5FU Treatment Research Articles

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimising administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterised for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumours from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumours, CTPS2 was the gene with the highest probability of differential expression (p=0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analogue, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p=0.072), while those with high expression did (p=0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance. See commentary: Biomarkers of 5-FU response in colon cancer

Antitumor effect of Croatian propolis as a consequence of diverse sex-related dihydropyrimidine dehydrogenase (DPD) protein expression

The aim of this study was to detect the antitumor properties of Croatian propolis in BALB/c male and female mice injected with 4T1 mammary carcinoma. Furthermore, the gender-dependence of this effect and the possible involvement of combined effect of propolis and 5-Fluorouracil (5FU) on dihydropyrimidine dehydrogenase (DPD) transcriptional and translational level, were determined. In combination with 5FU propolis treatment induced gender-related effects. The results of the study revealed that pretreatment of mice with propolis combined with 5FU treatment prolonged the suppressive effect of 5FU on tumor growth and reduced the number of metastasis only in male mice. Only males pretreated with propolis prior to 5FU administration had decreased DPD protein level indicating higher sensitivity to 5FU. Thus, benefitial effects of propolis in male tumor-bearing mice treated with 5FU might be explained by increased sensitivity to 5FU as the result of translationally downregulated DPD.

The YAP Transcriptional Co-Activator Is Not Required for Mouse Hematopoiesis, at Steady State or After 5FU Treatment.

AbstractAbstract 1592The Yes-associated protein (YAP) is a transcriptional co-activator that interacts with many transcription factors, including RUNX2, p73, ERBB4, PEBP2α, p53BP2, SMAD7 and the TEAD/TEF proteins. Recently, it has been established that YAP is the major downstream molecule of the evolutionarily conserved Hippo signaling pathway. First elucidated in Drosophila, the Hippo signaling pathway controls organ size by regulating apoptosis and proliferation. Beginning a kinase cascade, the MST1/MST2 kinases (fly ortholog: Hippo) cooperate with the WW domain-containing SAV1 (fly ortholog: Salvador) to phosphorylate and activate the LATS1/LATS2 kinases (fly ortholog: Warts). In turn, activated LATS1/LATS2 phosphorylate and inactivate YAP (fly ortholog: Yorkie). Inactivated YAP binds to 14-3-3 and is kept in the cytosol. In contrast, unphosphorylated YAP binds to TEAD family transcription factors (fly ortholog: Scalloped) and the complex transactivates genes including cell cycle regulators (e.g. cyclin) and cell death inhibitors (e.g. IAPs, inhibitor of apoptosis). Therefore, inactivation of MST or LATS kinase, or overexpression of YAP results in organ/tissue overgrowth characterized by excessive cell proliferation and diminished apoptosis in both fly and mammals. In two transgenic mouse models, overexpression of YAP1 in liver results in reversible increase in liver size due to increased proliferation and decreased cell death of hepatocytes.Several pieces of evidence suggest that YAP regulates stem cell self-renewal and differentiation. First, transcriptional profiling of mouse stem cells (hematopoietic, neuronal and embryonic) identified both YAP1 and TEAD2 as 2 of the only 14 transcription factors commonly expressed in all 3 types of stem cell. Second, in many adult human tissues (e.g. intestine, lung, pancreas), YAP1 is preferentially expressed in stem-progenitor cell compartments. Third, in mouse intestine and chick neural tubes, overexpression of YAP results in expansion of intestinal or neural stem-progenitor cells, as well as inhibition of progenitor cell differentiation in vivo. Finally, it has been shown that YAP expression decreases during mouse ES cell differentiation. Ectopic expression of YAP can maintain ES cell pluripotency and prevent differentiation both in vitro and in vivo. Given that YAP has a proven role in regulating stem-progenitor cells in multiple tissue and multiple organisms, we set out to investigate whether YAP also regulate hematopoetic stem-progenitors in mouse and human.In conditional YAP knockout mice with a specific deletion of YAP expression in the hematopoetic system, we found no abnormalities at steady state in hemtaopoietic lineages, as assessed by CBC (complete blood counts) or immunophenotypic analysis. In addition, the YAP-null hematopietic stem-progenitors (HSPCs) had no change in vitro hematopoietic colony-forming cells. Furthermore, 5-fluorouracil treatment did not reveal a significant difference in blood cell numbers or types, between wild type and YAP-null mice. Although these results in mice suggest that YAP appears not to be required in hematopoiesis, overexpression of YAP, which promotes stem-progenitor cell proliferation in other tissues, may still provide an excellent opportunity to drive HSPC expansion. Disclosures:No relevant conflicts of interest to declare.

