Expression of thymidylate synthase in salivary adenoid myoepithelial cells and its clinical significance / 南方医科大学学报

OBJECTIVE@#To evaluate the expression of thymidylate synthase (TS) in myoepithelial cells (MECs) of salivary adenoid tissues and explore its clinical significance.@*METHODS@#Immunohistochemical staining EnVision method was used to detect the expression of TS, P63, Calponin, CK5/6 and S-100 in 32 salivary gland specimens, including 10 non-neoplastic and salivary inflammation specimens, 11 mixed tumor specimens, 5 basal cell carcinoma specimens and 6 adenoid cyst carcinoma specimens. The specificity and sensitivity of TS as a specific molecular marker of salivary muscle epithelial cells were evaluated in comparison with P63, Calponin, CK5/6 and S-100.@*RESULTS@#The expression pattern of TS in all the salivary gland tissue specimens was identical with that of p63. TS and P63 both showed strong immunohistochemical expressions in MECs of salivary adenoid tissue specimens. Calponin, CK5/6, and S-100 showed cytoplasmic/membranous expressions in the MECs. In addition, TS exhibited weak or moderate cytoplasmic expression in a few salivary gland epithelial cells, cancer cells and scattered stromal cells, with negative expression in the cell nuclei. The expression of TS in the MECs of all the salivary adenoid specimens was highly consistent with those of P63, Calponin, CK5/6 and S-100 (>0.05) Except for CK5/6 expression in Salivary inflammation and Salivary gland specimens. Kappa>0.75. The specificity and sensitivity of TS as a molecular marker of MECs were both 100%.@*CONCLUSIONS@#TS is a new specific marker of MECs for differential diagnosis of salivary gland tumors.

Immunoexpression of TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers and its correlation with degree of differentiation, tumor staging and prognostic factors in colorectal adenocarcinoma: a retrospective longitudinal study

ABSTRACT BACKGROUND: There are cases of colorectal tumors that, although small, show more aggressive evolution than large tumors. This motivated us to study whether there are any proteins capable of alerting about these changes. The aim here was to correlate the immunoexpression of the TS, p53, COX2, EGFR, MSH6 and MLH1 biomarkers in tumors in patients with colorectal adenocarcinoma, with the degree of cell differentiation, tumor staging and clinical-pathological prognostic factors. DESIGN AND SETTING: Retrospective observational study at a public tertiary-level hospital. METHODS: We analyzed tissue-microarray paraffin blocks of tumor tissues that had been resected from 107 patients. We used Fisher's exact test to study associations between tumor differentiation/staging and the immunoexpression of biomarkers. We also used Kaplan-Meier estimation, the log-rank test and the adjusted Cox regression model to investigate the patients' overall survival (in months) according to biomarkers and disease-free interval. RESULTS: The degree of tumor differentiation and tumor staging were not associated with the biomarkers, except in cases of patients in stages III or IV, in which there was a correlation with MLH1 expression (P=0.021). Patient survival and disease-free interval were not associated with the biomarkers. CONCLUSION: There were no associations between the degree of tumor differentiation, staging, length of survival or disease-free interval and the immunoexpression of the TS, p53, COX2, EGFR or MSH6 tumor markers. In advanced cases of colorectal adenocarcinoma (stages III and IV), there was a higher percentage of MLH1-negative results.

Immunohistochemistry Biomarkers Predict Survival in Stage II/III Gastric Cancer Patients: From a Prospective Clinical Trial / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Identification 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 METHODS: This 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. RESULTS: Among 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. CONCLUSION: This 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.

