Thymidylate Synthase Gene Research Articles

The role of genetic polymorphisms in the thymidylate synthase (TYMS) gene in methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

Methotrexate (MTX) is an important drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). MTX is cytotoxic as it impairs DNA and RNA synthesis by inhibiting the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The association between genetic variants within the TYMS gene and MTX-induced toxicity has been studied, but results are inconsistent. We determined the role of three previously described variants within the TYMS gene and MTX-induced oral mucositis in a prospective cohort of Dutch children with ALL and performed a meta-analysis of the previous results. We analyzed the presence of a 28-base pair tandem repeat (rs34743033; 2R3R), a single nucleotide polymorphism present within the 28-base pair repeat on the 3R allele (rs2853542; 3RG>C) and a 6-base pair deletion (rs15126436; TTAAAG) within the TYMS gene in germline DNA of 117 pediatric patients with ALL. Oral mucositis was defined as grade≥3 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v.3.0. Data were analyzed for the individual rs34743033 (2R3R) and rs151264360 (6 bp deletion) polymorphisms, whereas rs2853542 (3RG>C) was combined with rs34743033 (2R3R) and analyzed according to predicted expression levels of TYMS: low expression (2R/2R, 2R/3RC and 3RC/3RC), median expression (2R/3RG and 3RC/3RG) and high expression (3RG/3RG). We performed a meta-analysis of the current literature on these polymorphisms in relation to oral mucositis using a fixed effects model. The 2R2R genotype (rs34743033) was not significantly associated with developing MTX-induced oral mucositis compared with the 2R3R/3R3R genotypes, which was confirmed in a meta-analysis [odds ratio (OR): 1.17 (0.62-2.19)]. Patients carrying the low-expression TYMS genotype (2R2R, 2R3RC, 3RC3RC) had an increased odds of developing MTX-induced oral mucositis [OR: 2.42 (0.86-6.80)], which did not reach statistical significance. The 6-bp deletion [rs151264360, OR: 0.79 (0.20-3.19)] was not associated with the development of MTX-induced oral mucositis. The TYMS 6-bp deletion and 2R3R polymorphism were not associated with MTX-induced oral mucositis. Validation studies in prospective cohorts are necessary to assess the possible role of the low-expression TYMS genotypes in relation to MTX-induced oral mucositis.

524P - Implications of thymidylate synthase gene polymorphisms, KRAS and BRAF mutations in the survival of patients with colorectal cancer treated with adjuvant chemotherapy

524P - Implications of thymidylate synthase gene polymorphisms, KRAS and BRAF mutations in the survival of patients with colorectal cancer treated with adjuvant chemotherapy

Polymorphism rs3819102 in thymidylate synthase and environmental factors: effects on lung cancer in Chinese population

PurposeLung cancer is the leading cause of cancer death worldwide, and the predominant risk factor for its development is smoking. Thymidylate synthase (TYMS) is a key enzyme in DNA synthesis that catalyzes the conversion of deoxyuridine monophosphate to dTMP. Rs931794, a single nucleotide polymorphism located in the TYMS gene, was suggested to be associated with cancer risk. MethodsTo analyze the interaction between rs3819102 and environmental factors on the risk of lung cancer in a Chinese population, single nucleotide polymorphismscan was used to genotype this polymorphism in 974 lung cancer cases and 1005 control subjects. ResultsThe frequencies of TT, CT, and CC genotypes of TYMS rs3819102 were 61.8%, 32.9%, and 5.3% in controls, and 53.8%, 38.4%, and 7.8% in cases, respectively. Compared with the TT genotype, the CT (odds ratio [OR], 1.380; 95% confidence interval [CI], 1.131-1.683), and CC (OR, 1.786; 95% CI, 1.213-2.644) genotypes were associated with an increased risk of lung cancer after adjustment for age, gender, smoking status, and family history. The C allele of rs3819102 is the risk allele for lung carcinogenesis in a dominant model (OR, 1.435; 95% CI, 1.188-1.735). In a stratified analysis, the risk effects of both the CT and CC genotypes of rs3819102 were more evident in subgroups of smokers and people without a family history of cancer. ConclusionThe rs3819102 polymorphism in TYMS might increase susceptibility to environmental factors and contribute to the risk of lung cancer. The C allele is a risk allele in lung carcinogenesis.