CDKN1C Modulates the Stress-Reponse of Hematopoietic Stem Cells Rendering Hematopoiesis Resistant to Chemotherapeutics

AbstractAbstract 3162The cyclin-dependent kinase inhibitor (CDKI) CDKN1C (p57) is a tumor suppressor gene with strong differential expression in both human and murine hematopoietic stem cells (HSC). Whereas the expression of other CDKIs is normal in most hematopoietic malignancies, p57 expression is silenced in 30 to 55% of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and B-Cell lymphoma patients suggesting a role as suppressor of hematopoietic cell transformation. p57 has a unique role in embryogenesis functioning in tissue-specific developmental programs to coordinate proliferation, differentiation and apoptosis. We have identified p57 as an early transcriptional target of the tumor suppressor TGFβ which is required for TGFβ-induced cytostasis of human CD34+ hematopoietic progenitor/stem cells; functions that provide a mechanistic basis for its expression being silenced in many aggressive human myeloid and lymphoid malignancies. To understand the role of p57 in HSC function, we used an engineered mouse strain deficient in p57. Using limiting-dilution HSC transplantation, we found that the fetal livers of p57-null mice have ∼4-fold fewer HSCs than their wild-type littermate controls. When we transplanted wt recipient mice with a mixture of p57-wt and p57-null fetal liver mononuclear cells (FLMC), we found that the p57-null hematopoietic cells were underrepresented in the blood of the recipient animals at steady-state. Both the limiting-dilution and competitive repopulation experiments indicate that the absence of p57 compromises HSC development. Strikingly, despite this quantitative deficit, we found that p57-null HSCs are qualitatively superior to p57-wt HSCs. Using serial transplantation, an assay of in vivo HSC self-renewal, we found that p57-null HSCs can be transplanted for two generations beyond which p57-wt HSCs are exhausted and unable to reconstitute hematopoiesis in the recipient animals. This result demonstrates under the tonic strain of serial transplantation, p57 normally serves to restrain HSC self-renewal. Importantly, mice with p57-null hematopoiesis are less sensitive to myelotoxic stress induced by the chemotherapeutics, 5-Fluorouracil (5FU) and cytarabine and have a shallower and shorter nadir following such treatment. Mice with p57-null hematopoiesis also recovered more briskly to the haemolytic agent phenylhydrazine suggesting that p57 serves a critical function restraining the stress-response of hematopoiesis. We next examined whether p57 might be involved in regulating the maintenance of LKSCD34- Flk2- long-term HSCs, LKSCD34+ Flk2- short-term HSCs, or LKSCD34+ Flk2+ MPPs (multipotent progenitors), LKSca-CD34+ FcRlow CMPs (common myeloid progenitors), LKSca- CD34+ FcR+ GMPs (granulocyte-monocyte progenitors) and LKSca- CD34- FcR- MEPs (megakaryocyte erythroid progenitors). Unlike in steady-state where the amount of cells in each subset in p57-null and wt bone marrow is comparable, we found significant variations in the p57-null bone marrow reconstitution after 5FU treatment. Furthermore when we administered 5FU to mice transplanted with mixtures of p57-null and p57-wt HSCs, we found that p57-null hematopoiesis contributed disproportionately to hematopoietic recovery and that this enhanced competition was durable. Our findings reveal a novel role of p57 to restrain HSC self-renewal during periods of hematopoietic stress. Deletion of p57 decelerates hematopoietic cells exhaustion due to serial transplantation and improves long-term engraftment, largely because of increased self-renewing divisions of HSCs in vivo. These results suggest that cells deficient in p57 have an advantage over p57-expressing cells only after hematopoietic stress suggesting that silenced p57 expression can serve to promote malignant hematopoiesis at the expense of normal hematopoiesis and contribute to chemotherapy resistance. Disclosures:No relevant conflicts of interest to declare.

Changes in 2′-deoxycytidine levels in various tissues of tumor-bearing mice

The nucleoside 2'-deoxycytidine (dCyd) increases in the plasma of cancer patients with poor prognoses. 5-fluorouracil (5FU) is one of the anti-cancer agents used in chemotherapy for patients whose plasma dCyd is elevated. We examined the free dCyd level in various tissues of mice, with and without tumors, and in mice with and without the administration of 5FU or of dCyd, and investigated the effects of dCyd in tumor-bearing animals. SP2/0-Ag14 mouse myeloma cells were transplanted subcutaneously into mice and 5FU or dCyd was administered intraperitoneally. Free dCyd was measured in blood and tissues by HPLC at two time points, once when mouse body weight was maximally decreased (1 day after the last administration of 5FU, day 16) and again when it returned to control level at 1 week after the last 5FU treatment (day 22). Results showed that in tumor-bearing mice, the level of dCyd (per g wet weight) increased in the spleen. The change in liver weight caused by the administration of 5FU correlated with the level of dCyd in the liver. Notably, the relative tumor volume and tumor weight was decreased in dCyd-treated animals in comparison with controls. In conclusion, the levels of dCyd were markedly altered in the tissues of the reticuloendothelial and lymphatic systems, such as liver and spleen, and dCyd apparently had the ability to inhibit tumor growth in the body.