Polimorfismo do gene timidilato sintase e nível plasmático total de homocisteína em um grupo de pacientes turcos com artrite reumatoide: relação com a atividade da doença e toxicidade ao metotrexato / Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity

Resumo Introdução: Relata-se que o polimorfismo do gene timidilato sintase (TS) e a homocisteína têm relação com o metabolismo do metotrexato (MTX), com achados conflitantes. O objetivo deste estudo foi determinar os níveis de homocisteína e a frequência de polimorfismos de repetição tripla (TS3R) e dupla (TS2R) do gene TS em um grupo de pacientes turcos com AR e avaliar sua associação com a toxicidade ao MTX e a atividade da doença. Métodos: Foram incluídos no estudo 64 pacientes com AR e 31 indivíduos no grupo controle, com média de 48,7 ± 12,5 e 46,2 ± 13,4 anos. Foram obtidas as características demográficas e foi registrado o número de pacientes que relataram efeitos adversos ao MTX no grupo AR. Foram analisados os níveis de homocisteína e os polimorfismos TS2R/TS3R. Foi determinada a distribuição de genótipos de acordo com a toxicidade ao MTX e a atividade da doença. Resultados: Os dados demográficos foram semelhantes entre os pacientes e controles. Todos faziam suplementação de ácido fólico a uma dose média de 5 mg/semana. Dos 64 pacientes, 36 apresentaram efeitos adversos ao tratamento com MTX. Encontrou-se uma frequência de polimorfismos TS2R e TS3R semelhante nos grupos AR e controle. Encontrou-se que os polimorfismos TS2R e TS3R eram semelhantes em pacientes com e sem eventos adversos relacionados com o MTX. O nível médio de homocisteína também foi similar em pacientes com e sem polimorfismo do gene TS, mas era mais elevado (12,45 μmol/L vs. 10,7 μmol/L) em pacientes com do que sem efeitos adversos relacionados com o MTX. O nível médio de homocisteína se correlacionou com o VHS no grupo AR. Conclusões: Os níveis de homocisteína podem afetar a atividade da doença e a toxicidade ao MTX, mas os polimorfismos 2 R e 3 R no gene TS não se correlacionaram com a toxicidade ao MTX em pacientes com AR que recebem suplementação de ácido fólico. São necessários mais estudos para esclarecer os polimorfismos em outras enzimas que podem ser responsáveis pela toxicidade ao MTX em pacientes com AR.

Abstract Background: The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Methods: Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years were enrolled for the study. Demographic characteristics were obtained and a number of patients with MTX-related adverse affects were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity was determined. Results: The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5 mg folic acid/week was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The respective frequency of TS2R and TS3R polymorphisms was found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45 μmol/L vs 10.7 μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. Conclusions: In conclusion, homocysteine levels might affect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible for the MTX toxicity in patients suffering from RA.

Polimorfismo 1494del6 del gen de la timidilato sintasa en población de la región central de Venezuela / Thymidylate synthase gen polymorphism 1494del6 in a population from the central region of Venezuela / Polimorfismo 1494del6 do gene timidilato sintetase na população da região central da Venezuela

La enzima Timidilato Sintasa (TS) codificada por el gen TYMS es inhibida por agentes quimioterapéuticos como 5-Fluorouracilo (5-FU) y Metotrexato (MTX). Varios estudios han postulado que existe una relación inversa entre los niveles de enzima y los beneficios terapéuticos de 5-FU y MTX. El gen TYMS presenta polimorfismos asociados con la expresión de TS; uno de ellos consiste en una inserción o deleción de 6 pb en la posición 1494 en 3’UTR (1494del6); la deleción se vincula con menor expresión enzimática. El objetivo del trabajo fue determinar el polimorfismo 1494del6 en 260 individuos sanos no consanguíneos de la región central de Venezuela. Luego de obtener el consentimiento informado, se tomó una muestra sanguínea para aislar ADN y amplificar mediante PCR una secuencia de 142/148 pb del gen TYMS. El producto fue digerido con Dra I y se realizó electroforesis en geles de poliacrilamida 8%, determinándose los genotipos y se compararon con los obtenidos en otras poblaciones. Se encontraron tres genotipos: -6pb/-6pb (15,8%), +6pb/-6pb (45,4%) y +6pb/+6pb (38,8%). Este último ha sido asociado con respuesta deficiente al tratamiento con 5-FU y MTX. Esta investigación es un paso inicial en la generación de datos de importancia farmacogenética en relación con gen TYMS en población venezolana...