Construction and characterization of thymidine auxotrophic (\u0394thyA) recombinant Lactobacillus casei expressing bovine lactoferricin

BackgroundLactobacillus casei (L. casei) is well known for its probiotic property in human and animals. Lactoferricin (Lfcin) polypeptide can effectively modulate host immune responses and have antimicrobial activity in vivo and in vitro. In order to develop a food-grade L. casei system constitutively expressing bovine Lfcin, this study constructed a thymidine auxotrophy (ΔthyA) recombinant L. casei.ResultsBased on the thymidylate synthase gene (thyA) insert site, LFEC(Lfcin expression cassette)was inserted into L. casei genome through homologous recombination, successfully expressed and could be stably inherited. The recombinant L. casei, ΔthyA L. casei-LFEC, is sensitive to chloramphenicol and limited when cultured without thymine. Meanwhile, ΔthyA L. casei-LFEC has both good antibacterial activity against Escherichia coli and Staphylococcus aureus and antiviral activity against porcine epidemic diarrhea virus (PEDV).ConclusionsWe successfully constructed a recombinant L. casei strain expressing Lfcin, ΔthyA L. casei-LFEC, which could only survive in the presence of thymine, and had excellent antimicrobial and antiviral activity with good genetic stability and sensitivity. This research provides a cost-effective alternative to the antibiotics with additional biological functions and wider applicability prospect. Using ΔthyA as the selectable mark instead of antibiotic to construct genetic engineering L.casei provides a safe and effective approach of feed additives in livestock raising.

Influence of TS (rs34743033) and RUNX1 (rs2014300) gene polymorphisms on survival outcomes of fluorouracil-based chemotherapy in Chinese advanced gastric cancer patients

BackgroundThis study aimed to explore the clinical correlation of single-nucleotide polymorphisms of thymidylate synthase (TS) and runt-related transcription factor 1 (RUNX1) in patients with postoperative stage II and III gastric cancer (GC).Patients and methodsSamples were obtained from 661 patients with postoperative stage II and III GC. TS (rs34743033) and RUNX1 (rs2014300) were genotyped in 261 patients who received postoperative basic platinum and fluorouracil chemotherapy regimens and 400 patients who did not accept chemotherapy.ResultsTS (rs34743033) variant genotypes significantly prolonged the median overall survival (OS) time compared to the patients who only received adjuvant chemotherapy (HR 1.604, 95% CI 1.068–2.410, p=0.021). Moreover, 3R/3R variant genotypes were demonstrated to have a positive effect on the OS of patients who received chemotherapy based on cisplatin (HR 1.754, 95% CI 1.041–2.954, p=0.031) compared to oxaliplatin. A stratification analysis indicated that 2R/3R and 2R/2R variant genotypes were associated with inferior survival in GC patients with intestinal-type tumors, tumor less than 5 cm in size, and poorly differentiated tumors (p<0.05). However, RUNX1 (rs2014300) AA genotypes markedly increased the risk of death in GC patients compared with the GG/GA genotypes (p=0.007), but no significant difference was observed between chemotherapy based on platinum. The stratification analysis showed that the GA/AA genotype was significantly associated with inferior survival in well to moderately differentiated tumors (HR 2.001, 95% CI 1.082–3.703, p=0.023).ConclusionThese preliminary results indicated that the two polymorphisms had a significant effect on postoperative adjuvant chemotherapy. TS (rs34743033) and RUNX1 (rs2014300) may be used as biomarkers to predict prognosis and select chemotherapy regimens in GC patients.

Antitumor effect of lapatinib and cytotoxic agents by suppression of E2F1 in HER2‑positive breast cancer.

The human epidermal growth factor receptor2 (HER2)‑targeting agent, lapatinib, combined with oral fluoropyrimidine capecitabine, has been previously demonstrated to be an effective treatment option for patients with trastuzumab‑resistant HER2‑positive metastatic breast cancer. To investigate the molecular mechanisms associated with the interactions between lapatinib and capecitabine, the effect of treatment with lapatinib and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K) inhibitors on the expression of E2F transcription factor 1 (E2F1) and thymidylate synthase (TS), which is associated with an increased response to 5‑fluorouracil (5‑FU)‑based chemotherapy, was determined in HER2‑positive breast cancer cells. The results of reverse transcription‑quantitative polymerase chain reaction demonstrated that administration of lapatinib and PI3K inhibitors decreased the mRNA expression of TS and E2F1, a transcription factor that promotes TS gene expression, in SKBR3 and T47D cell lines. Furthermore, treatment with lapatinib and PI3K inhibitors also suppressed the mRNA expression of ribonucleotide reductase M1 subunit (RRM1), an important determinant of gemcitabine resistance, and DNA topoisomerase II‑α (TOP2A), a molecular target of anthracyclines, in SKBR3 and T47D cell lines. Western blot analysis further demonstrated that the phosphorylation of Akt was inhibited by lapatinib, and the results of the MTT assay revealed that the combination of lapatinib with either 5‑FU or gemcitabine demonstrated synergistic antitumor effects, whereas a combinatory treatment of lapatinib with epirubicin, a typical anthracycline drug, exhibited antagonistic antitumor effects in HER2‑positive breast cancer cells. These results indicate that the synergistic antitumor effects exhibited by combinatory treatment of lapatinib with capecitabine may be induced via the suppression of E2F1‑mediated TS expression.

