High Thymidylate Synthase Expression Research Articles

TYMS Knockdown Suppresses Cells Proliferation, Promotes Ferroptosis via Inhibits PI3K/Akt/mTOR Signaling Pathway Activation in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by a grim prognosis and numerous challenges. The objective of our study was to examine the role of thymidylate synthase (TYMS) in TNBC and its impact on ferroptosis. The expression of TYMS was analyzed in databases, along with its prognostic correlation. TYMS positive expression was identified through immunohistochemistry (IHC), while real-time quantitative PCR (qRTPCR) was employed to measure TYMS mRNA levels in various cell lines. Western blotting was utilized to assess protein expression. Cell proliferation, mobility, apoptosis, and reactive oxygen species (ROS) levels were evaluated using CCK8, wound scratch healing assay, transwell assay, and flow cytometry, respectively. Additionally, a tumor xenograft model was established in BALB/c nude mice for further investigation. Tumor volume and weight were measured, and histopathological analysis using hematoxylin and eosin (H&E) staining was conducted to assess tumor tissue changes. IHC staining was employed to detect the expression of Ki67 in tumor tissues. High expression of TYMS was observed in TNBC and was found to be correlated with poor prognosis in patients. Among various cell lines, TYMS expression was highest in BT549 cells. Knockdown of TYMS resulted in suppression of cell proliferation and mobility, as well as promotion of apoptosis. Furthermore, knockdown of TYMS led to increased accumulation of ROS and Fe2+ levels, along with upregulation of ACLS4 expression and downregulation of glutathione peroxidase 4 (GPX4) expression. In vivo studies showed that knockdown of TYMS inhibited tumor growth. Additionally, knockdown of TYMS was associated with inhibition of mTOR, p-PI3K, and p-Akt expression. Our research showed that the knockdown of TYMS suppressed the TNBC progression by inhibited cells proliferation via ferroptosis. Its underlying mechanism is related to the PI3K /Akt pathway. Our study provides a novel sight for the suppression effect of TYMS on TNBC.

Sodium Butyrate Inhibits the Expression of Thymidylate Synthase and Induces Cell Death in Colorectal Cancer Cells.

The most commonly used chemotherapy for colorectal cancer (CRC) is the application of 5-fluorouracil (5-FU). Inhibition of thymidylate synthase (TYMS) expression appears to be a promising strategy to overcome the decreased sensitivity to 5-FU caused by high expression of TYMS, which can be induced by 5-FU treatment. Several compounds have been shown to potentially inhibit the expression of TYMS, but it is unclear whether short-chain fatty acids (SCFAs), which are naturally produced by bacteria in the human intestine, can regulate the expression of TYMS. Sodium butyrate (NaB) is the most widely known SCFA for its beneficial effects. Therefore, we investigated the enhancing effects on inhibition of cell viability and induction of apoptosis after co-treatment of NaB with 5-FU in two CRC cell lines, HCT116 and LoVo. This study suggests that the effect of NaB in improving therapeutic sensitivity to 5-FU in CRC cells may result from a mechanism that strongly inhibits the expression of TYMS. This study also shows that NaB inhibits the migration of CRC cells and can cause cell cycle arrest in the G2/M phase. These results suggest that NaB could be developed as a potential therapeutic adjuvant to improve the therapeutic effect of 5-FU in CRC.

Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors.

Cell division is driven by nucleic acid metabolism, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in nucleotide synthesis. As a result, thymidylate synthase has emerged as a critical target in chemotherapy. 5-Fluorouracil (5-FU) is currently being used to treat a wide range of cancers, including breast, pancreatic, head and neck, colorectal, ovarian, and gastric cancers The objective of this study was to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives (UD-1 to UD-5) and to evaluate their therapeutic potential in BC cells. One-pot organic synthesis processes using a single solvent were used for the synthesis of drug analogues of Uracil. Integrated bioinformatics using GEPIA2, UALCAN, and KM plotter were utilized to study the expression pattern and prognostic significance of TYMS, the key target gene of 5-fluorouracil in breast cancer patients. Cell viability, cell proliferation, and colony formation assays were used as in vitro methods to validate the in silico lead obtained. BC patients showed high levels of thymidylate synthase, and high expression of thymidylate synthase was found associated with poor prognosis. In silico studies indicated that synthesized uracil derivatives have a high affinity for thymidylate synthase. Notably, the uracil derivatives dramatically inhibited the proliferation and colonization potential of BC cells in vitro. In conclusion, our study identified novel uracil derivatives as promising therapeutic options for breast cancer patients expressing the augmented levels of thymidylate synthase.

