Thymidine Phosphorylase Expression Research Articles

Abstract 5352: Mesenchymal stem cell as delivery carrier for prodrug gene therapy against colorectal cancer cell

Abstract Gene-directed enzyme prodrug therapy (GDEPT) is an approach that delivers a suicidal gene to cancer cells for the expression of prodrug-activating enzyme which converts prodrug into cytotoxic drug. One of the major impediments of GDEPT is to have the therapeutic gene specifically target on cancer cells prior to the administration of prodrug. Among various gene delivery methods, mesenchymal stem cells (MSCs) have emerged as potential carriers for gene delivery. They exhibit remarkable tumor-tropic and low immunogenic capacities. In this study, MSCs was engineered to express thymidine phosphorylase (TP) which has the capability of converting the prodrug doxifluridine into toxic 5-fluorouracil. TP expression in the MSCs post-transfection of TP cDNA was confirmed by immunoblotting analysis and its activity was determined by spectrophotometric assay. Our results showed the anticancer effectiveness of human HT29 colorectal adenocarcinoma cells co-cultured with TP-expressing MSCs was enhanced with the addition of various dosages of doxifluridine. Citation Format: Nasrin Salehi, Ching-An Peng. Mesenchymal stem cell as delivery carrier for prodrug gene therapy against colorectal cancer cell. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2015-5352

Abstract 2569: Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer as a rationale for the innovative V-shoRT-R3 trial in locally advanced rectal cancer patients

Abstract We have recently demonstrated that the histone deacetylase-inhibitor (HDACi) vorinostat induces synergistic antitumour effects in combination with capecitabine by up-regulating, in vitro and in vivo, in colorectal cancer cells but not in ex vivo treated peripheral blood lymphocytes, the mRNA and protein expression of thymidine phosphorylase (TP), the key enzyme converting capecitabine to 5-FU (Di Gennaro, Brit J Cancer 2010). We confirmed a time and dose-dependent induction of TP mRNA and protein expression by several other HDACi, including valproic acid (VPA). We investigated potential antitumor interaction between capecitabine metabolite 5′-deoxy-5-fluorouridine (5′-DFUR) and several HDACi showing synergistic/additive antiproliferative and pro-apoptotic effects in all cancer cells tested, with good results with VPA. Interestingly, TP protein induction is achieved also at low doses of VPA (0.5-1 mM), corresponding to a plasma level between 50 and 100 μg/ml, easily reached in patients with normal anticonvulsant doses. Although at these doses VPA did not induce growth inhibition as single agents, a significant synergistic antitumor effect was still demonstrated in combination with 5′-DFUR, suggesting a specific mechanism of interaction. TP knockdown experiments confirmed a crucial role of TP protein modulation in the observed synergism. Radiotherapy further potentiated in colorectal cancer cells the antiproliferative, pro-apoptotic and DNA damage effects induced by 5′-DFUR/VPA combination, as demonstrated by clonogenic assay, Caspase-3 cleavage and γH2AX foci formation, respectively. On these bases we launched a phase I/II clinical study (V-ShoRT-R3 trial) to explore whether the addition of both VPA and capecitabine to short-course radiotherapy (SCRT) before optimal radical surgery, might increase the pathologic complete tumor regression rate in low-moderate risk rectal cancer patients (ClinicalTrials.gov number NCT01898104). Several biomarkers will be evaluated comparing normal mucosa with tumor and on blood samples. Tumor metabolism will be measured by 18FDG-PET at baseline and 11 days after the beginning of SCRT. Currently phase I clinical study is ongoing. We have also optimized a protocol to evaluate histones and proteins acetylation in peripheral blood mononuclear cells of recruited patients by flow cytometry, as pharmacodynamic/predictive specific marker of VPA HDACi activity and preliminary results will be presented. Citation Format: Manuela Terranova Barberio, Biagio Pecori, Serena Imbimbo, Alessandra Leone, Francesca Bruzzese, Maria Carmela Piccirillo, Paolo Delrio, Franco Bianco, Luigi Aloj, Antonio Sorrentino, Fabiana Tatangelo, Antonella Petrillo, Secondo Lastoria, Paolo Muto, Francesco Perrone, Antonio Avallone, Alfredo Budillon, Elena Di Gennaro. Synergistic antitumor interaction between valproic acid, capecitabine and radiotherapy in colorectal cancer as a rationale for the innovative V-shoRT-R3 trial in locally advanced rectal cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2569. doi:10.1158/1538-7445.AM2015-2569

Superior antitumor activity of trastuzumab combined with capecitabine plus oxaliplatin in a human epidermal growth factor receptor 2-positive human gastric cancer xenograft model.