3'-UTR Polymorphisms of MTHFR and TS Associated with Osteoporotic Vertebral Compression Fracture Susceptibility in Postmenopausal Women.

Postmenopausal osteoporosis is one of the most prominent diseases in postmenopausal women and it is increasing in prevalence with the aging population. Furthermore, osteoporosis and osteoporotic vertebral compression fractures (OVCFs) are related to mortality and decreased quality of life. Therefore, searching for biomarkers that are able to identify postmenopausal women who are at high risk of developing OVCFs is an effective strategy for improving the quality of life of patients and alleviating social and economic burdens. In this study, we investigated methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) gene polymorphisms in postmenopausal women with OVCF. We recruited 301 postmenopausal women and performed genotyping for the presence of MTHFR 2572C>A, 4869C>G and TS 1100C>T, 1170A>G. Genotyping was analyzed using the polymerization chain reaction restriction fragment length polymorphism assay. MTHFR 2572C>A and TS 1100C>T were associated with the prevalence of osteoporosis (MTHFR 2572CC versus CA+AA: odd ratio [OR] adjusted age, hypertention [HTN], and diabetes mellitus [DM] = 0.49, p = 0.012) and the occurrence of OVCFs (MTHFR 2572CC versus CA+AA: OR adjusted age, HTN, and DM = 0.38, p = 0.013; TS 1100CC versus CT+TT: OR adjusted age, HTN, and DM = 0.46, p = 0.02). Our novel finding is the identification of MTHFR and TS genetic variants that decrease susceptibility to OVCFs. Our findings suggest that polymorphisms in the MTHFR and TS genes are associated with susceptibility to osteoporosis and OVCFs in postmenopausal women.

Polymorphisms in folate metabolism genes are associated with susceptibility to presbycusis

AimPresbycusis or age related hearing loss is caused by several extrinsic and intrinsic factors that damage the auditory system. Gene polymorphisms in folate metabolism were found to play an important role in the etiology of presbycusis. The present study aimed to investigate the role of 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and thymidylate synthase (TYMS) gene polymorphisms in the onset of presbycusis in a South Indian population. Main methodsA total of 220 subjects confirmed with presbycusis along with 270 age and sex matched healthy controls visiting MAA ENT Hospitals, Hyderabad, India were enrolled for the study. Genotyping of MTHFR C677T (rs180133) and A1298C (rs1801131), MTR A2756G (rs1805087), TSER (rs1801136) and TS1494indel6 bp (rs16430) was carried out using PCR & PCR-RFLP methods. Key findingsThe ‘TT’ genotype of MTHFR C677T and ′152 bp/152 bp′ genotype of TS1494indel6 bp showed statistically significant risk for presbycusis while CC genotype of MTHFR A1298C, ‘2R/2R’ genotype of TSER at 3′UTR and 6 bp ins/6 bp ins of TYMS at 5′UTR were found to be protective. The T-A-A haplotype combination of MTHFR C677T, MTHFR A1298C and MTR A2756G as well as 3R- 152 bp of TYMS at 5′UTR and 3′UTR were also found to contribute significant risk for the onset of presbycusis. Further, the combination of SNP loci TSER: TS1494indel6 bp exhibited moderate linkage in presbycusis. SignificanceThe present pilot study identified the significant association of gene variants of MTHFR and TYMS with presbycusis. These findings aid in early diagnosis of hearing loss in the elderly population.

Association of thymidylate synthase and hypoxia inducible factor-1alpha DNA polymorphisms with pancreatic cancer.