The Predictive Value of ERCC1, RRM1, and Thymidylate Synthase in Advanced Malignant Pleural Mesothelioma Patients Treated with Platinum-Based Chemotherapy.

A rare yet severe neoplasia called malignant pleural mesothelioma (MPM) typically manifests itself in advanced stages. Despite some improvements in the treatment of patients with MPM, this malignancy continues to have a detrimental prognosis . The primary goal of the present study was to assess the associatin between ERCC1, RRM1, and thymidylate synthase (TS) expression and disease outcome in patients with malignant pleural mesothelioma (MPM) treated with either pemetrexed plus cisplatin or gemcitabine plus cisplatin. This prospective cohort study was done on ninety-one advanced MPM patients. The patients received either pemetrexed plus platinum (55 of 91) or gemcitabine plus platinum chemotherapy (36 of 91). Tissue biopsy was taken at time of diagnosis. Immunohistochemistry was used to assess the levels of ERCC1, RRM1, and TS transcription in tissue biopsies (paraffin embedded). Based on the findings, 70% of patients with low expression of TS had low expression of ERCC1, and 68% of patients with high expression of TS had high expression of ERCC1, suggesting a significat association between ERCC1 expression and TS expression (p=0.005). However, there was no relation between ERCC1 and RRM1 expression patterns (p= 0.296). In patients underwen platinum-based theraphy (n 91), a significant correlation was detected between low ERCC1 median High-scoring and longer progression time (TTP;9.6 v 5.3 months;P< .001) or overall survival (OS) (OS;18.1 v 9.1 months; P<0.001). A significant correlation was found between low TS protein expression and longer time progression (TTP; 11.8 v 5.4 months; P< .001) or OS (OS; 19.8 v 8.5 months; P <0.001) in patients undergoing pemetrexed plus platinum chemotherapy (n 55). Low RRM1 expression was accompanied by high progression free survival (TTP; 10.6 v 3.8 months) and OS (OS; 20.6 v 8.6 months) in patients receiving gemcitabine plus platinum chemotherapy. Based on the multivariate test results, the independent variables significantly affecting OS were tumor stage (HR: 2.3; 95% CI: 1.1-4.9; p= 0.021) and ERCC1 (HR: 2.7; 95% CI: 1.7-4.3; p < 0.001). Decreased TS protein expression can be indicative of greater responsivness to pemtrexed and of longer TTP and OS in individuals with advanced MPM (locally progressed or metastatic) who are receiving pemetrexed-based chemotheraphy. Low ERCC1 expressions in individuals with advanced MPM can predict increased PFS and OS, as well as a better responsivness to platinum-based chemotherapy. In patients with progressed MPM receiving gemcitabine plus cisplatin chemotherapy, lower RRM1 expression was associated with a better prognosis, longer PFS, and longer OS.

FOXM1-induced TYMS upregulation promotes the progression of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of primary liver cancer and one of the major causes of cancer-related death. Thymidylate synthase (TYMS) catalyzes the methylation of deoxy guanosine to deoxy thymidylate, which is a crucial gene for DNA repair and replication. Thus, TYMS was reported to be closely associated with developing a variety of tumors, but it has been poorly studied in HCC.Materials and methodsWe used the cell counting kit-8 (CCK-8), BrdU, and CFSE assay to measure cell proliferation. The flow cytometry assay and the TUNEL assay were used for assessing cell apoptosis. The flow cytometry assay was used to analyze the cell cycle. The Transwell invasion assay and the wound healing assay were conducted to determine the invasive ability of the cells. RT-qPCR and Western blot analyses were performed to evaluate the mRNA and protein expression levels of specific genes, respectively.ResultsTYMS was found to be upregulated in both HCC cells and patient samples. High expression of TYMS was associated with an unfavorable prognosis in HCC patients based on the TCGA-LIHC dataset. Cell proliferation, apoptosis, and invasion assays revealed that TYMS promoted the proliferation and invasion of HCC cells as well as inhibited apoptosis. In addition, TYMS is a downstream target of FOXM1. TYMS knockdown reversed the 5-FU resistance caused by FOXM1 overexpression and re-sensitized HCC cells to 5-FU treatment.ConclusionThis study suggested that TYMS serves as an oncogene in HCC, and targeting the FOXM1-TYMS axis may help improve the survival of HCC patients as well as provide new insights for treating advanced HCC patients.