Pregnancy in women undergoing hemodialysis: case series in a Southeast Brazilian reference center / Gestação em mulheres em tratamento hemodialítico: série de casos em um centro de referência do Sudeste do Brasil

PURPOSE: To describe maternal and neonatal outcomes in pregnant women undergoing hemodialysis in a referral center in Brazilian Southeast side. METHODS: Retrospective and descriptive study, with chart review of all pregnancies undergoing hemodialysis that were followed-up at an outpatient clinic of high- risk prenatal care in Southeast Brazil. RESULTS: Among the 16 women identified, 2 were excluded due to follow-up loss. In 14 women described, hypertension was the most frequent cause of chronic renal failure (half of cases). The majority (71.4%) had performed hemodialysis treatment for more than one year and all of them underwent 5 to 6 hemodialysis sessions per week. Eleven participants had chronic hypertension, 1 of which was also diabetic, and 6 of them were smokers. Regarding pregnancy complications, 1 of the hypertensive women developed malignant hypertension (with fetal growth restriction and preterm delivery at 29 weeks), 2 had acute pulmonary edema and 2 had abruption placenta. The mode of delivery was cesarean section in 9 women (64.3%). All neonates had Apgar score at five minutes above 7. CONCLUSIONS: To improve perinatal and maternal outcomes of women undergoing hemodialysis, it is important to ensure multidisciplinary approach in referral center, strict control of serum urea, hemoglobin and maternal blood pressure, as well as close monitoring of fetal well-being and maternal morbidities. Another important strategy is suitable guidance for contraception in these women. .

OBJETIVOS: Descrever os resultados maternos e neonatais de mulheres grávidas que estavam em tratamento de hemodiálise em um centro de referência no Sudeste brasileiro. MÉTODOS: Estudo retrospectivo e descritivo, com revisão de prontuários de todas as gestações em hemodiálise, acompanhadas no pré-natal especializado da região Sudeste do Brasil. RESULTADOS: Entre as 16 mulheres identificadas, 2 foram excluídas devido à perda de seguimento. Das 14 descritas, a hipertensão foi a causa mais frequente de insuficiência renal crônica (50% dos casos). A maioria (71,4%) realizava tratamento de hemodiálise há mais de um ano e todas elas foram submetidas a 5 ou 6 sessões por semana. Onze mulheres tinham hipertensão crônica, 1 das quais também era diabética, e 6 eram fumantes. Em relação às complicações da gravidez, 1 das mulheres hipertensas desenvolveu hipertensão maligna (com restrição de crescimento fetal e parto prematuro com 29 semanas), 2 tiveram edema pulmonar agudo e 2 apresentaram descolamento prematuro de placenta. O tipo de parto foi cesariana em 9 mulheres (64,3%). Todos os recém-nascidos tiveram Apgar aos cinco minutos maior que 7. CONCLUSÕES: Para melhorar os resultados perinatais e maternos de mulheres em hemodiálise, é importante ter uma abordagem multidisciplinar em centro de referência, um controle rigoroso da uremia, hemoglobina e pressão arterial materna, bem como acompanhar de perto o bem-estar fetal e a morbidade materna. Outra estratégia importante é a orientação adequada para contracepção nessas mulheres. .

