Enzyme Thymidine Phosphorylase Research Articles

2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase inhibitors

Thymidine phosphorylase (TP) over expression plays an important role in several pathological conditions, such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. In this regard, a series of twenty-five 2-arylquinazolin-4(3H)-one derivatives 1–25 were evaluated for thymidine phosphorylase inhibitory activity. Six compounds 5, 6, 20, 2, 23, and 3 were found to be active against thymidine phosphorylase enzyme with IC50 values in the range of 42.9–294.6μM. 7-Deazaxanthine (IC50=41.0±1.63μM) was used as a standard inhibitor. Compound 5 showed a significant activity (IC50=42.9±1.0μM), comparable to the standard. The enzyme kinetic studies on the most active compounds 5, 6, and 20 were performed for the determination of their modes of inhibition, and dissociation constants Ki. All active compounds were found to be largely non-cytotoxic against the mouse fibroblast 3T3 cell line. This study identifies a novel class of thymidine phosphorylase inhibitors which may be further investigated as leads to develop therapeutic agents.

Allogeneic haematopoietic stem cell transplantation for mitochondrial neurogastrointestinal encephalomyopathy.

Haematopoietic stem cell transplantation has been proposed as treatment for mitochondrial neurogastrointestinal encephalomyopathy, a rare fatal autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase deficiency. We conducted a retrospective analysis of all known patients suffering from mitochondrial neurogastrointestinal encephalomyopathy who underwent allogeneic haematopoietic stem cell transplantation between 2005 and 2011. Twenty-four patients, 11 males and 13 females, median age 25 years (range 10-41 years) treated with haematopoietic stem cell transplantation from related (n = 9) or unrelated donors (n = 15) in 15 institutions worldwide were analysed for outcome and its associated factors. Overall, 9 of 24 patients (37.5%) were alive at last follow-up with a median follow-up of these surviving patients of 1430 days. Deaths were attributed to transplant in nine (including two after a second transplant due to graft failure), and to mitochondrial neurogastrointestinal encephalomyopathy in six patients. Thymidine phosphorylase activity rose from undetectable to normal levels (median 697 nmol/h/mg protein, range 262-1285) in all survivors. Seven patients (29%) who were engrafted and living more than 2 years after transplantation, showed improvement of body mass index, gastrointestinal manifestations, and peripheral neuropathy. Univariate statistical analysis demonstrated that survival was associated with two defined pre-transplant characteristics: human leukocyte antigen match (10/10 versus <10/10) and disease characteristics (liver disease, history of gastrointestinal pseudo-obstruction or both). Allogeneic haematopoietic stem cell transplantation can restore thymidine phosphorylase enzyme function in patients with mitochondrial neurogastrointestinal encephalomyopathy and improve clinical manifestations of mitochondrial neurogastrointestinal encephalomyopathy in the long term. Allogeneic haematopoietic stem cell transplantation should be considered for selected patients with an optimal donor.