Comprehensive expressional analysis of chemosensitivity-related markers in large cell neuroendocrine carcinoma of the lung.

ObjectivesVarious drug‐sensitivity markers have been reported to be associated with tumor progression and chemotherapy resistance. Detailed expression profiles of sensitivity markers for cytotoxic chemotherapy in pulmonary large cell neuroendocrine carcinoma (LCNEC) remain unclear. Herein, we aimed to clarify the correlation between the expression of drug‐sensitivity markers and clinicopathological features, prognostic impact, and status of tumor immunity in patients with LCNEC.MethodsWe retrospectively analyzed the correlation between clinicopathological features and the expression of drug‐sensitivity‐related markers, including vascular endothelial growth factor 2 (VEGFR2), thymidylate synthase (TS), tubulin beta 3 class III (TUBB3), topoisomerase I (Topo‐I), and Topo‐II in 92 surgically resected LCNEC samples. Furthermore, we examined the prognostic significance of expression of these and their correlation with the immune cell status.ResultsOverall, high expression of TS, TUBB3, VEGFR2, Topo‐I, and Topo‐II was detected in 50 (54%), 31 (34%), 23 (25%), 65 (71%), and 36 (39%) samples, respectively. Univariate and multivariate analyses revealed that advanced pathological T and N factors, positive lymphatic permeation, and Topo‐II expression were independent unfavorable prognosticators for recurrence‐free survival, and advanced pathological T and N factors, Topo‐II positive expression, and TS positive expression were independent unfavorable prognosticators for overall survival. In terms of correlation with immune cell status, higher expression of VEGFR2 was closely linked to negative PD‐L1 expression.ConclusionsThese findings suggest that elevated Topo‐II and TS expression may contribute to poor outcomes through protumoral biology in patients with LCNEC, and elevated VEGFR2 expression might negatively impact tumor immune reactions in LCNEC.

Prognostic and Predictive Role of Excision Repair Cross-complementation Group 1 and Thymidylate Synthase in Colorectal Carcinoma Patients Received FOLFOX Chemotherapy: An Immunohistochemical Study.

We aim to determine the frequency of thymidylate synthase (TS) and excision repair cross-complementation group 1 (ERCC-1) immunohistochemical (IHC) expression and its relationship with clinicopathologic variables in colorectal carcinoma (CRC) patients. In addition, we aim to assess the correlation between TS and ERCC-1 expression and the response of these cases to oxaliplatin and 5-fluorouracil chemotherapy (FOLFOX). Fifty-one CRC patients were prepared for IHC analysis of ERCC-1 and TS protein expression. All patients received oxaliplatin and 5-fluorouracil combined chemotherapy (FOLFOX) and were followed up for 24 months. The data analysis showed that high ERCC-1 and TS expression was significantly associated with early treatment failure (P=0.020 and 0.000). In contrast, TS immunoexpression affects the disease-free survival rate (P=0.010). The presence of deep tumor invasion, distant metastasis, lymph node metastasis, and high Dukes' classification were significantly statistically associated with early treatment failure (P=0.001, 0.000, 0.041, and 0.015, respectively). Our results showed that both ERCC-1 and TS are predictive factors for early treatment failure in CRC patients. TS protein is a prognostic factor for disease-free survival rates. This supports the theory that both ERCC-1 and TS can be used to improve chemotherapeutic outcomes in CRC patients. High expression of TS and ERCC-1 helps in the identification of cases that will get fewer benefits from FOLFOX chemotherapy. As an innovative strategy, in these cases, we can use alternative chemotherapeutic regimens or add an extra agent. In addition, Dukes' classification and lymph node metastasis are predictive factors for early treatment failure. Thus, all those values can be used to predict CRC patients with bad prognosis and those who will get fewer benefits from FOLFOX.

