropathy (4%). No grade >3 hemorrhagic events or hypertension cases were reported. OS was significantly higher in patients with the lowest TS mRNA levels [median, 29.6 months (95% CI, 26.2–32.9)] compared with those in patients with higher levels [median, 9.3 months (95% CI, 6.6–12.0); p = 0.0001]. TS expression (mRNA levels or protein expression) did not influence the treatment response.ConclusionOverall, PCB followed by maintenance pemetrexed and bevacizumab was effective and tolerable in Hispanic patients with non-squamous NSCLC. This regimen was associated with acceptable toxicity and prolonged OS, particularly in patients with low TS expression. We found a role for Ki67 and TS expression as prognostic factors.

Molecular Biomarker Study in a Randomised Phase III Trial of Irinotecan Plus S-1 versus S-1 for Advanced Gastric Cancer (GC0301/TOP-002)

AimsGastric cancer is a common and heterogeneous disease; however, global standard and biomarkers for selecting chemotherapy regimens have not been established. This study was designed retrospectively to identify molecular biomarkers for irinotecan plus S-1 (IRI-S) and S-1 therapy from subset analyses in GC0301/TOP-002, a randomised phase III trial for advanced gastric cancer. Materials and methodsParaffin-embedded primary tumour specimens were collected from 126 of 326 randomised patients in GC0301/TOP-002. The mRNA was measured for thymidylate synthase, dihydropyrimidine dehydrogenase, topoisomerase I, excision repair cross-complementing gene 1 (ERCC1) and thymidine phosphorylase; categorised into low and high to analyse their association with efficacy end points. ResultsThere was no significant difference in each mRNA between S-1 and IRI-S groups, whereas there were differences among some clinical characteristics. Multivariate analyses for overall survival showed that mRNA levels were not correlated with prognosis. By comparison, between IRI-S and S-1 arms, low thymidylate synthase, low ERCC1 and high thymidine phosphorylase were associated with better prognosis for IRI-S versus S-1 (hazard ratio = 0.653, 0.702 and 0.709, respectively; P < 0.15 for each interaction). ConclusionLow thymidylate synthase, low ERCC1 and high thymidine phosphorylase are candidates for predictive biomarkers for first-line treatment in advanced gastric cancer by IRI-S. Further study is warranted to confirm these results in other clinical trials and cohort studies.

Implications of Circadian Rhythm Regulation by microRNAs in Colorectal Cancer.

AimTo establish a connection between microRNA (miRNAs), circadian rhythm, and colorectal cancer patient survival.MethodsGenomic and clinical data were extracted from The Cancer Genome Atlas (TCGA) colorectal cancer database, and the expression levels of candidate miRNAs and a set of circadian rhythm-related genes (Per1, Per2, Per3, Bmal1), and genes associated with chemosensitivity (thymidylate synthase, dihydrofolate reductase) were assessed for any correlations among their expression. In addition, survival analyses specific to different colorectal cancer stages were performed to determine if these genes contribute to patient outcomes.ResultsSignificant inverse correlation between the expression of Per1 and that of miR-192 and miR-194 was observed. In survival analyses, high miR-192 and miR-194 correlate with better overall survival in Stage II patients, but worse survival in more advanced Stage III/IV patients. The expression of Per1, but Per2 or Bmal1, is marginally associated with patient survival for Stage II patients. Low thymidylate synthase expression correlates with better overall survival in Stage II patients but worse survival in Stage III/IV patients.ConclusionThis study establishes a foundation based on a large genomic database of colorectal cancer, for further investigation into the importance of regulatory mechanisms of circadian rhythm by miRNAs in colorectal cancer.

Intrahepatic cholangiocarcinoma and gallbladder cancer: distinguishing molecular profiles to guide potential therapy