Thymidylate synthase and hypoxia inducible factor-1α play a central role in the control of tumor progression. In the present study, we investigated the effect of three DNA polymorphisms within the thymidylate synthase gene and two within hypoxia inducible factor-1α on the prognosis of pancreatic cancer. A retrospective study was performed in 59 patients diagnosed with invasive ductal adenocarcinoma of the pancreas and 159 healthy volunteers. The studied DNA polymorphisms were a variable tandem repeat of 28 bp (rs45445694), a G/C single nucleotide polymorphism (rs34743033), and a deletion of 6 bp (ins1494del 6bp; rs34489327) within the thymidylate synthase gene and C1772T and G1790A single nucleotide polymorphisms within hypoxia inducible factor-1α (rs11549465 and rs11549467, respectively) . Variable tandem repeats were determined by specific polymerase chain reaction, whereas thymidylate synthase single nucleotide polymorphism G/C, ins1494del 6pb, and hypoxia inducible factor-1α polymorphisms were identified by polymerase chain reaction and RFLP. Thymidylate synthase and hypoxia inducible factor-1α genotype distributions in patients and healthy volunteers were determined. The impact of the polymorphisms on clinico-pathological variables, including survival, was also studied. The frequency of carriers of the variant del6bp allele was significantly higher among patients (70.0% vs 51.0% of healthy donors, P = 0.02); 42% of male patients were homozygous 2R/2R vs 13.6% of females (P = 0.03), but differences regarding gender were not observed among healthy volunteers. Concerning hypoxia inducible factor-1α C1772T and G1790A single nucleotide polymorphisms, the rates of variant T/T and A/A homozygous genotypes were significantly elevated among patients (18.6% vs 5.3%, P = 0.001, and 5.1% vs none, P = 0.021 respectively). In our study, the variant del14946bp allele within the thymidylate synthase gene, and TT and AA genotypes of C1772T and G1790A hypoxia inducible factor-1α single nucleotide polymorphisms were associated with the development of pancreatic cancer. The 2R/2R genotype of variable tandem repeat thymidylate synthase polymorphism might be a risk factor for pancreatic cancer in males.

Biological Containment of Genetically Modified Bacillus subtilis.

Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes.IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment.

Uridine triacetate for severe 5-fluorouracil toxicity in a patient with thymidylate synthase gene variation: Potential pharmacogenomic implications

Adverse drug reactions can be unpredictable. However, pharmacogenomic testing can help identify patients who may be more susceptible to the toxic effects of certain drugs. Genetic variations in the dihydropyrimidine dehydrogenase and thymidylate synthase genes have been shown to increase the risk of 5-fluorouracil toxicity. 5-Fluorouracil toxicity can be life threatening. Fortunately, there is treatment available for 5-fluorouracil toxicity, called uridine triacetate. Although, the indications for its use limit its administration to within 96 h of receiving 5-fluorouracil, we report a case of effective therapy in a patient started on uridine triacetate beyond the recommended 96 h, who was found to carry a thymidylate synthase gene variation but no dihydropyrimidine dehydrogenase mutations. This provides important implications for pharmacogenomic testing.

Isolation and characterization of a N4-like lytic bacteriophage infecting Vibrio splendidus, a pathogen of fish and bivalves.

A novel virulent bacteriophage, vB_VspP_pVa5, infecting a strain of Vibrio splendidus was isolated from a sea-cage aquaculture farm in Greece, and characterized using microbiological methods and genomic analysis. Bacteriophage vB_VspP_pVa5 is a N4-like podovirus with an icosahedral head measuring 85 nm in length and a short non-contractile tail. The phage had a narrow host range infecting only the bacterial host, a latent period of 30 min and a burst size of 24 virions per infected bacterium. Its genome size was 78,145 bp and genomic analysis identified 107 densely-packed genes, 40 of which could be annotated. In addition to the very large virion encapsulated DNA-dependent RNA polymerase which is the signature of the N4-like genus, an interesting feature of the novel phage is the presence of a self-splicing group I intron in the thymidylate synthase gene. A tRNAStop interrupted by a ~2.5kb open reading frame–containing area was also identified. The absence of genes related to lysogeny along with the high efficacy observed during in vitro cell lysis trials, indicate that the vB_VspP_pVa5 is a potential candidate component in a bacteriophage cocktail suitable for the biological control of V. splendidus in aquaculture.

Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis.

A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM). In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10−9. Genetic disruption of the canonical mismatch (MMR) and guanine oxidized (GO) repair pathways increased the Tmpr frequency of mutation by ~2–3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10−7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.