Predictive Potential of PD-L1, TYMS, and DCC Expressions in Treatment Outcome of Colorectal Carcinoma.

Colorectal carcinoma (CRC) is a malignancy of epithelial origin in the large bowel. The elucidation of the biological functions of programmed cell death ligand-1 (PD-L1), thymidylate synthase (TYMS), and deleted in colorectal cancer (DCC) biomarkers including their roles in the pathophysiology of CRC - has led to their applications in diagnostic and chemo-pharmaceutics. We investigated whether PD-L1, TYMS, and DCC protein expression in CRC tumors are predictive biomarkers of treatment outcome for CRC patients. The expressions of PD-L1, TYMS, and DCC were evaluated by immunohistochemistry (IHC) in 91 paraffin-embedded samples from patients who underwent colectomy procedure in Hospital Serdang, Selangor, Malaysia. There was high expression of DCC in most cases: 84.6% (77/91). PD-L1 showed low expression in 93.4% (86/91) of cases and high expression in 6.6% (5/91) of cases. Low and high expressions of TYMS were detected in 53.8% (49/91) and 46.2% (42/91) of the CRC cases, respectively. There was a significant association between the TYMS expression and gender (P<0.05); the expression of TYMS was observed at a high level in 76.2% of males and in 23.8% of females. The mean overall survival (OS) was 100months for the CRC patients evaluated. The OS for patients with high expression of PD-L1 was 22months. Patients with high expression of TYMS and DCC showed OS of 90 and 96months, respectively. The results from this study suggest that PD-L1, TYMS, and DCC expression could be used as biomarkers to stratify CRC patients who could benefit from adjuvant therapy.

EP1.01-71 Thymidylate Synthase and Folate Receptor Alpha Expression as Potential Biomarkers for Efficacy of Pemetrexed in NSCLC

Predictive biomarkers for chemotherapy in advanced non-squamous NSCLC are lacking. Thymidylate synthase (TS) and folate receptor alpha (FRA) are target enzymes for pemetrexed. Here we investigated TS and FRA expression and their role as a prognostic and predictive biomarker for efficacy of pemetrexed-based chemotherapy. We performed immunohistochemistry on pre-treatment tumour specimens for TS and FRA expression and correlated it with patient’s demographic and clinical characteristics, treatment responses and survivals in a retrospective training cohort of patients with advanced non-squamous NSCLC treated with pemetrexed-based chemotherapy. Similar analysis for validation was performed in a prospective cohort of patients participating in a randomized control trial “to compare efficacy and safety of pemetrexed-carboplatin versus paclitaxel-carboplatin as induction regimen in advanced non-squamous NSCLC”. Chi-square test was used to co-relate TS and FRA expression with clinico-pathological characteristics. Kaplan-Meier methods, Log-rank test and Cox proportional-hazards model were used for survival analysis. In the retrospective training cohort median age was 57 (26-70) years with male predominance (ratio=2:1). TS and FRA expression were evaluable in 55 and 47 patients respectively. In this cohort TS and FRA expression didn’t co-relate with best overall response rates (ORR), however, low TS expression and positive FRA expression were associated with improved progression free survival (PFS), albeit non-significant. In the prospective validation cohort median age was 52 (28-65) years with 70% males. In this cohort TS and FRA expression were analysed in 113 and 97 patients respectively. High TS expression was significantly associated with better ORR in patients treated with paclitaxel-carboplatin (p=0.04) but there was no co-relation of TS expression with response rates in pemetrexed-carboplatin group. Younger patients (age<40 years) had more TS ‘low or negative’ status (p=0.04). High TS expression was associated with ALK positivity (p=0.02), bone metastases (p=0.01) and brain metastases (p=0.002). Positive FRA expression was associated with EGFR positivity (p=0.004) and liver metastases (p=0.020). Positive FRA expression was associated with improved PFS in patients treated with pemetrexed-carboplatin (median: 9.23 versus 4.27 months, p=0.01), paclitaxel-carboplatin (median: 10.87 versus 6.47 months, p=0.08) as well as improved OS (median: OS not reached versus 10.13 months, p=0.01), especially in patients treated with pemetrexed-carboplatin (median: 18.73 versus 10.46 months, p=0.08). In conclusion, the results from this study suggest that TS or FRA expression doesn’t predict efficacy of Pemetrexed, however, high TS expression may predict better ORR in patients receiving paclitaxel-carboplatin. FRA expression may serve as a prognostic factor in patients receiving chemotherapy irrespective of the regimen.