In the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastroesophageal junction cancer, it has been reported that the combination of trastuzumab with capecitabine plus cisplatin, or with 5-fluorouracil (5-FU) plus cisplatin, significantly increased overall survival compared with chemotherapy alone (ToGA trial). In addition, adjuvant therapy with capecitabine plus oxaliplatin (XELOX) improved the survival of patients who received curative D2 gastrectomy (CLASSIC trial). However, the efficacy of the combination of trastuzumab with XELOX for patients with HER2-positive gastric cancer remains unknown. The aim of this study, was to investigate the efficacy of the combination of trastuzumab with XELOX in a HER2-positive human gastric cancer xenograft model. Combination treatment with these three agents (trastuzumab 20 mg/kg, capecitabine 359 mg/kg and oxaliplatin 10 mg/kg), was found to exhibit a significantly stronger antitumor activity in NCI-N87 xenografts compared with either trastuzumab or XELOX alone. In this model, treatment with trastuzumab alone or trastuzumab plus oxaliplatin enhanced the expression of thymidine phosphorylase (TP), a key enzyme in the generation of 5-FU from capecitabine in tumor tissues. In in vitro experiments, trastuzumab induced TP mRNA expression in NCI-N87 cells. In addition, NCI-N87 cells co-cultured with the natural killer (NK) cell line CD16(158V)/NK-92 exhibited increased expression of TP mRNA. When NCI-N87 cells were cultured with CD16(158V)/NK-92 cells in the presence of trastuzumab, the mRNA expression of cytokines reported to have the ability to induce TP was upregulated in tumor cells. Furthermore, a medium conditioned by CD16(158V)/NK-92 cells also upregulated the expression of TP mRNA in NCI-N87 cells. These results suggest that trastuzumab promotes TP expression, either by acting directly on NCI-N87 cells, or indirectly via a mechanism that includes trastuzumab-mediated interactions between NK and NCI-N87 cells. Therefore, the combination of trastuzumab with XELOX may be a potent therapy for HER2-positive gastric cancer.

Relationship between biodistribution of a novel thymidine phosphorylase (TP) imaging probe and TP expression levels in normal mice.

Thymidine phosphorylase (TP) is a key enzyme in the pyrimidine nucleoside salvage pathway and its expression is upregulated in a wide variety of solid tumors. In mice, we previously observed high and specific accumulation levels of our TP imaging probe, radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) not only in high-TP-expressing tumors, but also in the liver and small intestine. To clarify the reason for the high accumulation levels of radioiodinated IIMU in the liver and small intestine, we investigated the expression levels of TP in mice in comparison with the biodistribution of radioiodinated IIMU ((123)I-IIMU). BALB/cCrSlc mice were injected with (123)I-IIMU, and the radioactivity levels [%ID/g (normalized to a mouse of 25 g body weight)] in the tissues of interest were determined 0.5, 1, 3 and 24 h after the injection (n = 5, each time point). To determine the expression levels of TP, BALB/cCrSlc and ddy mice (n = 3/each strain) were euthanized, and the heart, liver, lung, spleen, kidney, stomach, small intestine, large intestine and brain were collected. The mRNA and protein expression levels of TP in these organs were examined by quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. In BALB/cCrSlc mice administered (123)I-IIMU, markedly high radioactivity levels were observed in the liver [1.568 ± 0.237 (%ID/g)] and small intestine [0.506 ± 0.082 (%ID/g)], whereas those in the other tissues were fairly low [<0.010 ± 0.003 (%ID/g)] 30 min after the injection. The highest expression levels of TP mRNA were also observed in the liver and small intestine among the tissues tested. Immunoblotting showed intense immunoreactive bands of the TP protein for the liver and small intestine, whereas no notable bands were detected for other tissues. Similar expression profiles of TP mRNA and protein were observed in ddy mice. We confirmed TP expression in various tissues of mice at the mRNA and protein levels: high TP expression levels were observed in the liver and small intestine. These high TP expression levels are consistent with the high accumulation levels of (123)I-IIMU in these tissues. Our results may provide important information about the physiological accumulation of (123)I-IIMU, which may be useful for the clinical diagnostic imaging of TP.

