Expression and therapeutic targeting of dopamine receptor-1 (D1R) in breast cancer.

Patients with advanced breast cancer often fail to respond to treatment, creating a need to develop novel biomarkers and effective therapeutics. Dopamine (DA) is a catecholamine that binds to five G protein-coupled receptors. We discovered expression of DA type-1 receptors (D1Rs) in breast cancer, thereby identifying these receptors as novel therapeutic targets in this disease. Strong to moderate immunoreactive D1R expression was found in 30% of 751 primary breast carcinomas, and was associated with larger tumors, higher tumor grades, node metastasis and shorter patient survival. DA and D1R agonists, signaling through the cGMP/protein kinase G (PKG) pathway, suppressed cell viability, inhibited invasion and induced apoptosis in multiple breast cancer cell lines. Fenoldopam, a peripheral D1R agonist that does not penetrate the brain, dramatically suppressed tumor growth in two mouse models with D1R-expressing xenografts by increasing both necrosis and apoptosis. D1R-expressing primary tumors and metastases in mice were detected by fluorescence imaging. In conclusion, D1R overexpression is associated with advanced breast cancer and poor prognosis. Activation of the D1R/cGMP/PKG pathway induces apoptosis in vitro and causes tumor shrinkage in vivo. Fenoldopam, which is FDA (Food and Drug Administration) approved to treat renal hypertension, could be repurposed as a novel therapeutic agent for patients with D1R-expressing tumors.

The Cost Effectiveness of Docetaxel and Active Symptom Control versus Active Symptom Control Alone for Refractory Oesophagogastric Adenocarcinoma: Economic Analysis of the COUGAR-02 Trial.

BACKGROUND: The COUGAR-02 trial recently showed survival and quality-of-life benefits of docetaxel and active symptom control (DXL + ASC) over active symptom control (ASC) alone in patients with refractory oesophagogastric adenocarcinoma. AIM: The aim of this study was to conduct an economic evaluation conforming to National Institute for Health and Care Excellence (NICE) technology appraisal guidance to evaluate the cost effectiveness of DXL + ASC versus ASC from the perspective of the English National Health Service (NHS). METHODS: Cost-utility analyses were conducted using trial data. Utility values were captured using the EQ-5D completed by patients at 3- and 6-weekly intervals, while resource use was captured using nurse-completed report forms and patient reports. Incremental cost-effectiveness ratios (ICERs) were calculated and the main outcome was cost per incremental quality-adjusted life-year (QALY). Nonparametric bootstrapping was conducted to capture sampling uncertainty and to generate a cost-effectiveness acceptability curve (CEAC). The analysis horizon was the trial period (median follow-up 12 months) and no modelling or discounting of future costs and benefits was conducted. RESULTS: Average costs were £9352 and £6218 for DXL + ASC and ASC, respectively, and average QALYs were 0.302 and 0.186, respectively. This yielded an ICER of £27,180 for DXL + ASC. DXL + ASC had a 24 % chance of being cost effective at a £20,000 QALY threshold (lambda) and a mean net monetary benefit of -£821; this rose to 59 % and £332 when the threshold was raised to £30,000. If NICE end-of-life criteria are applied, the probability of cost effectiveness increases to 90 % (at lambda = £50,000). Results were robust to sensitivity analyses. CONCLUSIONS: DXL + ASC is likely to be cost effective if an end-of-life premium is applied. Further research should determine the impact of different utility measurement strategies and different chemotherapy delivery modes on estimates of cost effectiveness.

Docetaxel versus active symptom control for refractory oesophagogastric adenocarcinoma (COUGAR-02): an open-label, phase 3 randomised controlled trial.