Abstract 2556: Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks

Abstract Background: TAS-102 is a combination of an antineoplastic thymidine-based nucleoside analogue, trifluridine (FTD), and a thymidine phosphorylase inhibitor, tipiracil (TPI), at a molar ratio of 1:0.5. FTD is the active antitumor component of TAS-102, and its triphosphate form is incorporated into the DNA in tumor cells. TPI, a potent inhibitor of the FTD-degrading enzyme thymidine phosphorylase, maintains adequate plasma concentrations of orally administered FTD, thereby potentiating its antitumor activity. In an international multicenter randomized double blind Phase III study (RECOURSE), TAS-102 administration resulted in a significant improvement in overall survival and progression-free survival and a favorable safety profile compared to placebo treatment in patients with metastatic colorectal cancer refractory to standard chemotherapies. In this study, we investigated the mechanism of FTD-induced cytotoxicity in colorectal cancer and other cell lines. Method: The colorectal cancer cell lines RKO and HCT-116, the subline HCT-116 p53−/−, and the lung adenocarcinoma cell line A549 were used. Cells were treated with FTD or FdUrd using the IC50 values for 12 to 72 h. Molecules involved in DNA damage and the cell cycle were evaluated by western blot and RT-PCR. Cell cycle analysis and DNA strand-breaks were evaluated by flow cytometry and alkaline- and neutral-comet assays. Results: FTD and FdUrd activated similar DNA damage response pathways via transient phosphorylation of Chk1 at Ser345, followed by accumulation of p53 and p21 in HCT-116 cells. A similar biochemical response was observed using the IC50 concentration of FdUrd or FTD in RKO and A549 cells. FTD repressed G2 to M phase progression and the cells accumulated in G2 phase 72 h after treatment, with a 35% and 29% decrease in the CDK1 and CCNB1 mRNA levels compared to the control. The G2 phase arrest by FTD was commonly induced in each p53 proficient cell lines (HCT-116, RKO, and A549). In contrast, FTD-induced G2 phase arrest was not observed when p53 expression was suppressed. Furthermore, the p53-dependent G2 phase arrest by FTD was associated with the downregulation of Cyclin B1, both through its transcriptional repression and proteasomal degradation. Although FdUrd induced numerous single- and double-strand DNA breaks, FTD did not induce detectable DNA strand breaks. FTD was massively incorporated into DNA (41.4 pmol/μg DNA), whereas less FdUrd was incorporated (0.6 pmol/μg DNA) 72 h after treatment. Conclusion: Our analyses revealed that FTD induced sustained G2 phase arrest and few DNA strand breaks, in spite of massive incorporation into DNA. These results suggest that the mechanism of FTD action is unique at the cellular level, resulting in the clinical efficacy of TAS-102. Citation Format: Kazuaki Matsuoka, Makoto Iimori, Shinichiro Niimi, Hiroshi Tsukihara, Sugiko Watanabe, Shinichi Kiyonari, Mamoru Kiniwa, Koji Ando, Eriko Tokunaga, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks. [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 2556. doi:10.1158/1538-7445.AM2015-2556

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.

Chemical synthesis, molecular modelling, and evaluation of anticancer activity of some pyrazol-3-one Schiff base derivatives

A series of twelve pyrazol-3-one Schiff`s base derivatives (5–17) were designed and synthesized by microwave-assisted chemical synthesis. Their purity was confirmed by melting point and HPLC and their chemical structures were determined by FT-IR, UV, 1H, and 13C-NMR spectroscopic techniques. In silico docking of the synthesized compounds inside the active site of thymidine phosphorylase was performed using two molecular modeling programs. The compounds were tested in vitro on calf thymus DNA to study the interaction with DNA using a spectrophotometer. Some of the pyrazol-3-one Schiff`s base derivatives showed close match interaction with DNA. The tested compounds were also studied by application to angiogenic enzyme thymidine phosphorylase (TP, E.C. 2.4.2.4), carcinoma cell lines including both human breast (MCF-7) and human lung cell lines (A549). The lead compound 2 in the series caused inhibition of thymidine phosphorylase in the micro molar range (IC50 of 28 ± 2 µM) and was able to retard growing of breast carcinoma cells. Our results indicate that pyrazol-3-one Schiff base derivatives are promising lead compounds for the development of more active antitumor agents and exhibit their highest cytotoxic effect on breast carcinoma cell line.

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.

Regulation and novel action of thymidine phosphorylase in non-small cell lung cancer: crosstalk with Nrf2 and HO-1.

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

Epicardial coronary artery spasm as cause of capecitabine-induced tako tsubo cardiomyopathy