Influence of thymidylate synthase gene polymorphisms on high-dose methotrexate-related toxicities in childhood acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the influence of thymidylate synthase (TS) gene polymorphisms on high-dose methotrexate (HD-MTX)-related toxicities in childhood acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>A total of 73 children who were diagnosed with ALL between March 2011 and March 2013 were included into this study. Genomic DNAs were extracted from their peripheral blood. And then the genotypes of TS 5'-UTR were determined by direct DNA sequencing after PCR. The toxicity response of 73 patients receiving HD-MTX chemotherapy were observed and recorded, and plasma MTX concentrations at 42-48 hours after chemotherapy were measured.</p><p><b>RESULTS</b>The main HD-MTX-related toxicities of 73 patients receiving HD-MTX chemotherapy were neutropenia, decreased hemoglobin level, thrombocytopenia, liver toxicity, mucosal damage, and gastrointestinal reactions. There were no significant differences in the incidence rate of HD-MTX-related toxicities between children with different TS 5'-UTR genotypes after chemotherapy (P>0.05). TS 5'-UTR genotype was not significantly correlated with plasma MTX concentrations at 42-48 hours after chemotherapy (P>0.05).</p><p><b>CONCLUSIONS</b>TS gene polymorphisms have no influence on the incidence of HD-MTX-related toxicities in childhood ALL.</p>

Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania ( Viannia ) and its evaluation as a drug target / Caracterización bioquímica de la enzima bifuncional dihidrofolato reductasa-timidilato sintasa de Leishmania ( Viannia ) y su evaluación como blanco molecular

Introduction. Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania ( Viannia ) species . Materials and methods. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. Results. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed K m and V max values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H 2 F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity ( Ki = 22.0 µM) than trimethoprim ( Ki = 33 µM) and pyrimethamine ( Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. Conclusion. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Introducción. La dihidrofolato reductasa (DHFR) se ha utilizado como blanco molecular en tratamientos antibacterianos, anticancerígenos y antipalúdicos. También, actúa como blanco molecular en Leishmania ; sin embargo, no existen reportes de la enzima bifuncional en especies de Leishmania ( Viannia ). Materiales y métodos. Se ha aislado y expresado en Escherichia coli el gen que codifica para la enzima bifuncional DHFR y la timidilato-sintasa (TS) de Leishmania braziliensis . La enzima recombinante se purificó y caracterizó, y se evaluó el efecto inhibitorio de algunos antifolatos, así como de cuatro alcaloides aporfínicos. Resultados. El gen se compone de aproximadamente 1.560 pb y codifica un péptido de 58 kDa que contiene los dominios DHFR y TS ligados en una sola cadena polipeptídica. La enzima recombinante DHFR-TS, utilizando el dihidrofolato (H2F) como sustrato, presentó valores de K m y V max de 55,35 ± 4,02 (media ± el error estándar de la media) y de 0,02 ± 5,34 x 10 -4 , respectivamente. La enzima rDHFR-TS de L. braziliensis presentó valores de Ki para los antifolatos en el rango de micras. El metotrexato fue el inhibidor más potente de la actividad enzimática ( Ki =22,0 mM) en comparación del trimetoprim ( Ki =33 mM) y la pirimetamina ( Ki =68 mM). Estos valores de Ki son significativamente más bajos en comparación con los obtenidos para los alcaloides aporfínicos. Conclusión. Los resultados muestran el efecto inhibitorio de los antifolatos sobre la actividad enzimática, lo cual indica que la rDHFR-TS de L. braziliensis podría ser un modelo para estudiar moléculas antiprotozoarias potenciales.

Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology / 中南大学学报(医学版)

OBJECTIVE@#To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy.@*METHODS@#The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology.@*RESULTS@#The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS.@*CONCLUSION@#High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

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

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 of pemetrexed. CONCLUSION: 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.