Can localised 19F magnetic resonance spectroscopy pharmacokinetics of 5FU in colorectal metastases predict clinical response?

Background5-Fluorouracil remains widely used in colorectal cancer treatment more than 40 years after its development. 19F magnetic resonance spectroscopy can be used in vivo to measure 5FU’s half-life and metabolism to cytotoxic fluoronucleotides. Previous studies have shown better survival associated with longer 5FU tumour half-life. This work investigated 5FU pharmacokinetics in liver metastases of colorectal cancer.MethodsA total of 32 subjects with colorectal cancer undergoing 5FU treatment, 15 of whom had liver metastases, were examined in a 1.5T MRI scanner, using a large coil positioned over the liver. Non-localised spectra were acquired in 1-min blocks for 32 min after injection of a 5FU bolus. The 5FU half-life was measured in each subject, and averaged spectra were examined for the presence of fluoronucleotides. Associations with progression-free survival were assessed.ResultsNo association was observed between 5FU half-life, tumour burden and survival. Half-lives were all shorter than those associated with improved survival in the literature. Remarkably, in the group with liver metastases, high levels of fluoronucleotides were associated with poorer survival; this counterintuitive result may be due to the higher levels of fluoronucleotides (whose level is higher in tumour tissue than in normal liver) in patients with higher tumour burdens.ConclusionsIt is recommended that future studies use chemical shift imaging at higher field strengths to better resolve tumour from normal liver. Non-localised spectroscopy retains prognostic potential by enabling straightforward detection of fluoronucleotides, which are present at very low concentrations distributed throughout the tissue.

FGFR1 Is Required for and Elucidates Multiple Mechanisms of HSC Mobilization.

Abstract 2532Poster Board II-509Though fibroblast growth factor receptor 1 (FGFR1) is known to be expressed in hematopoietic stem cells (HSCs), and FGF ligands can support their in vitro culture, the function of FGFR1 in HSCs in vivo has not been elucidated. We examined mouse models in which Fgfr1 is conditionally deleted in adult hematopoietic tissues. While they exhibit normal hematopoiesis, FGFR1-null HSCs fail to mobilize to peripheral tissues in response to 5-fluorouracil (5FU) and AMD3100; however, they mobilize at levels similar to control animals when treated with granulocyte-colony stimulating factor (G-CSF). Further investigation revealed that FGFR1-null HSCs cannot migrate in response to the CXCR4 ligand SDF-1 in vitro, and after 5FU treatment they display diminished expression of CXCR4 in bone marrow at time points prior to mobilization. Additionally, HSCs lacking FGFR1 cannot be expanded in culture, indicating they bear proliferation defects. Together, these data support a novel role for FGFR1 in mobilization of HSCs and indicate that activation of FGF signaling may distinguish stress-responsive HSC mobilization from more physiologic mechanisms triggered by cytokines such as G-CSF. Disclosures:No relevant conflicts of interest to declare.

Induction of Cytoplasmic Accumulation of p53: A Mechanism for Low Levels of Arsenic Exposure to Predispose Cells for Malignant Transformation

Although epidemiologic studies have linked arsenic exposure to the development of human cancer, the mechanisms underlying the tumorigenic role of arsenic remain largely undefined. We report here that treatment of cells with sodium arsenite at the concentrations close to environmental exposure is associated with the up-regulation of Hdm2 and the accumulation of p53 in the cytoplasm. Through the mitogen-activated protein kinase pathway, arsenite stimulates the P2 promoter-mediated expression of Hdm2, which then promotes p53 nuclear export. As a consequence, the p53 response to genotoxic stress is compromised, as evidenced by the impaired p53 activation and apoptosis in response to UV irradiation or 5FU treatment. The ability of arsenite to impede p53 activation is further demonstrated by a significantly blunted p53-dependent tissue response to 5FU treatment when mice were fed with arsenite-containing water. Together, our data suggests that arsenic compounds predispose cells to malignant transformation by up-regulation of Hdm2 and subsequent p53 inactivation.