Cardiotoxicity of fluoropyrimidines like the orally administered capecitabine and the intravenous 5-fluorouracil (5-FU) is a well-known clinical challenge [1, 2]. The cardiotoxic effects of both agents seem to occur through the same mechanisms [3]. However, capecitabine-induced cardiotoxicity is less common because it obtains its pharmacological active form through a three-step enzymatic conversion, while the last step is catalyzed by the enzyme thymidine phosphorylase, which is more active in neoplastic tissues [4, 5]. But this enzyme is also actively expressed in arteriosclerotic plaques, which is thought to be one potential mechanism leading to coronary side effects, especially in patients with a history of coronary artery disease [3]. Until today, numerous reports have shown cardiac side effects of fluoropyrimidines. However, there is no explicit evidence of capecitabine as a direct trigger for tako tsubo cardiomyopathy (TTC). Amongst other causes, coronary artery spasm is discussed as a potential cause of TTC [6]. Epicardial vasospasm is a widely accepted hypothetical pathomechanism of 5-FU-associated cardiac side effects [7, 8]. Therefore, fluoropyrimidines could be plausible triggers for TTC and the following case will strengthen this possible connection. An 81-year-old female with metastatic breast cancer was admitted to our emergency unit with acute chest pain and severe dyspnoea. The symptoms began at about 4 p.m., shortly after a 1-h workout as part of a diabetes group training. On the same day, in the morning at about 9 a.m., the patient had received the first dose of capecitabine (1,500 mg). The patient did not report any extraordinary emotional stress, but had a history of obstructive coronary artery disease. Five years ago she had undergone percutaneous coronary intervention in the left anterior descending coronary artery (LAD). The patient further suffered from insulin-dependent diabetes mellitus type 2, hypercholesterolemia, hypertension and peripheral vascular disease. On admission the symptoms were relieved by sublingual nitroglycerin, but recurred after a few minutes. The electrocardiogram (ECG) showed sinus rhythm with ST-segment elevations, which had not been detected in a routine ECG 3 weeks ago (Fig. 1a). High sensitive cardiac troponin T concentration was 282 pg/ml (n \ 14 pg/ml). The patient was treated with acetylsalicylic acid (500 mg intravenous), clopidogrel (600 mg orally) and heparin (5,000 units intravenous) because of the suspicion of acute ST-elevation myocardial infarction. No abnormalities were discovered by physical examination. Blood pressure was 140/63 mm/Hg. Cardiac catheterization was performed immediately. Ventriculography showed apical dyskinesia Electronic supplementary material The online version of this article (doi:10.1007/s00392-013-0636-4) contains supplementary material, which is available to authorized users.

2,5-Disubstituted-1,3,4-oxadiazoles: thymidine phosphorylase inhibitors

A series of 2,5-disubstituted-1,3,4-oxadiazoles (1–16) were synthesized via microwave irradiation and screened against thymidine phosphorylase enzyme. Four members of the series have demonstrated thymidine phosphorylase inhibitory activities with IC50 values between 37 and 320 μM. Pyridinyl-containing 1,3,4-oxadiazoles exhibited an enhanced inhibitory potential, while phenyl analogs did not show substantial inhibitory potential. Compound 9, a dipyridinyl residue having an IC50 value of 37 ± 0.76 μM was found to be the most potent in the series superior to standard inhibitor 7-deazaxanthine (IC50 = 39.28 ± 0.76 μM).

Synthesis, in vitro evaluation of thymidine phosphorylase inhibitory activity, and in silico study of 1,3,5-triazin-2,4-dione and its fused analogues

Based on structural similarities with the reference compounds, a series of 1,3,5-triazin-2,4-dione and their fused analogues was designed, synthesized and their in vitro thymidine phosphorylase inhibitory potential was evaluated. The monocyclic analogues were found to be inactive. Among the different fused derivatives synthesized, compounds having keto group (C=O) at C7/C4 and thioketo group (C=S) at C5/C2 position showed TP inhibitory activity comparable to positive control, 7-deazaxanthine (7-DX) (IC50 value = 42.63 μM). Molecular docking of the target compounds into the enzyme thymidine phosphorylase was performed to illustrate the important structural information on the plausible ligand–enzyme-binding interactions.