Effects of RNA interference and nolatrexed on thymidylate synthase expression and cell proliferation of human colorectal carcinoma LOVO cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of the RNAi and the chemotherapy drugs nolatrxed on the expression of thymidylate synthase(TS) and the growth of the colorectal carcinoma LOVO cells.</p><p><b>METHODS</b>The siRNA was constructed targeting the human TS gene, and then transfected into the human colorectal cancer LOVO cells. RT-PCR and Western blot technique were used to observe the TS gene and protein expression levels, and MTT was used to detect cell proliferation after silencing the TS gene. In addition, siRNA and nolatrxed were applied to the LOVO cells to observe the TS protein expression and cell growth.</p><p><b>RESULTS</b>TS siRNA significantly reduced the expression of TS gene and protein in LOVO cells, and inhibited cell growth. The IC50 value of LOVO cells was (1.46±0.25) μmol/L in TS siRNA combined with nolatrexed group, (6.81±0.31) μmol/L in the negative control group, and (6.47±0.43) μmol/L in the single nolatrexed group. After treatment of TS siRNA combined with nolatrexed on LOVO cells for 36 hours, the apoptosis index was higher than that in single TS siRNA and nolatrexed[(62.12±0.89)% vs.(21.56±0.67)% and(40.51±0.83)%, both P<0.05].</p><p><b>CONCLUSION</b>TS siRNA can partly suppress the expression of TS gene in LOVO cells, inhibit cell proliferation, promote cell apoptosis and enhance cell sensitivity to apoptosis induced by nolatrexd.</p>

Thymidylate synthase genetic polymorphisms and cancer risk: a meta-analysis of 37 case-control studies / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Several studies have evaluated the association between polymorphisms of thymidylate synthase (TS) and cancer risk in diverse populations but with conflicting results. By pooling the relatively small samples in each study, it is possible to evaluate the association using a meta-analysis.</p><p><b>METHODS</b>A comprehensive search was conducted to identify all case-control studies on TS on a 28-bp tandem repeats in 5'untranslated region (5'UTR) and a 6-bp insertion (ins) and deletion (del) mutation in 3'UTR of the gene and cancer risk. Meta-analysis was conducted using a fixed and random effect model.</p><p><b>RESULTS</b>Our meta-analysis on a total of 13 307 cancer cases and 18 226 control subjects from 37 published case-control studies showed no significant association between the risk of cancer and the 5'UTR 28-bp tandem repeats polymorphism (3R/3R vs. 2R/2R: OR = 1.06, 95%CI, 0.93 - 1.20) or the 3'UTR 6-bp ins/del polymorphism (del6/del6 vs. ins6/ins6: OR = 0.93, 95%CI, 0.81 - 1.08) with significant between-study heterogeneity. In the cancer type- and ethnic subgroup-stratification analyses, we did not find any association between TS polymorphisms and cancer risk either.</p><p><b>CONCLUSION</b>TS 5'UTR 28-bp tandem repeats and 3'UTR 6-bp ins/del polymorphisms may not be associated with cancer risk.</p>

A practical approach for assessing chemosensitivity in colorectal cancer cell lines by comparative analysis of cell viability and thymidylate synthase mRNA expression / 대한외과학회지

PURPOSE: The purpose of this study is to suggest a probable problem in chemosensitivity tests performed in practice and to speculate on practicable measures for more accurate chemosensitivity evaluation. METHODS: Three colorectal cancer cells (RSC, RRC1, and RRC2) were treated with 5-fluorouracil (5-FU). Inhibition percentage (%inhibition) of cancer cells and relative quantitation of thymidylate synthase (TS) mRNA were measured on day 2, day 5 after replacement of 70% media on day 2, day 7, and day 3 after replacement of all media on day 7. Doses that produced 50% inhibition (Dm) were calculated to evaluate drug effect. Relative quantitation of TS mRNA and correlations between TS mRNA levels and 5-FU concentrations were analyzed. RESULTS: RRC1 was more resistant than RRC2 on day 7, but Dm value of RRC2 increased three days after replacement of media from 12.3 to 18.1. Mean TS mRNA levels of RSC on D2 and D7 were significantly lower than those of RRC1 and RRC2, respectively (P = 0.004, P = 0.004 on D2; P = 0.010, P = 0.006 on D7). TS mRNA levels in RRC1 were significantly reversely correlated with 5-FU concentrations on day 2 (correlation coefficient = -0.867, P = 0.015). On the other hand, correlations were not significant in RRC2 (r = 0.067). CONCLUSION: Evaluating %inhibition of cancer cells at one point in chemosensitivity tests seems to be inadequate in determining chemotherapeutic regimens. Multilateral approaches, such as trials evaluating cancer cell survival before and after media replacement and correlations between TS mRNA levels and 5-FU concentrations, needs to be implemented for the practical application of chemosensitivity tests.