235. Anticancer Effectiveness of Mesenchymal Stem Cells Transfected With Thymidine Phosphorylase for Colorectal Cancer Cells

Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells with a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. One of the major bottlenecks of GDEPT is to have the therapeutic gene specifically target on cancer cells prior to the administration of prodrug. Among various gene delivery methods, mesenchymal stem cells (MSCs) have emerged as potential cellular vehicles for gene delivery. They exhibit remarkable tumor-tropic and low immunogenic capacities. In this study, MSCs was engineered to express thymidine phosphorylase (TP) which has the capability of converting prodrug 5’-deoxy-5-fluorouridine into toxic 5-fluorouracil. TP expression in the MSCs post-transfection of TP cDNA was confirmed by immunoblotting analysis and its activity was determined by spectrophotometric assay. Our results showed the anticancer effectiveness of human HT29 colorectal cells co-cultured with TP-expressing MSCs was enhanced with the addition of various dosages of 5’-deoxy-5-fluorouridine.

279. Efficient and Safe Lentiviral Vector-Mediated Hematopoietic Stem Cell Gene Therapy in MNGIE Mice

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase(TP) resulting in systemic thymidine(Thd) and deoxyuridine(dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogenic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp−/-Upp−/- double knockout mice, a model for MNGIE disease, was investigated. At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco = 96±4 nmoles/h/mg), and in SFFV recipient mice 400-fold higher (450±5 nmoles/h/mg). Consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months) showed on average 1.2-fold wild type TP activity levels increase in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice. This was sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). The LV integration profile in bone marrow cells of primary recipients was analyzed; LVs displayed the expected tendency to integrate within highly expressed genes and the integration pattern did not differ from that of other SIN-LV vectors in other disease models (primary immune deficiencies and lysosomal enzyme storage disorders). Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

OP45 – 3024: Efficient lentiviral vector-mediated hematopoietic stem cell gene therapy in MNGIE mice

Objective Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase (TP) resulting in systemic thymidine (Thd) and deoxyuridine (dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogeneic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Application of lentiviral vector therapy. Methods Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp-/-Upp-/- double knockout mice, a model for MNGIE disease, was investigated. Results At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco=96±4 nmoles/h/mg), compared to a 400-fold increase observed in SFFV recipient mice (450±5) and consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months post treatment) showed on average 1.2-fold TP wild type activity levels in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice, sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). Conclusion Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

Pilot study investigating the prognostic significance of thymidine phosphorylase expression in patients with metastatic breast cancer: a single institution retrospective analysis.

BackgroundThe thymidine phosphorylase (TP) enzyme is expressed in higher levels in cancer tissue when compared with normal tissue. It is involved in the intratumoral activation of widely prescribed pyrimidine-derived antimetabolites such as 5′-deoxy-5-fluorouridine and capecitabine (Xeloda®). The purpose of this study was to determine the clinical correlation between TP expression in tumor tissue and the clinical outcome of capecitabine-based therapy in patients with locally advanced (stage III) or metastatic breast cancer (stage IV).MethodsThe following variables were analyzed as potential determinants of benefit from a capecitabine-based therapy: TP expression, estrogen receptor (ER) and progesterone receptor (PR) status, human epidermal growth factor receptor-2 status, and Ki67 status. This was accomplished by immunohistochemical analysis of paraffin-embedded cancer tissues from 18 patients with breast cancer treated with at least one cycle of capecitabine. Clinical outcome was measured as time to progression.ResultsTP staining intensities in both the invasive and in situ components in patients with lobular and ductal carcinomas were reported. Higher levels of TP in the invasive component were expressed in ER-negative tumors when compared with ER-positive tumors (P<0.05). The ER-positive group expressing lower levels of TP had a median time to progression of 13 months compared with the ER-negative group expressing higher levels of TP which had a median time to progression of 7.5 months (P=0.14).ConclusionPatients with ER-positive tumors expressing lower levels of TP exhibit a longer time to progression when compared with patients with ER-negative tumors. Consequently, tumor TP expression does not seem to predict the outcome of capecitabine-based chemotherapy.

Relative mRNA expression of prostate-derived E-twenty-six factor and E-twenty-six variant 4 transcription factors, and of uridine phosphorylase-1 and thymidine phosphorylase enzymes, in benign and malignant prostatic tissue.