Is surgical debridement and topical 5 fluorouracil the optimum treatment for woodworkers’ adenocarcinoma of the ethmoid sinuses? A case‐controlled study of a 20‐year experience

Woodworkers' adenocarcinoma of the ethmoid sinuses is an extremely rare occupational malignancy with a locally aggressive course. Treatment with surgical exenteration and topical 5 fluorouracil (5FU) packs has become standard treatment over the last 10 years in this ENT unit. This study presents level 2 evidence that 5FU provides improved survival over previous management with primary radiotherapy and salvage craniofacial resection. Retrospective case series with historical control group. District General Hospital. The records of 31 consecutive patients with the disease were analysed. Five patients died prior to treatment. One patient was treated with surgery alone and therefore excluded. Twenty-five patients were included in the analysis. Fourteen were treated with primary radiotherapy and 11 with surgery and topical 5FU. Disease free survival was measured using Kaplan-Meier survival analysis. Five-year disease free survival improved from 50% with primary radiotherapy to 86% with surgery and 5FU. This improvement is statistically significant (P = 0.03). Topical 5FU treatment improves survival of Woodworker's adenocarcinoma of the ethmoid sinuses. This finding may be useful in the treatment of other locally aggressive sinonasal malignancies.

Induction of cell cycle arrest and apoptosis by 5-fluorouracil in laryngeal cancer cells containing HPV16 E6 and E7 oncoproteins

ObjectiveTo investigate the cytotoxic effect of 5-fluorouracil (5Fu) on HPV16-associated laryngeal cancer cells. Design and methodsCytotoxicity assay and TUNEL assay were used to assess the effect of 5Fu on laryngeal cancer cells transfected with HPV16 E6 or E7. Results5Fu induced apoptosis in the cells either with or without HPV16 in a dose- and time-dependent manner. 5Fu caused the accumulation of active pRb and p21WAF1/CIP1, together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu. 5Fu also presented some effects on the E6 and E7 oncoproteins. ConclusionsHPV16 E6 and E7 oncoproteins do not prevent 5Fu-medicated apoptosis and G1/S cell arrest in laryngeal cancers. The sensitivity of 5Fu treatment is associated with the decrease of Bcl-2 and/or the increase in Bak and p21WAFI/CIP1.

The P47 GTPase Lrg-47 Links Host Defense and HSC Proliferation.

Hematopoietic stem cells (HSCs) are largely quiescent, self-renewing cells that give rise to all adult blood lineages. They have a remarkable capacity to respond to proliferative stimuli from a variety of insults, exiting their quiescent phase and undergoing a period of self-renewal and differentiation in order to restore hematopoietic homeostasis. Yet, while some molecular controls on the cycle of HSC activation and quiescence have been elucidated, little is known about the mechanisms linking this process to natural signals, such as the stress of an immune response to chronic infection. Previous work in our laboratory examined gene expression changes in a cycle of activation, expansion, and return to quiescence in HSCs stimulated by the chemotherapeutic agent 5-fluorouracil (5FU). One family of genes identified by this approach encodes the interferon-inducible p47 GTPases, which has been previously characterized solely in the context of the immune response to intracellular infections. Interestingly, mice deficient for one member of this family, Lrg-47, had been observed to develop a major hematopoietic defect in the face of infectious challenge. This combination of findings led us to test the novel hypothesis that Lrg-47 might regulate HSC function in the face of either chemical or pathogenic stress. We found that, while Lrg-47 −/ − mice have largely normal blood counts under homeostasis, these animals exhibited impaired hematopoietic recovery from stress in the form of sublethal irradiation or 5FU treatment. Furthermore, we show that HSCs deficient in Lrg-47 are profoundly compromised in functional repopulation assays, even when given extreme advantages over competitor marrow. We also observed, in BrdU labeling studies, that Lrg-47 −/ − HSCs display dysregulated HSC proliferation, with substantially increased turnover at baseline. We found that this inappropriate proliferation has a functional consequence, as Lrg-47 −/ − mice have a delayed expansion of the HSC compartment following 5FU treatment. Finally, in order to investigate the response of stem and progenitor populations to infectious stimuli, we challenged mice by infection with Mycobacterium avium. Phenotypic and functional assays demonstrated that, while wild-type progenitors expand dramatically in response to infection, Lrg-47 −/ − mice show a stark defect. Our findings thus point to a dual role for Lrg-47 - first as an inhibitor of HSC proliferation in the steady state, and second as a key regulator of HSCs in the response to injury. Overall, our results establish a link between the response to infection and HSC activation, and demonstrate a novel function for a member of the p47GTPase family. Additionally, as Lrg-47 has been chiefly known as an effector of IFNgamma signalling, our results suggest a possible mechanism for the inhibitory effects of this cytokine on hematopoiesis, with implications for human diseases with interferon-related pathogenesis. Most generally, our findings reveal an unexpected intersection point between the fields of stem cell biology and the immune response to pathogens, and suggest that defects in HSC function should be considered as possible determinants of host susceptibility to infection.