Interferon-αEnhances 5′-Deoxy-5-Fluorouridine-Induced Apoptosis by ERK-Dependant Upregulation of Thymidine Phosphorylase

5-Florouracil (5-FU) is the basic agent used in the treatment of gastric cancer. Capecitabine, a prodrug of 5-FU, displays increased antitumor efficacy compared with 5-FU in the clinic. 5′-Deoxy-5-fluorouracil (5′-DFUR), the metabolite of capecitabine, is converted to 5-FU by the enzyme thymidine phosphorylase (TP), which is present at high concentrations in human tumors. In this study, we investigated the effect of interferon-α (IFN-α) on the sensitivity of gastric cancer cells to treatment with 5′-DFUR and its relationship with TP expression. Preincubation of gastric cancer cells with IFN-α enhanced 5′-DFUR-induced apoptosis via IFN-α-mediated upregulation of TP. The depletion of TP with small interfering RNA (siRNA) obviously inhibited IFN-α-induced upregulation of TP expression and thus prevented apoptosis induced by IFN-α and 5′-DFUR. Treatment with IFN-α and combined IFN-α and 5′-DFUR treatment were also associated with concomitant activation of ERK signaling. Treatment with the ERK inhibitor PD98059 or depletion of ERK with siRNA partially reversed IFN-α-induced upregulation of TP expression, thus partially preventing apoptosis induced by IFN-α and 5′-DFUR. Taken together, our study shows that IFN-α enhanced 5′-DFUR-induced apoptosis in gastric cancer cells by upregulation of TP expression, which is partially regulated by activation of ERK signaling.

The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera

Erythrocyte encapsulated thymidine phosphorylase (EE-TP) is under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a fatal metabolic disorder resulting from an inherited deficiency of the enzyme thymidine phosphorylase. We report here the development and validation of a sensitive electrochemiluminescent (ECL) bridging immunoassay to support Good Laboratory Practice (GLP)-compliant preclinical safety studies of EE-TP in the mouse and dog. Affinity-purified rabbit anti-E. coli thymidine phosphorylase (TP) antibody was used as a calibrator standard with an effective working range of 2.5–7500ng/mL. The minimum required dilution (MRD) for both mouse and dog sera was 1:10. The mean analytical recoveries for anti-TP antibodies spiked into serum at 70ng/mL and 7000ng/mL were 117.9% and 93.2%, respectively for mouse, and 112.0% and 104.3%, respectively for dog. The intra-assay precision (coefficient of variation, CV) ranged between 1.1% and 8.0% in mouse serum, and 1.9% and 2.5% in dog serum. Inter-assay precision ranged between −1.6% and 6.7% in mouse serum, and −13.0% and −2.5% in dog serum. Assay cut-point/screening cut-point correction factors were 201.37 and 44.4, respectively for mouse and dog sera. For future analysis of positive test samples, less than 37.12% (mouse) and 31.41% (dog) inhibition of the assay signal in the confirmation assay will confer anti-TP antibody specificity. Assay drift and hook effects (prozone) were not observed. The intra-assay and inter-assay accuracy for robustness were within ±20%.

Mitochondrial Neurogastrointestinal Encephalomyopathy: Novel Pathogenic Mutations in Thymidine Phosphorylase Gene in Two Italian Brothers

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, MIM 603041) is an autosomal recessive multisystem disorder occurring due to mutations in a nuclear gene coding for the enzyme thymidine phosphorylase (TYMP). Clinical features of MNGIE include gastrointestinal dysmotility, cachexia, ptosis or ophthalmoparesis, peripheral neuropathy, diffuse leukoencephalopathy, and signs of mitochondrial dysfunction in tissues. We report the clinical and molecular findings in two brothers in whom novel TYMP gene mutations (c.215-13_215delinsGCGTGA; c.1159 + 2T > A) were associated with different clinical presentations and outcomes.

The Thymidine Phosphorylase Inhibitor 5′-O-Tritylinosine (KIN59) Is an Antiangiogenic Multitarget Fibroblast Growth Factor-2 Antagonist