Prostate cancer is the most frequent urological tumor, and the second most common cancer diagnosed in men. Incidence and mortality are variable and appear to depend on behavioral factors and genetic predisposition. The prostate-derived E-twenty-six factor (PDEF) and E-twenty-six variant 4 (ETV4) transcription factors, and the thymidine phosphorylase (TP) and uridine phosphorylase-1 (UP-1) enzymes, are reported to be components of the pathways leading to tumorigenesis and/or metastasis in a number of tumors. The present study aimed to analyze the mRNA expression levels of these proteins in prostatic cancerous and benign tissue, and their association with clinical and pathological variables. Using quantitative reverse transcription polymerase chain reaction, the mRNA expression levels of PDEF, ETV4, TP and UP-1 were studied in 52 tissue samples (31 of benign prostatic hyperplasia and 21 of prostate adenocarcinomas) obtained from patients treated by transurethral resection of the prostate or by radical prostatectomy. Relative expression was assessed using the ∆-CT method. Data was analyzed using Spearman's tests for correlation. P<0.05 was considered to indicate a statistically significant difference. The results revealed that PDEF, ETV4, UP-1 and TP were expressed in 85.7, 90.5, 95.2 and 100% of the prostate cancer samples, and in 90.3, 96.8, 90.3 and 96.8% of the benign samples, respectively. PDEF and ETV4 exhibited a significantly higher relative expression level in the tumor samples compared with their benign counterparts. The relative expression of TP and UP-1 did not differ significantly between benign and cancerous prostate tissues. The relative expression of TP was moderately and significantly correlated with the expression of ETV4 in the benign tissues. The relative expression of UP-1 was significantly lower in T3 compared with T1 and T2 cancers. These findings indicate that PDEF, ETV4, TP and UP-1 are typically expressed in benign and malignant prostatic tissues. Further studies are necessary to define the role of these proteins as therapeutic targets in prostate cancer.

Predictive markers of the efficacy of colon adenocarcinoma treatment by 5-fluoropyrimidines determined by means of immunohistochemistry method

5-fluorouracil (5-FU) has remained the treatment of choice in the adjuvant and palliative setting of colon cancer (CC).The purpose of this study was to evaluate the expression of the thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPYD)in CC patients treated with 5-fluorouracil (5-FU) to determine their predictive and prognostic significance. Immunohistochemistry (IHC) analysis of the expression of proteins was performed on tumour tissue of 104 patients with stage IV colon cancer. All patient was treated withfluoropyrimidine/antifolate-based therapy (FOLFOX, De Gramont, FOLFIRI). Usedmultivariate logistic regression analyses we found the level of expression of proteins thymidine phosphorylase (TP), thymidylate synthase (TS) as predictors efficiency 5 - fluoropyrimidine with adenocarcinoma of the colon. The value of expression of TP and TS has a statistically significant effect (p = 0,006 and p = 0.0036) on time to disease progression, respectively.Our data demonstrated the efficacy of the 5-fluoropyrimidine chemotherapy colon adenocarcinoma may be utilized evaluation of expression of TS and TP.

Thymidine phosphorylase activates NFκB and stimulates the expression of angiogenic and metastatic factors in human cancer cells

Thymidine phosphorylase (TP) promotes angiogenesis and metastasis, and confers resistance to anticancer agents in some cancer cell types. We previously reported that TP stimulates the expression of interleukin (IL)-8 in human KB cancer cells by an unknown mechanism. A mutation in the nuclear factor (NF)κB binding site of the IL-8 promoter suppressed promoter activity in KB/TP cells that overexpress TP. Specifically inhibiting NFκB by using BY11-7082 also suppressed TP-induced IL-8 promoter activity and IL-8 expression. Moreover, TP overexpression led to the activation of NFκB and an upregulation in the expression of its target genes, and increased phosphorylated IKKα/β protein levels, while promoting IκBα degradation as well as p65 phosphorylation and nuclear localization. The activation of NFκB in KB/TP cells was suppressed by the antioxidants N-acetylcysteine and EUK-8. In addition, in gastric cancer tissue samples, the expression of the NFκB-regulated genes, including IL-8, IL-6, and fibronectin-1 was positively correlated with TP expression. These findings indicate that reactive oxygen species mediated NFκB activation by TP increases the expression of genes that promote angiogenesis and metastasis in gastric cancer.

4-Methylumbelliferone inhibits ovarian cancer growth by suppressing thymidine phosphorylase expression.