High thymidylate synthase gene expression predicts poor outcome after resection of hepatocellular carcinoma.

IntroductionPrognosis after resection of hepatocellular carcinoma (HCC) is highly variable. Compared to clinicopathologic factors, the use of molecular markers to predict outcome has not been well studied. We investigated the prognostic importance of thymidylate synthase (TS) gene expression and polymorphisms in patients after resection of HCC.MethodsPatients who underwent complete resection of HCC for whom tissue was available were identified. TS gene expression level and polymorphisms were determined in HCC specimens. Prognostic factors were evaluated using Kaplan-Meier curves and Cox proportional hazard models.ResultsThe study included 67 patients. In univariate analysis, variables that negatively influenced survival included TNM stage, microvascular invasion, and high TS expression. For the high TS expression group, median survival was 54 months and 5-year actuarial survival was 47%. For the low TS expression group, median survival was not reached and the 5-year actuarial survival was 91%. In multivariate analysis, only high TS expression remained an independent predictor of poor survival (HR = 10.77, 95% CI 1.36–84.91; P = 0.02). TS gene polymorphisms were not associated with TS expression or overall survival.ConclusionsHigh TS expression predicts poor outcome after resection of HCC. Molecular markers might be robust predictors of patient outcome after resection of HCC.

Immunohistochemistry Biomarkers Predict Survival in Stage II/III Gastric Cancer Patients: From a Prospective Clinical Trial.

PurposeIdentification of biomarkers to predict recurrence risk is essential to improve adjuvant treatment strategies in stage II/III gastric cancer patients. This study evaluated biomarkers for predicting survival after surgical resection.Materials and MethodsThis post-hoc analysis evaluated patients from the CLASSIC trial who underwent D2 gastrectomywith orwithout adjuvant chemotherapy (capecitabine plus oxaliplatin) at the Yonsei Cancer Center. Tumor expressions of thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), and programmed death-ligand 1 (PD-L1) were evaluated by immunohistochemical (IHC) staining to determine their predictive values.ResultsAmong 139 patients, IHC analysis revealed high tumor expression of TS (n=22, 15.8%), ERCC1 (n=23, 16.5%), and PD-L1 (n=42, 30.2%) in the subset of patients. Among all patients, high TS expression tended to predict poor disease-free survival (DFS; hazard ratio [HR], 1.80; p=0.053), whereas PD-L1 positivity was associated with favorable DFS (HR, 0.33; p=0.001) and overall survival (OS; HR, 0.38; p=0.009) in multivariate Cox analysis. In the subgroup analysis, poor DFS was independently predicted by high TS expression (HR, 2.51; p=0.022) in the adjuvant chemotherapy subgroup (n=66). High PD-L1 expression was associated with favorable DFS (HR, 0.25; p=0.011) and OS (HR, 0.22; p=0.015) only in the surgery-alone subgroup (n=73). The prognostic impact of high ERCC1 expression was not significant in the multivariate Cox analysis.ConclusionThis study shows that high TS expression is a predictive factor for worse outcomes on capecitabine plus oxaliplatin adjuvant chemotherapy, whereas PD-L1 expression is a favorable prognostic factor in locally advanced gastric cancer patients.

Thymidylate synthase predicts poor response to pemetrexed chemotherapy in patients with advanced breast cancer.

Pemetrexed is a candidate chemotherapy regimen for anthracycline- and taxane-pretreated advanced breast cancer. However, to the best of our knowledge, no efficient treatment efficacy biomarkers have been identified. In the present study, the potential correlation between thymidylate synthase (TYMS) expression and clinical response to pemetrexed was examined in advanced breast cancer. A retrospective collection was performed by using 77 advanced breast cancer subjects, who received at least three cycles of pemetrexed treatment in the Second Hospital of Shandong University hospital. TYMS expression was detected using immunohistopathological staining. The correlations between TYMS and therapeutic efficacies of different chemotherapy treatment were analyzed. The objective response rate (ORR) and disease control was 31.17 and 64.94%, respectively. Immunohistochemical staining demonstrated that TYMS expression was observed in the cytoplasm and nuclei of breast cancer cells. High TYMS expression was observed in 32 specimens. Elevated TYMS expression was correlated with higher histological grade and lymph node metastasis (P<0.05). Furthermore, significantly higher TYMS expression was observed in treatment-resistant patients than response ones (P<0.05). Patients with low expression level of TYMS exhibit significantly higher ORR. Cox regression analysis indicated that elevated TYMS expression was a detrimental factor for pemetrexed treatment for advanced breast cancer patients. The present results suggested that TYMS expression levels predicts therapeutic sensitivity of pemetrexed chemotherapy in advanced breast cancer, indicating that it may be a useful biomarker to choose chemotherapy regimens.