BackgroundChemotherapy regimens for intrahepatic cholangiocarcinoma (ICC) and gallbladder adenocarcinoma (GC) remain interchangeable; however, response rates are frequently suboptimal. Biomarkers from ICC and GC patients were interrogated to identify actionable differences with potential therapeutic implications. MethodsFrom 2009 to 2012, pathological specimens from 217 ICC and 28 GC patients referred to Caris Life Sciences were evaluated. Specific testing by immunohistochemical analysis for 17 different biomarkers was performed. ResultsIn the collective cohort (n= 245), actionable targets included: 95% low thymidylate synthase (TS), 82% low ribonucleotide reductase subunit M (RMM) 1 and 74% low excision repair cross complementation group (ERCC) 1, indicating potential susceptibility to fluoropyrimidines/capecitabine, gemcitabine and platinum agents, respectively. Additional targets included TOPO1 (53.3% high, Irinotecan), MGMT (50.3% low, temozolomide), TOP2A (33% high, anthracyclines) and PGP (30.1% low, taxanes). Subgroup analysis by tumour origin demonstrated a differential biomarker expression pattern with a higher frequency of ICC tumours showing low levels of TS (99% versus 72%, P < 0.01), and RRM1 (85% versus 64%, P = 0.02) when compared with GC. Conversely a greater frequency of GC demonstrated high levels of TOPO1 (76% versus 50%, P = 0.02) versus ICC, indicating a potential increased benefit from irinotecan. DiscussionDifferences in the molecular profiles between ICC and GC provide evidence that the two are distinct diseases, requiring different treatment strategies to optimize a response.

Increased risk of severe fluoropyrimidine-associated toxicity in patients carrying a G to C substitution in the first 28-bp tandem repeat of the thymidylate synthase 2R allele.

The fluoropyrimidines act by inhibiting thymidylate synthase (TS). Recent studies have shown that patients' risk of severe fluoropyrimidine-associated toxicity is affected by polymorphisms in the 5'-untranslated region of TYMS, the gene encoding TS. A G>C substitution in the promoter enhancer region of TYMS, rs183205964 (known as the 2RC allele), markedly reduces TS activity in vitro, but its clinical relevance is unknown. We determined rs183205964 in 1605 patients previously enrolled in a prospective multicenter study. Associations between putative low TS expression genotypes (3RC/2RC, 2RG/2RC, and 2RC/2RC) and severe toxicity were investigated using univariable and multivariable logistic regression. Activity of TS and TYMS gene expression were determined in peripheral blood mononuclear cells (PBMCs) of a patient carrying genotype 2RC/2RC and of a control group of healthy individuals. Among 1,605 patients, 28 patients (1.7%) carried the 2RC allele. Twenty patients (1.2%) carried a risk-associated genotype (2RG/2RC, n=13; 3RC/2RC, n=6; and 2RC/2RC, n=1), the eight remaining patients had genotype 3RG/2RC. Early severe toxicity and toxicity-related hospitalization were significantly more frequent in risk-associated genotype carriers (OR 3.0, 95%CI 1.04-8.93, p=0.043 and OR 3.8, 95%CI 1.19-11.9, p=0.024, respectively, in multivariable analysis). The patient with genotype 2RC/2RC was hospitalized twice and had severe febrile neutropenia, diarrhea, and hand-foot syndrome. Baseline TS activity and gene expression in PBMCs of this patient, and a healthy individual with the 2RC allele, were found to be within the normal range. Our study suggests that patients carrying rs183205964 are at strongly increased risk of severe, potentially life-threatening, toxicity when treated with fluoropyrimidines.

Abstract 4335: Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed

Abstract Background: Approximately 40% of malignant pleural mesothelioma (MPM) patients respond to the combination treatment with the antifolate pemetrexed (PMX) and platinum compounds. Several studies highlighted the predictive role of thymidylate synthase (TS), but additional resistance mechanisms are poorly understood. This study aimed to evaluate the role of the reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) as potential biological markers of intrinsic PMX-resistance in MPM. Materials and methods: PCFT promoter methylation was evaluated by combined bisulfite restriction analysis as well as MethyLight PCR in 14 cell lines and 10 MPM tissues, while PCFT and RFC mRNA levels were studied by Real-Time-PCR in MPM specimens from a test and a validation cohort of 73 and 51 PMX-treated patients, respectively. PCFT protein expression was evaluated by immunohistochemistry in a tissue microarray with representative MPM core biopsies from 35 patients. Data were analyzed by t-test, Fisher/log-rank test and Cox proportional hazards models. Results: PCFT expression inversely correlated with promoter methylation and treatment with 5-Aza-2′-deoxycytidine (5-Aza-dC) induced its marked restoration in MPM cells. Patients with low PCFT mRNA levels had significantly shorter overall survival (OS), in both the test (median OS, 11.3 vs. 17.6 months, P = 0.01) and in the validation cohort (OS, 12.6 vs. 30.3 months, P = 0.02). Multivariate analysis, including age, sex, EORTC prognostic score and histology, confirmed PCFT prognostic relevance (HR, 2.1 and 2.4 in the test and validation cohorts, respectively). Low PCFT protein expression was also associated with significantly shorter OS (11.8 vs. 30.3 months, P = 0.02). Remarkably, the subgroup of patients with both low PCFT and high TS mRNA levels (30% of the patients in the test cohort) had the worse prognosis (median OS, 5.5 months, P&amp;lt;0.01), while no correlation was found in a series of 40 MPM patients not PMX-treated. No significant associations were found between RFC and clinical outcome. Conclusions: These results define PCFT as a novel prognostic factor for MPM patients treated with PMX. Prospective trials for the validation of the prognostic/predictive role of PCFT in MPM patients treated with PMX-based regimens are warranted. Furthermore preclinical data suggest that exposure to 5-Aza-dC might restore PCFT expression and result in efficient eradication of antifolate-resistant cells. Citation Format: Elisa Giovannetti, Paolo A. Zucali, Yehuda G. Assaraf, Niccola Funel, Maria Gemelli, Michal Stark, Leticia G. Leon, Zhanjun Hou, Matteo Perrino, Larry H. Matherly, Godefridus J. Peters. Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2015-4335