Genetic variation in genes involved in folate and drug metabolism in a south Indian population.

BACKGROUND: Genetic variations represented as single nucleotide polymorphisms (SNPs) vary across the world population. This genetic polymorphism (such as SNPs) plays an important role in pharmacogenomics. SNPs that affects cellular metabolism, by altering the enzyme activity, have an important role in therapeutic outcome. Allele frequencies in number of clinically relevant SNPs within south Indian populations are not yet known. Hence, we genotyped randomly selected unrelated south Indian subjects from different locations of south India representing the heterogeneous ethnic background of the population. MATERIALS AND METHODS: Common variants of MTHFD1, TYMS, SHMT1, MTR, MTRR, CBS and SULT1A1 gene polymorphisms were screened from healthy unrelated south Indian volunteers. Genotypes were determined using RFLP analysis of polymerase chain reaction-amplified products and confirmed by DNA sequencing. Chi-square test was performed to test for deviation from the Hardy-Weinberg equilibrium for each locus. RESULTS: Gene allele frequency for several polymorphisms in our study differed significantly between the populations of other nations reported for several of the SNPs. These results demonstrate that the populations in different geographic regions may have widely varying genetic allele frequencies for clinically relevant SNPs. CONCLUSION: The present study reports, for the first time, the frequency distribution of MTHFD1, TYMS, SHMT1, MTR, MTRR, CBS and SULTIA1 gene polymorphisms in a south Indian population. Population-specific genetic polymorphism studies will help in practicing pharmacogenomic principles in the clinics.

Associations between gene polymorphisms of thymidylate synthase with its protein expression and chemosensitivity to 5-fluorouracil in pancreatic carcinoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Thymidylate synthase (TS) is a key regulatory enzyme for de novo DNA synthesis. TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs, and its expression may be affected by gene polymorphisms. In this study, we investigated the associations between polymorphisms of the TS gene and its protein expression, and the implications on the efficacy of 5-fluorouracil (5-FU) in pancreatic cancer cells.</p><p><b>METHODS</b>Genotypes based on the 28-bp TS tandem repeat for pancreatic cell lines were determined by electrophoretic analysis of PCR products. A single nucleotide polymorphism (SNP) at nucleotide 12 of the second 28-bp repeat of the 3R allele was determined by nucleotide sequencing. The chemosensitivity of pancreatic carcinoma cells to 5-FU in vitro was evaluated using Cell Counting Kit-8 (CCK-8). TS protein expression was analyzed by Western blotting.</p><p><b>RESULTS</b>Seven pancreatic carcinoma cell lines had different genotypes in terms of the 28-bp TS tandem repeat, as follows: homozygous 2R/2R (T3M4 and BxPC-3 cells), heterozygous 2R/3R (AsPC-1, Capan-1, and SU86.86), and homozygous 3R/3R (PANC-1 and COLO357). The optical density ratio of genotypes 3R/3R, 2R/2R and 2R/3R was 1.393 ± 0.374, 0.568 ± 0.032 and 0.561 ± 0.056, respectively. Cells with the 2R/3R or 3R/3R genotypes were further analyzed for the G to C SNP at nucleotide 12 of the second 28-bp repeat of the 3R allele, yielding heterozygous 2R/3Rc (AsPC-1, Capan-1, and SU86.86), homozygous 3Rg/3Rg (COLO357) and homozygous 3Rc/3Rc (PANC-1). The optical density ratio of homozygous 3Rg/3Rg cells and homozygous 3Rc/3Rc cells was 1.723 ± 0.062 and 1.063 ± 0.134, respectively, and this difference was statistically significant (P < 0.05). Cells with the 2R/2R and 2R/3R genotypes of TS were hypersensitive to 5-FU in vitro as compared with those with the 3R/3R cells.</p><p><b>CONCLUSIONS</b>Polymorphisms in the TS gene influenced its protein expression and affected sensitivity of 5-FU in seven pancreatic cancer cell lines. Cells with the 3R/3R genotype had higher TS protein expression than the 2R/2R or 2R/3R genotypes. Cells of the 3R/3R genotype with high TS protein expression were shown lower 5-FU sensitivity than cells with the 2R/2R or 2R/3R genotypes. These data warrant large-scale clinical studies to assess the role of polymorphisms in the TS gene on its protein expression and chemosensitivity to 5-FU in pancreatic cancer.</p>