5'-O-Tritylinosine (KIN59) is an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase. Previous observations showed the capacity of KIN59 to abrogate thymidine phosphorylase-induced as well as developmental angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Here, we show that KIN59 also inhibits the angiogenic response triggered by fibroblast growth factor-2 (FGF2) but not by VEGF in the CAM assay. Immunohistochemical and reverse transcriptase PCR analyses revealed that the expression of laminin, the major proteoglycan of the basement membrane of blood vessels, is downregulated by KIN59 administration in control as well as in thymidine phosphorylase- or FGF2-treated CAMs, but not in CAMs treated with VEGF. Also, KIN59 abrogated FGF2-induced endothelial cell proliferation, FGF receptor activation, and Akt signaling in vitro with no effect on VEGF-stimulated biologic responses. Accordingly, KIN59 inhibited the binding of FGF2 to FGF receptor-1 (FGFR1), thus preventing the formation of productive heparan sulphate proteoglycan/FGF2/FGFR1 ternary complexes, without affecting heparin interaction. In keeping with these observations, systemic administration of KIN59 inhibited the growth and neovascularization of subcutaneous tumors induced by FGF2-transformed endothelial cells injected in immunodeficient nude mice. Taken together, the data indicate that the thymidine phosphorylase inhibitor KIN59 is endowed with a significant FGF2 antagonist activity, thus representing a promising lead compound for the design of multitargeted antiangiogenic cancer drugs.

Thymidine Phosphorylase Gene Expression in Stage III Colorectal Cancer

BackgroundThe thymidine phosphorylase (TP) enzyme has several tumor-promoting functions. The aim of this study was to explore TP gene expression in relation to clinical and histopathological data obtained from patients with stage III colorectal cancer.Methods and resultsTP gene expression was analyzed by real-time quantitative PCR in tumor and mucosa samples from 254 patients. TP gene expression in tumors correlated with lymph node staging, with higher expression relating to a higher number of positive nodes and a worse N-stage. Higher TP expression was also associated with a worse histological tumor grade. Patients with rectal cancer had significantly higher TP expression in mucosa and tumors compared with patients having colon cancer.ConclusionHigher intratumoral TP expression appears to be related to a worse N stage, and thus, with a worse prognosis. TP gene expression measured in a preoperative biopsy could be of interest in preoperative staging.

Increased Mean Corpuscular Volume of Erythrocytes during Capecitabine Treatment: A Simple Surrogate Marker for Clinical Response

Capecitabine, as all fluoropyrimidines, interferes with vitamin metabolism and may thus have an impact on hematopoiesis. It is metabolized to its active form 5-fluoruracil by the enzyme thymidine phosphorylase, which exists in higher concentrations in tumor tissue and liver than in normal tissues. In the study, we investigated the changes in mean corpuscular volume (MCV) of red blood cells and the possible correlation of these changes with the clinical outcome of capecitabine treatment in women with metastatic breast cancer. Data from 75 metastatic breast cancer patients were analyzed retrospectively. Capecitabine was used at a dose of 2500 mg/m² daily for 14 days of every 3-week period. Mean corpuscular volume of red blood cells and other parameters of complete blood count were recorded at the beginning of the treatment, in the ninth week, and periodically thereafter. Mean age was 51.5 ± 10.8 and 61.3% of the patients were premenopausal. Capecitabine was administered as the median 3rd line (min-max: 1-9) treatment and a median of 6 cycles (min-max: 1-24) for metastatic breast cancer. Median ΔMCV level (post-treatment values at ninth week - baseline) was 8. ΔMCV was ≥8 in 37 patients and <8 in 38 patients. The 35 of the 37 patients with ΔMCV level ≥8 and 25 of the 38 patients with ΔMCV level <8 had clinical benefit (complete response + partial response + stable disease) from capecitabine treatment (P = 0.02). However, the difference between progression-free survival of the patients with ΔMCV levels higher than 8 and those with ΔMCV levels lower than 8 according to Kaplan-Meier survival analysis was not statistically significant (6.7 and 4.3 months, respectively, P = 0.26). Additionally, median ΔMCV level was 9.1 (min-max: -2.4 to 24.9) among patients who had clinical benefit and 5.90 (min-max: -0.8 to 12.3) among nonresponders (P = 0.016). Capecitabine increases the mean corpuscular volume levels of red blood cells by a yet unidentified mechanism. Early increment of mean corpuscular volume levels is higher than 8, i.e. by the 9th week, might predict clinical benefit from the treatment.