Background4-Methylumbelliferone (4-MU), a hyaluronan (HA) synthesis inhibitor, has antitumor activity in cancer cells. However, few studies have focused on its effects on ovarian cancer. The aim of this study was to investigate the effects of 4-MU on ovarian cancer and to elucidate its mechanism of action.MethodsThe HRA human ovarian serous adenocarcinoma cell line was used in this study. The effects of 4-MU on cell proliferation, migration, and invasion were determined by using in vitro assays as well as an in vivo rat peritoneal carcinomatosis model. The expression of HA synthase (HAS), CD44 HA receptor, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (TP) mRNA in HRA cells was analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results4-MU administration inhibited the growth of peritoneal tumors and significantly prolonged survival. In vitro experiments showed that 4-MU inhibited HRA cell proliferation in a dose-dependent manner, while it did not affect HRA cell invasion and migration. 4-MU significantly decreased TP mRNA expression in HRA cells. On the other hand, since HAS2, CD44, and VEGF endogenous mRNA expression levels were very low in HRA cells, it was impossible to evaluate the effect of 4-MU treatment.ConclusionsThese results suggest that 4-MU exerts its antitumor effect on ovarian cancer through suppressing TP expression.

Thymidine phosphorylase mRNA expression may be a predictor of response to post-operative adjuvant chemotherapy with S-1 in patients with stage III colorectal cancer.

The aim of the present study was to investigate markers in surgically resected specimens of colorectal cancer that can be used to predict the response to chemotherapy. The mRNA expression levels of enzymes involved in 5-fluorouracil (5-FU) metabolism and folate metabolism were measured in formalin-fixed, paraffin-embedded tumor sections obtained from the primary tumors of 54 patients with resected stage II or III colorectal cancer who received S-1 for one year. The 5-FU metabolizing enzymes studied were thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase (TP). The folate metabolizing enzymes studied were folypolyglutamate synthetase, γ-glutamyl hydrolase and dihydrofolate reductase. The associations between the mRNA expression levels of these enzymes and clinical variables were investigated. Tumors were classified as exhibiting high or low expression as compared with the median mRNA expression level of each metabolizing enzyme defined as the cutoff value. The associations between the high and low expression levels of each enzyme and disease-free survival (DFS) were analyzed with the use of Kaplan-Meier curves and the log-rank test. DFS was not significantly associated with the relative mRNA expression level of any metabolizing enzyme in the study group as a whole, but there was a trend toward longer DFS in patients with high TP expression (P=0.066). In patients with stage III colorectal cancer, high TP expression was associated with significantly improved outcomes compared with low TP expression (P=0.039). These results indicate that the mRNA expression of TP, a metabolizing enzyme of 5-FU, is a significant predictor of response to post-operative chemotherapy with S-1 in patients with stage III colorectal cancer.

Translational Study for Mechanisms of Synergy and Toxicity of Tivozanib in Combination with Capecitabine in a Population-Based Murine Tumor Model

ABSTRACT Aim: Clinical trials combining vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) and capecitabine have been promising but often met with increased toxicities requiring dose reductions. Tivozanib is a highly selective VEGFR TKI with demonstrated antitumor activity. Translational studies were conducted using a genetically diverse population of tumor models to understand tivozanib+capecitabine potential synergistic responses and possible toxicities. Methods: The antitumor activity of tivozanib+capecitabine at various dose levels was assessed in multiple tumors that exhibited partial or no response to either agent alone. Tumor growth, cell proliferation, angiogenesis, and the effect of tivozanib on capecitabine conversion to 5-fluorouracil (FU) were investigated. Results: The combination of tivozanib+capecitabine enhanced antitumor activity of capecitabine and blocked inflammatory and angiogenic reactions triggered by capecitabine monotherapy. A subset of tumors exhibited strong synergistic response to the combination treatment and complete tumor growth inhibition was achieved at capecitabine doses reduced to as low as 25% of maximum tolerated dose. RT-PCR analysis of the treated tumors revealed that tivo significantly induced the expression of thymidine phosphorylase (TP), an enzyme for cape conversion in the body. Increased TP expression in tumors was associated with elevated local 5-FU concentration, suggesting a pharmacokinetic mechanism of synergy. In addition, TP expression was induced by tivozanib treatment in both the epidermis of treated mice and in cultured human keratinocytes suggesting increased capecitabine conversion in the skin. Conclusions: These results indicate tivozanib TP induction may be a key mechanism of synergistic response to the combination treatment and suggests clinical antitumor activity may be achieved with a reduced capecitabine dose. The elevated epidermal TP expression may provide a mechanistic explanation for increased Hand-Foot Syndrome observed in phase 1 trial patients treated with tivozanib+capecitabine (NCT01306630). Further exploration is warranted. Disclosure: J. Lin: AVEO Oncology employee • Stock • Salary Other financial relationships • None; G. Li: AVEO Oncology employee • Stock • Salary Other financial relationships • None; F. Jiang: AVEO Oncology employee • Stock • Salary Other financial relationships • None; L. Shen: AVEO Oncology employee • Stock • Salary Other financial relationships • None; J. Gyuris: AVEO Oncology employee • Stock • Salary Other financial relationships • None.