rTSbeta as a novel 5-fluorouracil resistance marker of colorectal cancer: a preliminary study

<p><b>INTRODUCTION</b>Colorectal cancer is the most common form of malignancy in Taiwan and the third leading cause of death from cancer, preceded only by lung and hepatic cancers. Colorectal cancer is typically treated by surgical intervention and/or chemotherapy and radiotherapy, if necessary. To date, 5-fluorouracil (5-FU) is the most commonly used anti-cancer chemotherapy drug. However, patients commonly experience resistance to the drug therefore limiting its efficiency. In this study, we measured the expression of rTSbeta in human colon cancer as a novel 5-FU resistance marker.</p><p><b>MATERIALS AND METHODS</b>We collected 172 colon cancer samples from 4 different hospitals (including 21 pairs of colon cancer biopsies and 151 pathologic slides of colon cancer). In vitro, we measured the cytotoxicity of 5-FU and 5-FU plus leucovorin in H630 and H630-1 colon cancer cell lines.</p><p><b>RESULTS</b>The results revealed that rTSbeta was expressed in 115 (66.9 %) pathology samples and that tumour expression was higher than in corresponding normal tissue. Survival rates of up to 5 years following treatment was significantly higher for patients without rTSbeta expression than for those with rTSbeta expression (P = 0.0023). In vitro, H630-1 (with rTSbeta overexpression) had significantly higher IC50 of 5-FU than did H630. IC50 of 5-FU decreased when leucovorin was added.</p><p><b>CONCLUSIONS</b>Results indicate a close relationship between rTSbeta expression and resistance to the drug 5-FU in human colorectal cancer. These results provide further evidence for rTSbeta expression as a novel 5-FU resistance marker of colorectal cancer.</p>

Thymidylate synthase expression and therapeutic effect analysis of pemetrexed in advanced lung adenocarcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of thymidylate synthase (TS) in patients with advanced lung adenocarcinoma and its relation with the therapeutic effect of pemetrexed.</p><p><b>METHODS</b>The clinicopathological data of 38 patients with stage IIIIB/IV lung adenocarcinoma receiving pemetrexed treatment were retrospectively analyzed. The tumor samples of the patients were collected for detecting TS expression using RT-PCR, and the therapeutic effect of the treatment was analyzed.</p><p><b>RESULTS</b>TS positivity was found in 26.32 (10/38) of the patients. TS positivity was not correlated to gender, TNM stage or PS score. The total response rate of pemetrexed treatment (CR+PR) was 34.21 (13/38) in these patients, and the rate was 39.29% (11/28) in TS-negative patients, as compared to 20.00% (2/10) in the positive patients (P=0.087). Patients with low TS expression had significantly higher control rate by pemetrexed treatment than those with TS overexpression [89.29% (25/28) vs 40.00% (4/10), P=0.002].</p><p><b>CONCLUSION</b>TS expression may serve as a potential indicator of chemosensitivity to pemetrexed in patients with advanced lung adenocarcinoma.</p>

Gastric Cancer Susceptibility according to Methylenetetrahydrofolate Reductase and Thymidylate Synthase Gene Polymorphism