P05.1 Efficacy and tolerability of miglustat in patients with Niemann – Pick C disease

and dysarthria. She had left sided dystonia since 6 years of age. Because of her fatigue she was bound toWheel chair for 4 years. The parents were not consangineous. Her examination revealed that she was cachectic, had total ophthalmoplegia, pitosis, left facial weakness. Tonus was increased on the left extremities, and had rigidity on the left upper extremity, deep tendon reflexes were hypoactive. Muscle strength was 3/5 on lower extremities. Bilateral babinsky was positive. Previously made tests were as follows; brain MRI; showed hyperintensity on basal ganglions especially on putamen and globus pallidus. MRI spectroscopy showed constant lactate peak on thalamus. MELAS; MERRF; NARP mutations were normal. Muscle biopsy was normal, EEG; normal. With the neurological findings she was thought to have MNGIE: BAER revealed bilateral moderate degree sensory-neural deafness, electromyography revealed demyelinating neuropathy on lower extremities, serum lactate level was normal. Thymidine phosphorylase enzyme activity was normal. Since Thymidine phosphorylase was normal she was reassesed and for Coeliac disease anti-tissue transglutaminase IgA (t-TGIgA) and antigliadin IgA and IgG (AGA IgA and IgG) titres were studied and found to be high. Small intestine biopsy revealed gluten-sensitive enteropathy stage III and she was diagnosed as Coeliac disease. After gluten-free diet her neurological findings improved. We presented this case to show the broad spectrum of neurologic findings in Coeliac Disease.

Phase I Studies of Drug Combinations

A better understanding of tumor biology has provided a large number of drug-able targets that are specific to the cancer cell or its environment. The ability to identify critical targets involved in cancer cell growth and survival is one of the reasons that we are witnessing a tremendous increase in the number of promising new drugs in oncology. This situation has created several challenges to the drug development process, including the need to study combinations of targeted agents that would be more effective and less susceptible to drug resistance compared with single agents. Although it is possible to conceive of many different drug combinations that might be studied in the phase I setting, not every combination will be of equal interest. The large number of new agents, coupled with the complexity and expense of performing clinical trials, makes it imperative to identify features of phase I combination trials that predict for a high likelihood of success when such regimens are evaluated in subsequent phase II and III trials. In this regard, the editors at Journal of Clinical Oncology (JCO) have placed higher priority on those phase I drug combination trials that have the characteristics outlined in Table 1. Implicit in these criteria is the need for investigators to have a compelling mechanistic rationale for studying a given drug combination, and to provide results that are interpretable and that will move the field forward. The study by Perotti et al in this issue of JCO illustrates some of the features that are considered to be important for high-profile phase I combination trials being considered for publication by JCO. The rationale for investigating the combination of the mammalian target of rapamycin (mTOR) inhibitor ridaforolimus and capecitabine was based on the additive effect of both agents in preclinical models, the clinical relevance of these two drug classes when used as single agents in the treatment of cancer, and the expectation that each agent might have nonoverlapping toxicities that would permit their safe administration in a phase I setting. In addition, the study included the performance of pharmacokinetic (PK) correlates to guard against serious interaction between the two agents that might compromise activity or enhance toxicity. The investigators also incorporated pharmacodynamic end points to demonstrate that the appropriate drug targets were inhibited at clinically achievable doses of each agent. Finally, the study provided evidence to suggest that this combination is of sufficient interest to justify continued investigation. Essentially, all of the major criteria listed in Table 1 were satisfied by this study, as well as the minor criterion of demonstrating clinical activity. The rationale behind testing the combination of ridaforolimus and capecitabine, clearly described by Perotti et al, related to the fact that capecitabine is a prodrug that is activated by the enzyme thymidine phosphorylase. A by-product of this reaction is 2-deoxy-Dribose, a molecule with proangiogenic activity. Ridaforolimus, by exerting its effects through a non–cross-resistant pathway, was hypothesized to counteract the proangiogenic effects of thymidine phosphorylase and thereby potentiate the antitumor properties of fluoropyrimidines. Ridaforolimus was given on days 1, 8, and 15, with capecitabine given on days 1 through 14 of a 4-week cycle. Importantly, and based on the experience with the combination of temsirolimus and fluorouracil, the investigators elected to maximize the dose of capecitabine, thereby potentially compromising the dose of the mTOR inhibitor. Ideally, the rationale for justifying this type of design should be explained thoroughly when reporting such studies. It appears that the investigators were anticipating mucositis with this combination and, therefore, defined dose-limiting toxicity to include at least grade 3 functional mucositis and failure to recover to grade 1 toxicity by day 28. Although this is a novel approach in defining dose-limiting toxicity that satisfies one of the minor criteria listed in Table 1, accepting such levels of severe toxicity might lead to difficulties when adopting this combination for more general use. In addition, the combination induced frequent and uncomfortable adverse effects (stomatitis, 69%; dermatitis, 44%; fatigue, 47%; anorexia, 37%; hemifacial spasm, 37%; diarrhea, 22%), even though these toxicities were reported to be mild. Continuous mild adverse effects of Table 1. Characteristics of High-Priority Phase I Drug Combination Trials for Consideration by JCO