N-cadherin Expression Level Distinguishes Reserved Versus Primed States of Hematopoietic Stem Cells.

Within the bone marrow, three hematopoietic stem cell niches have been identified; the osteoblastic niche (Arai et al., 2004; Calvi et al., 2003; Nilsson et al., 2001; Zhang et al., 2003), the vascular niche (Kiel et al., 2005), and the CAR cell niche (Sugiyama et al., 2006). The adhesion molecule N-cadherin has been found associated with the osteoblastic and CAR cell niches, implicating N-cadherin's function for hematopoietic stem cell (HSC) anchoring and signaling (Arai et al., 2004; Muguruma et al., 2006; Zhang et al., 2003). However, as of yet, a HSC population expressing N-cadherin has not been fully characterized. Therefore, we examined how N-cadherin expression in HSCs relates to their function and lifecycle. Unexpectedly, we found that doses of 5000 bone marrow cells expressing the highest level of N-cadherin (N-cadherinhi) failed to reconstitute hematopoietic lineages in irradiated recipient mice. An explanation for this engraftment failure came with detailed cell surface phenotyping which revealed that these N-cadherinhi cells were primarily Lineage+ and devoid of the characteristic hematopoietic stem pool, Lineage-Sca+cKit+ (LSK) cells. Instead, we found that Flk2-LSK HSCs express a gradient of N-cadherin which could be described as low (N-cadherinlo) to intermediate (N-cadherinint) levels (Figure1).FACS applications were used to isolate pure populations of these N-cadherinlo and N-cadherinint Flk2-LSK HSCs. Real time RT-PCR (N-cadherin primers crossed the intron between Exon 2 and 3).(Figure 2), microarray studies, and competitive reconstitution transplantation assays revealed that this N-cadherin division of Flk2- LSK HSCs formed two populations with very distinct properties. In transplantation assays the N-cadherinlo population more robustly reconstituted the hematopoietic system. Principle Component Analysis and gene ontology analysis of microarray data revealed that the N-cadherinlo cells expressed genes that may prime them to respond to signals and to mobilize. This data was confirmed with mobilization studies which showed that HSCs mobilized from bone marrow to spleen were predominantly N-cadherinlo. In contrast, the expression profile of N-cadherinint cells suggests they are more ‘reserved' and this population was also maintained with HSCs spared by 5-fluouracil (5FU) treatment. Our results suggest that differential N-cadherin expression reflects important functional distinctions between HSC subpopulations. N-cadherinlo HSCs, with their robust reconstitution efficiency and properties similar to mobilized HSCs, may have clinical relevance. [Display omitted] [Display omitted]

Cardiotoxicity of fluoropyrimidines in different schedules of administration: a prospective study

Cardiotoxicity associated with 5-Fluorouracil (5FU) administration has been infrequently reported in literature, albeit various series of acute coronary syndromes have recorded a low but definite incidence of the above toxicity. In the present study, patients undergoing 5FU-based and oral capecitabine (Xeloda-based chemotherapy were tested for the potential development of cardiac-related symptoms during their administration. Six hundred and forty-four patients entered the study. Those experiencing any cardiac-related symptoms during 5FU infusion or oral capecitabine were subjected to ECG and serum cardiac enzymes determination. If cardiotoxicity was confirmed, 5FU infusion or oral capecitabine were interrupted, sublingual nitrates administered and cardiac monitoring initiated, while patients with >two-fold enzyme elevation were followed in a coronary care unit for at least 72 h. Cases with acute myocardial infarction were excluded from further 5FU or oral capecitabine treatment. Overall 26 patients (4.03%) developed symptoms and/or ECG abnormalities due to 5FU and capecitabine. Patients with continuous 5FU infusion presented a higher incidence of cardiotoxicity [14/209; 6.7%, 95% confidence interval (CI) = 3.3-10.1%] than the remaining (7/317; 2.3%, 95% CI = 0.8-3.3%) (P < 0.012). Specifically, an increased incidence of cardiac-related events was encountered in patients with continuous 24-h 5FU + LV infusion for 5 days (12.5%, 95% CI = 2.3-22.7%) rather than in patients with the same schedule without LV (5.3%, 95% CI = 1.95-8.67%) (P < 0.027), as well as in patients with short 5FU + LV administration (2.4%, 95% CI = 0.9-3.9%) (P < 0.019). Overall, 3/54 patients (5.5%, 95% CI = -0.6-11.1%) on oral capecitabine developed cardiac-related events. Seven out of the 20 patients suffered an acute myocardial infarction, 6 developed ischemia only, while 4 more patients had ECG consistent with coronary vasospasm and 3 with conduction disturbances, of which one subsequently died. Patients administered oral capecitabine had a similar incidence of cardiac-related events; 1/22 (4.5%) patients with advanced breast cancer and 2/32 (6.2%) with colorectal cancer. The present study supports the toxic effect of 5-FU on the myocardium, which is largely schedule-dependent, whereas a low but finite risk of such toxicity has been observed with oral capecitabine. A high level of alertness is required when using fluoropyrimidines (i.v. 5FU or oral capecitabine), while their toxic effect on the coronary endothelium and myocardium merits further investigation.