Prognostic significance of thymidylate synthase in postoperative non-small cell lung cancer patients.

The aim of the present study was to investigate the clinicopathologic/prognostic significance of thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT), and thymidine phosphorylase (TP) proteins in postoperative non-small cell lung cancer (NSCLC) patients. Microarray slides from a set of 178 NSCLC patients were used for the detection of TS, OPRT, and TP expression by immunohistochemistry. The correlation between clinicopathologic factors and protein expression of three proteins was analyzed. Ninety seven carcinomas (57.4%) were TS-positive, 90 carcinomas (53.9%) were OPRT-positive, and 102 carcinomas (69.4%) were TP-positive. Compared with the TS-positive patients, the overall survival (OS) was significantly lower in the TS-negative patients (hazard ratio [HR] =1.766, 95% confidence interval [CI] =1.212–2.573, P=0.003). Significant differences between TS-positive and TS-negative patients was also observed in the following stratified analyses: 1) adenocarcinoma subgroup (HR =2.079, 95% CI =1.235–3.500, P=0.006); 2) less than 60-year-old subgroup (HR =1.890, 95% CI =1.061–3.366, P=0.031); 3) stage II/III subgroup (HR =1.594, 95% CI =1.036–2.453, P=0.034); and 4) surgery plus adjuvant therapy subgroup (HR =1.976, 95% CI =1.226–3.185, P=0.005). However, the OS was not significantly correlated with OPRT or TP protein expression. This study demonstrates that the TS level in tumor tissues may be a useful marker to predict the postoperative OS in NSCLC patients.

Thymidine phosphorylase expression is associated with time to progression in patients with metastatic colorectal cancer.

Background5-Fluorouracil (5-FU) is the cornerstone of chemotherapeutic treatment for patients with colorectal cancer. The enzyme thymidine phosphorylase (TP) catalyzes the conversion of 5-FU to its active metabolite, 5-fluoro-2’-deoxyuridine. TP is expressed in tumour epithelial cells and stromal cells, particularly in tumour-associated macrophages. These macrophages may affect sensitivity to chemotherapy. Previously, we identified TP as a predictive factor in microdissected tumour samples of patients with advanced colorectal cancer. In the present study, we analysed TP expression in tissues and associated stromal cells from patients with advanced colorectal cancer and associated TP levels to tumour response and time-to-event variables during first-line chemotherapy treatment. We also investigated the association between serum TP levels at the time of surgery and gene expression in primary tumour tissues.MethodsThis study included 125 patients with metastatic colorectal cancer treated with first-line 5-FU-based chemotherapy. To quantify TP gene expression levels in tumour tissues, real-time polymerase chain reaction was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). TP protein concentration in matched serum samples was determined using an enzyme-linked immunosorbent assay system (USCN Life Science Inc.).ResultsThe tumour response rate was 31%, and 30% of patients exhibited stable disease. No associations between TP expression level and age or gender were observed. Levels of TP mRNA in mucosa and tumours were positively correlated (r = 0.41, p < 0.01). No correlation between TP expression and tumour response rate was observed. Time to progression was significantly longer in patients with high TP expression (p < 0.01). Serum TP protein levels were not associated with tumour response or time-to-event variables and did not correlate with gene expression in tumour tissues.ConclusionsHigh TP gene expression in non-microdissected tumour tissues of patients with advanced colorectal cancer correlates with longer time to progression, which could be related to treatment. These results are in contrast to previous studies where microdissected tumour cells were analysed and may be due to the presence of adjacent stromal cells. Serum TP protein expression does not correlate to TP gene expression in tissues of patients with advanced colorectal cancer.