PURPOSE: The genetic polymorphism and intracellular activity of methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) is clinically associated with carcinogenesis and biological therapeutic effect in gastrointestinal malignancies. We aimed to elucidate the susceptibility of gastric cancer according to MTHFR and TS gene polymorphism. METHODS: This study was designed as a hospital-based case-control study in a single institute. The gastric cancer group (n=300) for the study was diagnosed at first time as tubular adenocarcinoma, and the control group (n=100) was diagnosed as no malignancy in the endoscopic biopsy. The genetic polymorphism of TS and MTHFR were confirmed by PCR. RESULTS: The MTHFR mutant type had a more than 2-fold increased risk of developing gastric cancer (RR: 2.341). But, only heterozygote type (677CT) revealed significantly higher susceptibility compared to wild type (RR: 2.581). In TS gene genotype, the mutant genotype rate (2R/3R and 3R/3R) was significantly higher in gastric cancer group compared to control group (P=0.008), and the mutant type had a more than 3-fold increased risk of developing gastric cancer (RR: 3.222). In combined MTHFR and TS, 677CT+2R/3R and 677CT+3R/3R there was more than a 3-fold increased risk rate of developing gastric cancer compared with other combinations (RR, 3.474 in 677CT with 2R/3R; RR, 3.895 in 677CT with 3R/3R). CONCLUSION: This study shows a significant association between the MTHFR and TS polymorphisms and susceptibility to gastric cancer, providing a genetic basis. The polymorphisms study of two genes could be applied as susceptibility markers, clinically, for gastric cancer.

Polymorphisms of thymidylate synthase gene detected by RT-PCR-denaturing gradient gel electrophoresis in children with acute leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Thymidylate synthase (TS) catalyses the conversion of deoxy-uridylate to deoxy-thymidylate and is a key enzyme for DNA synthesis. TS is the target enzyme of 5-fluorouracil (5-FU) and involved in folate metabolism. TS gene polymorphisms play an important role in the efficiency of fluorouracil activity in vivo. This study investigated the allelic frequencies and distribution characters of single-nucleotide polymorphisms within the coding region (cSNPs) of TS gene in Chinese children with acute leukemia (AL) and normal control children in order to explore the possible relationship between the cSNP in human TS gene and chemotherapeutic effects of 5-fluorouracils.</p><p><b>METHODS</b>Bone marrow samples from 53 children with AL and peripheral blood samples from 115 normal children were obtained to prepare complementary DNAs (cDNAs). The cDNAs were analyzed for the polymorphisms in TS gene by reverse transcriptase (RT)-polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The distributive difference of each genotype between AL children and control children was evaluated.</p><p><b>RESULTS</b>A polymorphism 381 A>G (E127E) in the coding region of TS gene was firstly identified in the Chinese population. The 381 A>G allelic frequency in AL children and control children was 12.3% and 13.5% respectively (P>0.05), which were similar to that in the International SNP Bank (12.3%). The allelic frequency of cSNPs was not associated with the susceptibility to AL.</p><p><b>CONCLUSIONS</b>A polymorphism 381 A>G (E127E) in TS gene was successfully identified in children using RT-PCR-DGGE combined with DNA sequencing. There was no significant difference in the allelic frequency of cSNPs in AL children and normal children.</p>

Biomarkers for Colorectal Cancer Treatment / 대한소화기학회지

Individualized tailored therapy is a currently pursuing direction for improving the outcome of patients with colorectal cancer. Targeted therapy is the potential strategy to reach this goal by evaluating status of the presumed targets and their related effector molecules and by maximizing the efficacy of chemotherapeutic agents with less toxicity in individual patient. Numerous hurdles should be overcome, however, because therapeutic outcome can be affected by multiple components; tumor characteristics such as somatic mutations at the DNA, RNA, and protein levels; patient characteristics like germline genetic polymorphisms in enzymes linked to drug metabolism; and environmental factors that include diet and physical activity. Currently, large numbers of potential biomarkers have been proposed but have not yet accomplished supporting evidences for their routine usage in clinics. Therefore, clinical trials driven by molecular targets and relevant biomarkers for the understanding of the conflicting data are needed to make markers available in clinical practice.