Phase II trial of continuous/metronomic 5-fluorouracil infusion plus long acting Octreotide in advanced neuroendocrine carcinoma

15003 Background: The administration of cytotoxic drugs on a metronomic schedule has shown antiangiogenetic properties. Well differentiated neuroendocrine carcinomas are notoriously highly vascularized and may be responsive to metronomic chemotherapy. A phase II study was designed to test the activity of protracted 5-fluorouracil (5FU) infusion in association with and long-acting release (LAR) octreotide in patients with advanced/metastatic neuroendocrine carcinoma. Patients and Methods: Twenty-nine patients (pts), with metastatic or locally advanced well differentiated neuroendocrine carcinoma not amenable to surgery with radical intent and not previously submitted to systemic treatments were enrolled. Treatment consisted of 5FU intravenous continuous infusion (200 mg/m2 daily) in association with Octreotide LAR (20 mg once every 4 weeks). 5FU treatment was administered until progression or for a maximum of 9 months. Octreotide LAR was administered until progression. Patients were followed for toxicity, objective response, symptomatic response, biochemical response, time to progression and survival. Results: According to RECIST criteria a partial response was observed in 7 patients (24.1%), a stable disease in 20 pts (69.0%) and a progression disease in only 2 pts (6.9%). A biochemical (chromogranin A) response was observed in 11/25 assessable patients (44.0%), 9/15 symptomatic patients (60.0%) obtained symptom relief. Post-treatment surgery was attempted in three patients, one of them was radically resected. The median time to disease progression was 25 months (range 2–69 months), median overall survival was not reached. Hematologic toxicity was rare and mild [grade 2 neutropenia in 2 pts (6.9%)]. Six cases (20.7%) suffered from grade 2–3 hand-foot syndrome. No patient withdrew from the study because of adverse events. Conclusions: Continuous/metronomic 5FU infusion plus octreotide LAR is well tolerated and shows promising activity in patients with advanced well differentiated neuroendocrine carcinoma. Additional studies with this combination are warranted. No significant financial relationships to disclose.

Should DPD analysis be required prior to prescribing fluoropyrimidines?

Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the metabolic catabolism of chemotherapeutic agent 5-fluorouracil (5FU) and its derivatives, including capecitabine. Numerous genetic mutations have been identified in the DPD gene locus ( DPYD), with a few key variants having functional consequences on enzymatic activity. Deficiencies in DPD activity have been shown to cause 5FU-treated cancer patients to experience severe drug-related toxicities, often requiring extensive medical intervention. We review the performance of assays that assess DPD and DPYD status, with an emphasis on the robustness for routine clinical applications. None of the current strategies are adequate to mandate routine DPD testing prior to starting a fluoropyrimidine-based therapy. However, further research and technological improvements will hopefully allow prospective identification of potentially toxic patients, in order to reduce the number of patients with severe, life-threatening side effects to 5FU treatment.

Monitoring chemotherapeutic response in RIF-1 tumors by single-quantum and triple-quantum-filtered 23Na MRI, 1H diffusion-weighted MRI and PET imaging