Angiogenic factor thymidine phosphorylase associates with angiogenesis and lymphangiogenesis in the intestinal-type gastric cancer

As an angiogenic factor, thymidine phosphorylase (TP) expression in primary tumours has been thought to be a risk factor for lymph node (LN) and hepatic metastasis in patients with gastric adenocarcinoma. However, the molecular basis for the induction of metastasis by TP is largely unknown. We aim to elucidate the role of TP expression in gastric cancer neovascularisation and LN metastasis.The angiogenic and lymphangiogenic activity (CD31, D2-40, Ki-67, VEGFC, VEGFR3) and expression status of TP were detected in 103 resected human gastric carcinoma samples by immunohistochemistry. The influence of TP expression on neovascularisation and cancer cell invasion was further comparatively investigated in two groups of nude mice intraperitoneally injected with TP overexpressing MKN-45 cells (MKN-45/TP) and control cells (MKN-45/CV). In gastric cancer tissues, we found that high TP expression and various angiogenic and lymphangiogenic activities were significantly associated with poor prognostic outcomes. In addition, TP expression was also found to be associated with neovascularisation activity of gastric cancer tissues. In vivo, the MKN-45/TP group exhibited significantly increased infiltrating tumour nodules and neovascularisation activity compared to the MKN-45/CV group. TP could strongly influence gastric cancer progression via the dual activities of angiogenesis and lymphangiogenesis.

Thymidine phosphorylase is both a therapeutic and a suicide gene in a murine model of mitochondrial neurogastrointestinal encephalomyopathy.

Suicide gene therapy (SGT) is a promising strategy for treating cancer. In this work, we show that thymidine phosphorylase (TP) deficiency, the underlying genetic defect in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), presents an opportunity to apply SGT using capecitabine, a commonly used prodrug that is converted into 5-fluorouracil by TP. Using an immortalised B-lymphoblastoid cell line from a patient with MNGIE, the tumourigenic EL-4 cell line, lentiviral vectors encoding TP and a double knockout (Tymp(-/-)Upp1(-/-)) murine model, we found that EL-4 cell-derived TP(+) tumours were exquisitely sensitive to capecitabine and generated a significant local bystander effect. In addition, we detected a spontaneous cytolytic immune response in a significant fraction of the animals surviving more than 20 days after termination of the therapy. These data indicate that, in individuals lacking TP expression, TP is a highly specific suicide gene, which can be used to treat tumours that could hypothetically arise in MNGIE patients undergoing gene therapy, as these tumours will likely originate from the gene-modified cells and will be selectively targeted by capecitabine. These observations have important implications for gene therapy for MNGIE.

Molecular patterns in deficient mismatch repair colorectal tumours: results from a French prospective multicentric biological and genetic study.

Background:To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours.Methods:This prospective multicentric study involved 251 stage I–III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12–13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4–9), CpG island methylation phenotype status, ploidy, S-phase, LOH.Results:The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR.Conclusions:Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy.

Comparison of site-specific gene expression levels in primary tumors and synchronous lymph node metastases in advanced gastric cancer

Many malignant tumors consist of heterogeneous subpopulations of cells. This heterogeneity is associated with genetic characteristics. However, it remains unclear whether gene expression levels differ among specific sites of tumors in gastric cancer. We studied differences in gene expression levels among specific sites of primary tumors and synchronous lymph node metastases, using formalin-fixed, paraffin-embedded specimens resected surgically from 48 patients with previously untreated advanced gastric cancer. Specimens were obtained by laser-captured microdissection from five regions: (1) nonneoplastic mucosa, (2) surface layer (mucosa) of the primary tumor (surface sections), (3) middle layer (submucosa) of the primary tumor (middle sections), (4) the deepest layer of the primary tumor (muscularis propria or deeper) at the site of deepest invasion (deep sections), and (5) level 1 synchronous lymph node metastasis (lymph node metastases). Expression levels of the following target genes were determined by quantitative real-time polymerase chain reaction: thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF1α). TP, DPD, EGFR, and HIF1α gene expression levels were significantly higher in deep sections than in surface sections. TP, EGFR, VEGF, and HIF1α gene expression levels were significantly higher in lymph node metastases than in surface sections. TP, DPD, EGFR, VEGF, and HIF1α gene expression levels were positively correlated with the specific samples harvested from the tumors. Our results show that the expression levels of some genes in tumor cells can change in specific sites of tumors and can become higher in association with tumor progression.