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.

ERCC1 and Thymidylate Synthase as Prognostic Biomarkers in Malignant Mesothelioma

The standard of care for malignant pleural mesothelioma (MPM) is a combination of platinum compounds and Pemetrexed. Still, the median survival of MPM patients is low (about 12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts by removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), which is involved in DNA synthesis.Pleural biopsies were collected from 140 MPM patients (98 males and 42 females, mean age 68 years) before treatment initiation. Average disease specific survival was 13 months (range 1‐60). mRNA was extracted from formalin fixed paraffin blocks, reverse transcribed and assayed by qRT‐PCR. Immunohistochemistry with anti‐ERCC1 and anti‐TS mAbs was scored semi‐quantitatively.Of 140 patients, 102 were treated, the remaining were not because of bad performance status or advanced age. Patients with low ERCC1 protein expression survived longer (24.3 vs 12.5 months, P=.03). Likewise, patients with low levels of TS mRNA had better survival (P=.01); there was no advantage, however, in the treated sub‐group, suggesting that Pemetrexed treatment and low TS expression had no additive value.ERCC1 protein expression and TS gene expression both seem important prognostic markers; we suggest their evaluation in routine biopsies from MPM patients.

Prognostic and predictive significance of thymidylate synthase protein expression in non-small cell lung cancer: a systematic review and meta-analysis.

It remains controversial whether thymidylate synthase (TS) protein expression is associated with survival for patients with non-small cell lung cancer (NSCLC). To evaluate prognostic and predictive significance of tumor TS protein level in NSCLC. Electronic searches were performed for relevant studies in PubMed, EMBASE, Web of Science, and Chinese Biomedical Literature Database. Hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS) were pooled for meta-analysis. Subgroup and sensitivity analyses were performed. Publication bias was evaluated by funnel plot and Begg's test. Twenty-four studies, including 2280 patients, were eligible. This analysis showed that patients with low TS expression had statistically significantly longer OS and PFS than those with high TS (HR=0.51 and HR=0.49, respectively). Based on TS-targeted drug use status, TS expression was significantly associated with OS in pemetrexed (HR=0.42) and 5-Fluorouracil subgroups (HR=0.34), but not in no TS-targeted drug subgroup. There were similar results for PFS analyses. Sensitivity analysis indicated that the results were robust. Begg's test did not reveal any publication bias. Low TS protein expression is a favorable predictive factor for better OS/PFS in NSCLC patients treated with TS-targeted drugs. Prognostic value of TS protein expression needs further validation.

Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location impacts the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the mRNA expression of proteins involved in major signaling pathways, including tumor growth (EGFR), angiogenesis (VEGFR2), DNA repair (ERCC1) and fluoropyrimidine metabolism (TS). FFPE tumor specimens from 431 advanced CRC patients were analyzed. The presence of 7 different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by RT-PCR. BRAF mutations were significantly more common in the proximal colon (p<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared to distal and proximal colon tumors (p=0.001), and increased TS levels compared to distal colon cancers (p=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR, and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

Abstract 1684: Histone deacetylase inhibition enhances Pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer models