The effects of 5-fluorouracil (5FU, 150 mg/kg, ip) on subcutaneously implanted radiation-induced fibrosarcoma (RIF-1) tumors were monitored by in vivo (1)H MRI to evaluate the water apparent diffusion coefficient (ADC), by single-quantum (SQ) and triple-quantum-filtered (TQF) (23)Na MRI to evaluate compartmental Na(+) content and by positron emission tomography (PET) to evaluate 2-[(18)F]fluoro-2-deoxy-d-glucose (FDG) uptake in the tumor. The MRI experiments were performed on untreated control and treated mice once before and then daily for 3 days after treatment. The PET experiments were performed on separate groups of age- and tumor-volume-matched animals once before and then 3 days after treatment. Tumor volumes significantly decreased in treated animals 2 and 3 days posttreatment. At the same time points, in vivo MRI measurements showed an increase in both total tissue SQ (23)Na signal intensity (SI) and water ADC in treated tumors while control tumors showed no change in these parameters. TQF (23)Na SI and FDG uptake were significantly lower in treated tumors compared with control tumors 3 days after 5FU treatment. The correlated increases in total tissue (23)Na SI and water ADC following chemotherapy reflect an increase in extracellular space, while the lower TQF (23)Na SI and FDG uptake in treated tumors compared with control tumors suggest a shift in tumor metabolism from glycolysis to oxidation and/or a decrease in cell density.

Is thymidylate synthase a reliable predictor for response and survival during hepatic arterial infusion for hepatic metastases from colorectal cancer?

Sir, Decreased levels of the target enzyme thymidylate synthase (TS) have been repeatedly associated with superior clinical outcome in gastrointestinal cancers, including stomach cancer. In the study by Goekkurt et al (2006), patients who possessed a TS 5′ genotype associated with low TS mRNA expression levels showed a trend for superior survival time compared with those having TS genotypes associated with high TS mRNA expression. A significant association between clinical outcome to 5-fluorouracil (5FU)-based chemotherapy in colorectal cancer and TS polymorphisms was demonstrated when both TS polymorphisms within the 5′ untranslated (UTR) region were contemporarily analysed (Marcuello et al, 2004). TS gene amplification and overexpression can lead to resistance to TS-targeting chemotherapeutic drugs (5FU and fluorodeoxyuridine (FUDR)) (Copur et al, 1995; Davies et al, 1999; Wu and Dolnick, 2003). Moreover, by hepatic arterial infusion (HAI) it is possible to reach lower TS mRNA and higher ribonucleotide reductase activity than with systemic chemotherapy (SC) (Kubota et al, 2002). HAI therapy could generate higher intracellular levels of 5FU and FUDR metabolite, fluorodeoxyuridylate, locally (Gonen et al, 2003a). Among 135 patients randomly assigned to receive HAI vs systemic bolus 5FU and leucovorin, overall survival was significantly longer for HAI vs systemic treatment (P=0.003), as was time to hepatic progression (P=0.03) (Kemeny et al, 2006). By contrast, time to extrahepatic progression was significantly shorter in the HAI group (P=0.02). In a previous study, Gonen et al (2003b) reported that patients with resectable TS overexpressing (TS+) liver metastases from colorectal cancer have better overall survival (OS) when treated by HAI plus SC rather than by SC alone. On the contrary, patients with TS-negative metastatic colorectal cancer treated with SC plus HAI had similar OS compared with SC alone. More interestingly, in the study by Kemeny et al (2006), liver biopsies in the HAI group, although based on small numbers (40 patients), demonstrated that for patients with TS levels in tumour ⩾4 the median OS was 14 months, while for those with levels less than 4, median OS was 24 months. As previously reported (Alimonti et al, 2003), we think that, regarding patients treated with SC, it could be interesting to ascertain the role of the thymidine phosporylase (TP), the first enzyme involved in the metabolic activation pathway of FU to fluorodeoxyribonucleotides. Conversely, continuous HAI of FUDR generates FUDR monophosphate via uptake and activation by thymidine kinase and, unlike i.v bolus 5FU treatment, levels of TP would not likely be of predictive value. Preoperative biopsies and resection specimens from patients with stage II/III rectal carcinoma receiving neoadjuvant 5FU-based chemoradiotherapy have been recently studied for TS and TP protein expression by immunohistochemistry, and results have been compared with histopathologic tumour regression. A significant association was observed between high TS expression in tumour biopsies as well as resection specimens and nonresponse of the tumour to therapy (P=0.04), but low TP expression in the resection specimens was significantly associated with lack of response (P=0.02) (Jakob et al, 2005). However, it is still controversial whether TP expression in tumour is an independent factor that adds to TS positivity in worsening the prognosis of patients with metastatic colorectal cancer. Interindividual variation in the activity of metabolising enzymes can affect the extent of pyrimidine prodrug activation, acting on the efficacy of chemotherapy treatment (Maring et al, 2005). Patients with low levels of TP could be unable to properly metabolise FU administered by i.v. systemic infusion. A repeat polymorphism within the 5′ UTR, that alters TS expression, has been correlated with response and survival in colorectal cancer patients receiving 5FU in several studies (Iacopetta et al, 2001). Thus, the role of germline polymorphisms (uridine monophosphate kinase (UMPK), orotate phosphoribosyl transferase (OPRT), TS, dihydropyrimidine dehydrogenase (DPD), and methylene tetrahydrofolate reductase (MTHFR)), tumour-specific somatic mutations and gene/protein expression levels (OPRT, UMPK, TS, DPD, uridine phosphorylase, uridine kinase, TP, thymidine kinase, deoxyuridine triphosphate nucleotide hydrolase) must be taken into account in explaining variation in anti-tumour efficacy and toxicity of 5F.