Study on the correlation between thymidylate synthase expression level and postoperative chemotherapy in gastric cancer

Objective To investigate the expression of thymidylate synthase (TS), and the correlations with the clinicopathologcal factors and the prognosis of postoperative fluorouracil chemo-therapy in gastric cancer. Methods 42 patients with gastric cancer who underwent radical gastrectomy were collected from April 2011 to April 2014 in Lu′an Hospital of Anhui Medical University, and the chemotherapy regimen was Cisplatin+ Capecitabine or Cisplatin+ Teggio. The levels of TS in tumor tissues of 42 patients with gastric cancer were detected by immunohistochemical method. The relationships between TS expression level, clinical features and the prognosis of patients treated with fluorouracil chemotherapy were analyzed. Results In 42 patients with gastric cancer, 19 patients (45.2%) have TS high expressions, 23 patients (54.8%) have TS low expressions. There were no significant correlations between the expression of TS and age, sex, clinical stage, differentiation degree, tumor location, depth of invasion, lymph node metastasis, vascular tumor thrombus and nerve bundle invasion (all P>0.05). There were significant differences between low TS expression and high TS expression in median progression-free survival and 3-year survival rate of patients [35 months vs. 17 months, 73.9% (17/23) vs. 42.1% (8/19)], and the differences were statistically significant (χ2=4.788, P=0.029; χ2=4.369, P=0.037). In patients with stage Ⅲ gastric cancer, the median progression-free survival of patients with low TS expression was significantly prolonged compared to that with high TS expression (33 months vs. 9 months), and the difference was statistically significant (χ2=4.731, P=0.030). Conclusion TS expression levels have no significant correlations with the clinicopatho-logcal factors, but closely relate to the prognosis of gastric cancer patients treatment with fluorouracil chemotherapy. The chemotherapy prognosis of TS high expression is worse than that of TS low expression in gastric cancer. Key words: Thymidylate synthase; Stomach neoplasms; Chemotherapy, adjuvant; Fluorouracil

Trifluridine/tipiracil overcomes the resistance of human gastric 5-fluorouracil-refractory cells with high thymidylate synthase expression.

Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is a combination of the antineoplastic thymidine analog, FTD, and thymidine phosphorylase inhibitor, TPI (molar ratio 1:0.5). FTD/TPI was approved in Japan, the United States, and the European Union for the treatment of unresectable advanced or recurrent colorectal cancer. We evaluated the in vitro and in vivo efficacy and mechanisms of action of FTD and FTD/TPI against 5-fluorouracil (5-FU)-resistant MKN45/5FU, MKN74/5FU, and KATOIII/5FU human gastric cancer cells overexpressing thymidylate synthase (TS) and their respective parent cell lines. MKN45/5FU and KATOIII/5FU cells were not cross-resistant to FTD, whereas MKN45/5FU cells were 3.7-fold more resistant than the parental cells in vitro. FTD was also incorporated into genomic DNA in a concentration-dependent manner in 5-FU-resistant and parental cells. Additionally, deoxyuridine monophosphate levels in MKN45/5FU cells after 24-h FTD treatment were 3.0-fold higher than those in parental cells, and FTD treatment for 72 h induced G2/M arrest in MKN45/5FU cells, unlike the S phase arrest in MKN45 cells. Thus, TS-overexpressing MKN45/5FU cells, but not MKN74/5FU and KATOIII/5FU cells, showed partial cross-resistance to FTD. However, FTD/TPI (administered orally twice a day) exhibited antitumor activity to the same extent in MKN45 and MKN45/5FU xenograft mouse models, overcoming in vitro cross-resistance to FTD. DNA incorporation rather than TS inhibition seems to be the main action of FTD under these in vivo conditions. Thus, FTD/TPI is a promising chemotherapeutic agent against gastric cancers recurring following 5-FU therapy.