Abstract LB-254: Thymidylate synthase gene polymorphisms is negatively associated with gastric cancer survival

Abstract Background: Gastric cancer (GC) is a major public health problem in China and its carcinogenesis has not been fully understood. It was found that genetic variation played an important role in the etiology. Thymidylate synthase (TYMS) is a critical target for cancer chemo-therapy, which functions in DNA replication, synthesis and repair. However, the results of the relationship between TYMS and cancer is unclear. To find the genomic susceptibility in TYMS, bio-markers are necessary for preventing and understanding the etiology of gastric cancer. Objectives: The research explores the relationship between TYMS and survival time of 876 Chinese gastric cancer patients. Methods: A retrospective cohort of 876 surgically resected gastric cancer patients with clinicopathologic and follow up information were collected. The genotypes of SNPs were examined by SNaPshot method. The median survival time (MST) for each categorical variable and survival curve were estimated by Kaplan-Meier method. The overall survival was analyzed by univariate and multivariate Cox proportional hazard model. Results: TYMS was significantly associated with survival time. The study found eight samples (1.0%) with the novel allele (4R). In univariate analysis, the group of including 4R allele had lowest survival condition (P value = 0.006). The hazard ratio of individual with 4R allele was 2.48 (95% CI: 1.10-5.58) after controlling age, gender, smoking, drinking and TNM stage. Conclusion: Our study found a novel allele (4R) in TYMS in gastric cancer patients. Key words: Gastric cancer; TYMS; overall survival Citation Format: Jinfei Chen, Maggie Haitian Wang, Benny Chung-Ying Zee. Thymidylate synthase gene polymorphisms is negatively associated with gastric cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-254. doi:10.1158/1538-7445.AM2017-LB-254

Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways.

Antibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010–2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.

A study of thymidylate synthase gene expression as a biomarker for the treatment of esophageal adenocarcinoma.

e15563 Background: Expression of thymidylate synthase (TS), a target of fluoropyrimidine-based (5-FU) chemotherapy, has prognostic value in colorectal adenocarcinoma. Its value as a biomarker in treatment of esophageal adenocarcinoma (EAC) is unknown. With widespread adoption of CROSS trial protocol for treatment of EAC, 5-FU is frequently omitted from regimens. We retrospectively analyzed the association between TS protein expression and clinical outcomes in EAC. Methods: TS expression levels were measured by immunohistochemistry in formalin-fixed paraffin-embedded specimens from 35 primary EAC patients from 2 institutions (range 5/2006-1/2016); at time of diagnosis and surgical resection (33 neoadjuvant chemoradiation and 2 surgery alone). TS expression higher than 1+, 5% was read as positive. Mean follow up was 1323 days (range 263-3677). TS expression correlated with treatment response and survival in patients treated with and without 5-FU therapy (log-rank test, Kaplan-Meier method). Results: In the cohort of 35 patients, 21 patients (60%) had pretreatment positive TS expression. Overall survival (OS) for TS negative patients was 1216 days versus 974 days for TS positive (p = 0.15). In the 23 patients treated with 5-FU, TS-negative patients had a trend towards longer overall survival compared to TS-positive patients, mean OS (1272 days versus 743 days, p = 0.08). In the group of 12 patients not treated with 5-FU, 2 patients showed negative TS staining. Neoadjuvant chemoradiation was performed in 33/35 patients. Downstaging was observed in 69% (9/13) of patients with negative pre-treatment TS levels versus 55% (11/20) in pre-treatment TS positive patients (p = 0.48). In 23 patients treated with 5FU, 9/12 (75%) were downstaged in the TS-negative cohort versus 6/11 (45%) in the TS-positive cohort (p = 0.21). In the 5FU treatment group, complete response was seen in 2 patients with negative TS staining. Conclusions: Low TS expression in EAC patients trended towards improved survival and tumor downstaging, particularly in patients treated with neoadjuvant 5-FU and platinum based therapy. Prospective data and greater patient numbers may be necessary to demonstrate statistical significance.

Transcriptomic and Metabolomics Profiling of Phage-Host Interactions between Phage PaP1 and Pseudomonas aeruginosa.