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM's molecular targets) expression. Histone deacetylase inhibitors (HDACi) modulate the expression of multiple genes, including thymidylate synthase (TS), one of the main targets of Pemetrexed. Since high levels of TS have been linked to clinical resistance to Pemetrexed in non-small cell lung cancer (NSCLC), we investigated the molecular and functional effects of combined Pemetrexed and ITF2357, a class I/II HDACi currently in clinical trials as anti-cancer agents .ITF2357 reduced TS mRNA and protein expression, potentially affecting sensitivity to Pemetrexed. When the two drugs were used in combiation, their sequence of administration was critical in determining the pharmacologic response: in both NSCLC cell lines and LCSC, a strikingly synergistic reduction in cell viability was observed with the sequence Pemetrexed followed by ITF2357, in both adenocarcinoma and squamous cell carcinoma models. Cell death induced by the combination involved both apoptosis and autophagy. Genetic and pharmacological approaches provided an interesting link between these two pathways and showed that sequential Pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential Pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival. Overall, this data provide a strong rationale for the clinical development of sequential schedules employing Pemetrexed followed by HDAC inhibition in NSCLC. Citation Format: Daniela Trisciuoglio, Marianna Desideri, Teresa De Luca, Marta Di Martile, Chiara Gabellini, Adriana Eramo, Ruggero De Maria, Michele Milella, Donatella Del Bufalo. Histone deacetylase inhibition enhances Pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer models. [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 1684. doi:10.1158/1538-7445.AM2014-1684

Cisplatin Plus Pemetrexed (Cp) Versus Cisplatin Plus Gemcitabine (Cg) According to Thymidylate Synthase Expression in Non-Squamous Nsclc: a Biomarker-Stratified Randomized Phase Ii Trial

ABSTRACT Aim: JMDB trial showed CP was significantly superior to CG in non-squamous NSCLC, while CP was inferior in squamous NSCLC. Thymidylate synthase (TS) is one of target proteins of pemetrexed and the efficacy of pemetrexed-based chemotherapy could depend on TS expression. The primary endpoint is to determine the predictive value of TS in non-squamous NSCLC in terms of objective response rate (RR) by testing on the interaction between treatment arm and TS positivity. Methods: Main inclusion criteria are non-squamous NSCLC excepting for large cell neuroendocrine carcinoma, one or more measurable lesions, and no prior chemotherapy for advanced NSCLC. Written consents were obtained from eligible patients, and TS expression was collected by IHC. The patients with more than 10% of tumors expressing TS were grouped as a TS(+) and those with 10% or less were grouped as a TS(-). After being stratified as TS(+) or TS(-), patients were randomized to either CP or CG arms by 1: 1 fashion. CP or CG was administered until disease progression or unacceptable toxicity with maximum 6 cycles. Response evaluation was done every 2 cycles during treatment and every 2 months after completion of study chemotherapy. One-sided alpha of 0.1 was used for testing on the interaction term. Otherwise, all p-values are two-sided. Results: For 315 evaluable patients, the RR of CP and CG were 47.0% and 21.1% in TS (-) group, whereas 40.3% and 39.2% in TS (+) group (interaction P = 0.008). The RR of CP and CG arms evaluated by independent reviewers were 38.6% and 21.1% in TS(-) group, and 40.3% and 48.1% in TS(+) group (interaction P = 0.007). The median PFS of CP and CG were 6.4 and 5.5 months in TS (-) group (P = 0.013), whereas 5.9 and 5.3 months in TS (+) group (P = 0.64) (interaction P = 0.07). The median OS of CP and CG were not different in TS (-) group (not reached vs. 28.3 months, P = 0.86) and in TS (+) group (19.0 vs. 14.4 months, P = 0.36) (interaction P = 0.31). TS-negativity was an independent prognostic factor for OS (HR = 0.64, 95% CI 0.45-0.90). Conclusions: In terms of RR and PFS, clinical benefits of CP chemotherapy compared to CG were more prominent in TS (-) group than in TS (+) group, suggesting that TS can be used as a predictive biomarker. Furthermore, given that low TS expression was associated with prolonged OS irrespective of chemotherapeutic regimens, TS expression can also serve as a prognostic biomarker. Disclosure: All authors have declared no conflicts of interest.

Differential thymidylate synthase expression in lung cancer adenocarcinoma subtypes.

e18539 Background: Lung adenocarcinoma (ADC) is characterized by lower Thymidylate Synthase (TS) levels compared to squamous carcinoma and high sensitivity to Pemetrexed, an antifolate targeting TS...