Astrocyte-elevated gene-1 confers resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase expression.

Previous studies have suggested that astrocyte-elevated gene-1 (AEG-1) contributes to the mechanisms of resistance to various chemotherapeutics. In this study, we investigated whether AEG-1 expression level correlated with that of thymidylate synthase (TS), as higher TS expression is known to be associated with the resistance to pemetrexed chemotherapy in patients with advanced lung adenocarcinoma. Using pemetrexed-resistant lung adenocarcinoma PC-9 cell line, we demonstrated that transfection of AEG-1 siRNA lowered TS expression and decreased pemetrexed IC50 value. In contrast, overexpression of AEG-1 was associated with increased expression of TS and higher pemetrexed IC50 value. Immunohistochemical staining of clinical biopsy samples showed that patients with lower AEG-1 expression had longer overall survival time. Moreover, analysis of repeated biopsy samples revealed that an increase in the TS level from baseline to disease progression was significantly associated with the elevation of AEG-1 expression. In conclusion, our data demonstrated that TS expression might be regulated by AEG-1 and that increased expression of these proteins contributes to lung cancer disease progression and may be associated with the development of resistance to pemetrexed.

Вплив інтратуморальної експресії тимідилатсинтази на виживаність хворих на рак підшлункової залози, які отримували гемци­табін і тегафур в ад’ювантному лікуванні

Рак підшлункової залози (РПЖ) входить в першу четвірку в структурі онкологічної смертності. Комбінації фторпіримідинів і гемцитабина давно і активно використовуються в паліативної хіміотерапії РПЖ. У ад'ювантном режимі такі комбінації використовуються рідше, було показано, що вони можуть впливати на довгострокову виживаність, але ефект значно відрізняється серед пацієнтів з різним фенотипом пухлини. Метою нашого дослідження було вивчення взамосвязі рівня експресії тіміділатсінтази (TS) у хворих РПЖ, які отримують ад'ювантному гемцитабін + тегафур (ГемТег), і їх загальної виживаності. У дослідження були залучені 62 пацієнта, яким була виконана радикальна резекція з приводу РПЖ. Aд'ювантная хіміотерапія за схемою ГемТег починалася не раніше 2 тижнів після операції. У всіх випадках протоковая аденокарцинома підшлункової залози була верифікована при рутинному гістологічному дослідженні, після чого імуногістохімічно визначали експресію TS. Дані загального виживання були проаналізовані шляхом побудови кривих Каплана-Майера. Виживання в групах порівнювали за допомогою log-rank тесту. Медіана виживання хворих з високою експресією TS на 14 місяців більше, ніж у хворих з низькою експресією TS (36 і 12 соотв.). Перевага в виживання хворих з високою експресією TS є статистично значущим (р = 0,005). Високий рівень імуногістохімічної експресії TS в пухлинах хворих РПЖ може позитивно впливати на прогноз хворих, які отримують поліхіміотерапію за схемою ГемТег в ад'ювантному режимі.

Integrating TYMS, KRAS and BRAF testing in patients with metastatic colorectal cancer.