Genomic Copy Number Changes Affecting the Thymidylate Synthase (TYMS) Gene in Cancer: A Model for Patient Classification to Aid Fluoropyrimidine Therapy

Thymidylate synthase (TS) is an important target for 5-fluorouracil (5FU)-based therapy. The TS polymorphic 5'-untranslated region tandem repeat sequence is under investigation to guide 5FU treatment, yet current protocols omit consideration of copy number changes at the TS locus. We surveyed the TS tandem repeat sequence and found copy number changes in gastrointestinal cancers. Ten of 12 informative cases had loss of heterozygosity (LOH), whereas two others and an additional cell line had a novel TS genotype, allelic imbalance at the TS locus due to polysomy. Experimentally, we studied a diploid colorectal cancer line heterozygous at TS to mimic three common TS genotypes of cancers. Using genetic engineering, we deleted the short tandem repeat (two repeats) allele and retained the long (three repeats) allele to produce artificial LOH at the TS gene; the TS(+/-) line had a reduced TS protein expression and was hypersensitive to 5FU and 5-fluoro-2'-deoxyuridine in vitro as compared with syngeneic control lines. We linked this sensitivity directly to the reduced TS expression by introducing exogenous TS cDNA expression into the TS(+/-) line (i.e., increased TS copies). Our model predicts that the 5FU sensitivity of a tumor is modified by aneuploidy producing copy number changes of TS alleles by one or more of the following: LOH, amplification, and, as presented here, copy number changes due to polysomy. The data suggest that TS copy number in a patient's tumor may be a dominating variable affecting 5FU responsiveness.

5-Fluorouracil (5FU) treatment does not influence invasion and metastasis in microsatellite unstable (MSI-H) colorectal cancer

Microsatellite instability is a recognised pathway of colorectal carcinogenesis responsible for about 15% of all sporadic colorectal cancers. Recent evidence has suggested that these tumours may not have the same response as microsatellite stable colon cancers to 5-fluorouracil (5FU)-based chemotherapy. The response to 5FU in four microsatellite unstable (MSI-H) cell lines was examined by cell viability assays and invasion assays. Flow cytometry was used to assess the effect of 5FU on MSI-H cell lines. In vivo response to 5FU was assessed by intraperitoneal injection of 5FU or control to 80 nude mice that had received intrasplenic injections of an MSI-H cell line KM12C prior to commencing treatment. There was inhibition of cell growth in MSI-H cell lines when treated with 5FU. There was no difference in invasiveness in the MSI-H cell lines when treated with 5FU. Primary tumours formed in 27 of the untreated and 25 of the 5FU treated mice (p=NS). There was a 36% reduction in splenic weight in those mice treated with 5FU (p<0.03). Metastases formed in 5 of the untreated and 9 of the treated mice (p=0.12). 5FU treatment of MSI-H tumours results in a reduction in growth but does not result in a reduction in invasion or metastasis.

Methyl-Group Metabolism and the Response of Colorectal Cancer to 5-Fluorouracil

The major mechanism of action of 5-fluorouracil (5FU)-based therapies is thought to be inhibition of thymidylate synthase (TS). This enzyme catalyzes synthesis of the thymidine nucleotide precursor using a methyl group provided by a folate cofactor. In addition to TS activity levels, various elements of methyl-group metabolism could also be predictive for the response of colorectal cancer (CRC) to 5FU. These include the activity of enzymes involved in folate metabolism, the concentrations of intracellular folate intermediates, and surrogate markers of aberrant methyl-group metabolism, such as DNA methylation and microsatellite instability. The factors of age, gender, common genetic variants, and diet have been shown to influence both systemic and tumor methyl-group metabolism. This has important implications for the prediction of toxicity and response to 5FU, respectively. Identification of predictive factors within the methyl-group metabolism pathway should assist in targeting 5FU treatment to the most responsive CRC patient groups. This is particularly important for early-stage disease where conclusive demonstration of a survival benefit from 5FU in the overall CRC group has thus far proven difficult.