The basic biology of bacteriophage–host interactions has attracted increasing attention due to a renewed interest in the therapeutic potential of bacteriophages. In addition, knowledge of the host pathways inhibited by phage may provide clues to novel drug targets. However, the effect of phage on bacterial gene expression and metabolism is still poorly understood. In this study, we tracked phage–host interactions by combining transcriptomic and metabolomic analyses in Pseudomonas aeruginosa infected with a lytic bacteriophage, PaP1. Compared with the uninfected host, 7.1% (399/5655) of the genes of the phage-infected host were differentially expressed genes (DEGs); of those, 354 DEGs were downregulated at the late infection phase. Many of the downregulated DEGs were found in amino acid and energy metabolism pathways. Using metabolomics approach, we then analyzed the changes in metabolite levels in the PaP1-infected host compared to un-infected controls. Thymidine was significantly increased in the host after PaP1 infection, results that were further supported by increased expression of a PaP1-encoded thymidylate synthase gene. Furthermore, the intracellular betaine concentration was drastically reduced, whereas choline increased, presumably due to downregulation of the choline–glycine betaine pathway. Interestingly, the choline–glycine betaine pathway is a potential antimicrobial target; previous studies have shown that betB inhibition results in the depletion of betaine and the accumulation of betaine aldehyde, the combination of which is toxic to P. aeruginosa. These results present a detailed description of an example of phage-directed metabolism in P. aeruginosa. Both phage-encoded auxiliary metabolic genes and phage-directed host gene expression may contribute to the metabolic changes observed in the host.

Effect of thymidylate synthase (TYMS) gene polymorphisms with methotrexate treatment outcome in south Indian Tamil patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease causing joint damage and significant functional impairment. Methotrexate (MTX) remains the mainstay for the treatment of RA. MTX inhibits several enzymes of the folate and nucleotide pathways. Thymidylate synthase (TYMS) is an important enzyme in the de novo pyrimidine pathway responsible for DNA replication. The two common gene polymorphisms analyzed in TYMS are 28-bp tandem repeat polymorphism and a 6-bp insertion/deletion polymorphism. The present study was carried out to find the role of these TYMS gene polymorphisms with clinical phenotype, treatment response, and MTX adverse events in 254 patients with RA of south Indian Tamil ethnicity. TYMS gene polymorphisms were analyzed by PCR. The allele frequencies of TYMS gene polymorphisms did not differ between good and non-responders. However, the TYMS 28-bp tandem repeat 3R allele was higher in non-responders than in patients undergoing remission [64 vs 51.11%, p=0.06, OR 0.58, 95% CI (0.34-1.00)]. The TYMS 6-bp deletion allele was higher in non-responders than good responders [78.20 vs 64.92%, p=0.06, OR 0.51 95% CI (0.27-0.98)]. TYMS 3R allele and TYMS 6-bp deletion allele may favor non-response to MTX in south Indian Tamils. TYMS gene polymorphisms did not influence MTX adverse events.

The role of pharmacogenetics in the new ESMO colorectal cancer guidelines.

The role of pharmacogenetics in the new ESMO colorectal cancer guidelines.

Role of thymidylate synthase as a predictor of neoadjuvant chemoradiation therapy in rectal cancer

Aim. To study the level of thymidylate synthase gene expression in biopsy material from patients with rectal cancer treated with neoadjuvant chemoradiation therapy. &#x0D; Methods. Analysis of treatment of 101 patients with locally advanced rectal cancer (T3-4, N0-2, M0) was conducted. Measurement of the level of thymidylate synthase gene expression was performed by polymerase chain reaction. To extract ribonucleic acid Qiagen (RNA easy FFPEkit; Qiagen,GmbH,Germany) reagents were used. &#x0D; Results. Cancer pathomorphosis stage TRG 1 was revealed in 23 (22.7%) patients, TRG 2 - in 31 (30.7%) patients, TRG 3 - in 32 (31.7%) patients, complete clinical response (i.e. complete tumor resorption TRG 4) - in 15 (14.9%) patients. Analysis of thymidylate synthase gene expression in patients with poor response to chemoradiation therapy showed that in 34.8% of cases high expression was revealed, while in 65.2% of cases - low expression. Among patients with complete clinical response significant prevalence of patients with high expression of thymidylate synthase gene was revealed - 73.3%, and in 26.7% of patients with pathomorphosis of grade 4 low expression of this gene was registered. In patients with tumor regression grade 2 and 3 no significant difference in patients’ ratio was revealed. The most prominent correlation according to the level of thymidylate synthase gene expression was found in patients without the response to conducted chemoradiation therapy (with prevalence of patients with low expression) and complete tumor regression (with prevalence of patients with high expression). &#x0D; Conclusion. There is inverse correlation between the grade of tumor regression and thymidylate synthase gene expression; evaluation of the level of thymidylate synthase gene expression and response of rectal cancer to neoadjuvant chemoradiation therapy requires further investigation both on molecular and clinical levels.