AIMTo investigate the impact of thymidylate synthase (TYMS), KRAS and BRAF in the survival of metastatic colorectal cancer (mCRC) patients treated with chemotherapy.METHODSClinical data were collected retrospectively from records of consecutive patients with mCRC treated with fluoropyrimidine-based chemotherapy from 1/2005 to 1/2007. Formalin-fixed paraffin-embedded tissues were retrieved for analysis. TYMS genotypes were identified with restriction fragment analysis PCR, while KRAS and BRAF mutation status was evaluated using real-time PCR assays. TYMS gene polymorphisms of each of the 3’ untranslated region (UTR) and 5’UTR were classified into three groups according to the probability they have for high, medium and low TYMS expression (and similar levels of risk) based on evidence from previous studies. Univariate and multivariate survival analyses were performed.RESULTSThe analysis recovered 89 patients with mCRC (46.1% de novo metastatic disease and 53.9% relapsed). Of these, 46 patients (51.7%) had colon cancer and 43 (48.3%) rectal cancer as primary. All patients were treated with fluoropyrimidine-based chemotherapy (5FU or capecitabine) as single-agent or in combination with irinotecan or/and oxaliplatin or/and bevacizumab. With a median follow-up time of 14.8 mo (range 0-119.8), 85 patients (95.5%) experienced disease progression, and 63 deaths (70.8%) were recorded. The 3-year and 5-year OS rate was 25.4% and 7.7% while the 3-year progression-free survival rate was 7.1%. Multivariate analysis of TYMS polymorphisms, KRAS and BRAF with clinicopathological parameters indicated that TYMS 3’UTR polymorphisms are associated with risk for disease progression and death (P < 0.05 and P < 0.03 respectively). When compared to tumors without any del allele (genotypes ins/ins and ins/loss of heterozygosity (LOH) linked with high TYMS expression) tumors with del/del genotype (low expression group) and tumors with ins/del or del/LOH (intermediate expression group) have lower risk for disease progression (HR = 0.432, 95%CI: 0.198-0.946, P < 0.04 and HR = 0.513, 95%CI: 0.287-0.919, P < 0.03 respectively) and death (HR = 0.366, 95%CI: 0.162-0.827, P < 0.02 and HR = 0.559, 95%CI: 0.309-1.113, P < 0.06 respectively). Additionally, KRAS mutation was associated independently with the risk of disease progression (HR = 1.600, 95%CI: 1.011-2.531, P < 0.05). The addition of irinotecan in 1st line chemotherapy was associated independently with lower risk for disease progression and death (HR = 0.600, 95%CI: 0.372-0.969, P < 0.04 and HR = 0.352, 95%CI: 0.164-0.757, P < 0.01 respectively).CONCLUSIONThe TYMS genotypes ins/ins and ins/LOH associate with worst prognosis in mCRC patients under fluoropyrimidine-based chemotherapy. Large prospective studies are needed for validation of our findings.

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

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

P2.03a-027 A Phase I Study of the Non-Receptor Tyrsine Kinase Inhibitor (NKI) Bosutinib in Combination with Pemetrexed in Patients with Advanced Solid Tumors: Topic: Clinical Trials

The combination of cytotoxic chemotherapy with Src signaling pathway inhibitors represents a potential novel strategy to improve tumor response. Preclinical data suggests that thymidylate synthase (TS) and Src act via a common pathway and their overexpression has prognostic significance. Ceppi et al, explored the concept that Src inhibitors could be synergistic in combination with pemetrexed. Using immunohistochemical detection, Src kinase activation, evaluated by a phosphospecific antibody, was associated with higher TS expression in tumor specimens. Src-inhibition synergistically enhanced pemetrexed-cytotoxicity in human A549 lung cancer cells. Treatment with a Src inhibitor prevented up-regulation of TS mRNA and protein levels induced by pemetrexed that increased resistance to treatment. This suggests that Src represents a potential target to improve the efficacy of TS-inhibiting agents mainly in treatment of metastatic adenocarcinoma of the lung and malignant mesothelioma. Bosutinib is a NKI that inhibits the Abelson and Src kinases approved to treat CML. Hypothesis. Bosutinib can be safely administered in combination with pemetrexed with the MTD determined. Objectives. 1) Determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of bosutinib combined with pemetrexed, 2) Estimate the tumor response rate (RR), progression-free survival (PFS) and overall survival (OS) for patients with selected advanced cancers treated with this combination, 3) Explore potential biomarkers for treatment selection and response, including expression of Src kinase, phosphorylated Src kinase, and expression of potential modifying genes including K-ras, and 4) Determine the effect on the pharmacokinetics of each drug. This is a single institution phase I dose-escalation study for patients with selected advanced solid malignancies eligible to receive pemetrexed and ECOG performance status 0-2. Sequential dose cohorts of 3-6 patients will be treated. The starting dose of bosutinib is 200 mg daily in addition to pemetrexed 500 mg/m2 (Table 1).Tabled 1Dose level (cohort)number of patientsBosutinib dose (mg)Pemetexed dose (mg/m2)-23-6100400-13-610050013-620050023-630050033-640050043-6500500Expansion cohort10-12MTD500 Open table in a new tab not applicable. not applicable.

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.