Simple oral therapy with capecitabine (CAPE) and cyclophosphamide (CPA) for metastatic breast cancer (MBC).

1006 Background: There is continued interest in oral agents for the treatment of MBC as many patients (pts) prefer oral, home-based therapy to IV clinic-based regimens. The SWOG evaluated a simple oral combination cytotoxic regimen for MBC in a phase II trial, S0430. There is a dual scientific basis for combining CAPE with CPA. 1) CAPE is converted to 5-FU in tumors, utilizing the enzyme thymidine phosphorylase (TP). Preclinical and clinical studies show that TP in tumors is increased by CPA. 2) Metronomic regimens may inhibit angiogenesis by continuously exposing the tumor endothelial cells to cytotoxic drug. Methods: Eligible pts had MBC, 0-2 prior chemo regimens, and if ER+ had progressed on endocrine therapy in the metastatic setting. Pts had RECIST- measurable disease or CA 15-3/CA 27-29 > 2X ULN. Pts received CPA 100 mg po qd days 1-14 and CAPE 1,500 mg po bid days 8-21, cycles repeated every 21d. Doses were not adjusted for BSA. Results: 112 pts enrolled between 8/15/2005 and 9/1/2007. 16 were ineligible. Toxicity (tox) was mild, with no treatment-related deaths. Grade 4 tox included thrombosis/embolism (1 pt). Grade 3 tox included HFS (7 pts), fatigue, diarrhea, and dehydration (2 pts each). RECIST RR (80 pts), and PFS, OS (96 pts) are listed in the Table, in context with recently reported trials of other CAPE combinations. Conclusions: This therapy did not meet prespecified criteria of interest, a 42% RR. However, RR and PFS of CAPE/CPA compare favorably with CAPE monotherapy or combination therapy with bevacizumab or sorafenib, especially given more highly pretreated and ER- pts in S0430. If either becomes standard Rx with CAPE, we believe that a randomized trial comparing these to the SWOG regimen is warranted, as the toxicity and cost of CPA is lower. RIBBON-1 RIBBON-2 SOLTI S0430 CAPE combinator Placebo/bevacizumab Placebo/bevacizumab Placebo/sorafenib Cyclophosphamide # of pts 206/409 47/97 114/115 96 Prior regimens 0 100% E 54%/43% 54% 1 E 100% 45%/57% 32% 2 E E E 13% Receptors ER or PR + NR NR 68%/77% 59% ER- NR NR 29%/17% 41% HER2 + E E E 6% RECIST RR 23.6%/35.4% NR 30.7%/38.3% 36% (95% CI 26-48%) PFS (mo.) 5.7 /8.6 4.1/6.9 4.1/6.4 5.9 (95% CI 3.9-8.0) OS (mo.) 21.2/29.0 NR NR 19.6 (95% CI 13.5-21.9) Abbreviations: E, excluded; NR, not reported. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech, Roche Roche Genentech, Roche