BACKGROUND: Second-line chemotherapy for patients with oesophagogastric adenocarcinoma refractory to platinum and fluoropyrimidines has not shown benefits in health-related quality of life (HRQoL). We assessed whether the addition of docetaxel to active symptom control alone can improve survival and HRQoL for patients. METHODS: For this open-labelled, multicentre trial, we recruited patients aged 18 years or older from 30 UK centres. Patients were eligible if they had an advanced, histologically confirmed adenocarcinoma of the oesophagus, oesophagogastric junction, or stomach that had progressed on or within 6 months of treatment with a platinum-fluoropyrimidine combination. Patients could have an Eastern Cooperative Oncology Group performance status of 0-2. We randomly assigned patients using a central, computerised minimisation procedure to receive docetaxel plus active symptom control, or active symptom control alone (1:1; stratified by disease status, disease site, duration of response to previous chemotherapy, and performance status). Docetaxel was given at a dose of 75 mg/m(2) by intravenous infusion every 3 weeks for up to six cycles. The primary endpoint was overall survival, analysed by intention to treat. This is the report of the planned final analysis. This study is an International Standardised Randomised Controlled Trial, number ISRCTN13366390. FINDINGS: Between April 21, 2008, and April 26, 2012, we recruited 168 patients, allocating 84 to each treatment group. After a median follow-up of 12 months [IQR 10-21]) and 161 (96%) deaths (80 in the docetaxel group, 81 in the active symptom control group), median overall survival in the docetaxel group was 5.2 months (95% CI 4.1-5.9) versus 3.6 months (3.3-4.4) in the active symptom control group (hazard ratio 0.67, 95% CI 0.49-0.92; p=0.01). Docetaxel was associated with higher incidence of grade 3-4 neutropenia (12 [15%] patients vs no patients), infection (15 [19%] patients vs two [3%] patients), and febrile neutropenia (six [7%] patients vs no patients). Patients receiving docetaxel reported less pain (p=0.0008) and less nausea and vomiting (p=0.02) and constipation (p=0.02). Global HRQoL was similar between the groups (p=0.53). Disease specific HRQoL measures also showed benefits for docetaxel in reducing dysphagia (p=0.02) and abdominal pain (p=0.01). INTERPRETATION: Our findings suggest that docetaxel can be recommended as an appropriate second-line treatment for patients with oesophagogastric adenocarcinoma that is refractory to treatment with platinum and fluoropyrimidine. FUNDING: Cancer Research UK.

The role of multi-disciplinary teams in decision-making for patients with recurrent malignant disease.

BACKGROUND: It is mandatory in many countries for decisions for all new patients with cancer to be made within multi-disciplinary teams (MDTs). Whether patients with disease recurrence should also routinely be discussed by the MDT is unknown. AIM: This study investigated the role of an upper gastro intestinal (UGI) MDT in decision-making for patients with disease recurrence. DESIGN: A retrospective review of prospectively kept MDT records (2010 to 2011) was performed identifying patients discussed with recurrence of oesophagogastric cancer. Information was recorded about: i) why an MDT referral was made, ii) who made the referral and iii) the final MDT recommendation. Implementation of the MDT recommendation was also examined. PARTICIPANTS: All patients discussed with recurrence of cancer at a central UGI cancer MDT were included. RESULTS: During the study 54 MDT meetings included discussions regarding 304 new patients and 29 with disease recurrence. Referrals to the MDT for patients with recurrence came from outpatient clinics (n=19, 65.5%) or following emergency admission (n=10). Most referrals were made by the surgical team (n=25, 86.2%). MDT recommendations were best supportive care (n=11, 37.9%), palliative chemotherapy (n=9, 31.0%), stent (n=5, 17.2%), palliative radiotherapy (n=3, 10.3%) and further surgery (n=1, 3.4%), with 25 (86.2%) of these implemented. CONCLUSION: UGI MDTs focus on new referrals and only a small proportion of patients with recurrent disease are re-discussed. Many patients go on to receive further treatments. Whether such patients are optimally managed within the standard MDT is uncertain, however, and warrants further consideration.

Gefitinib versus vinorelbine in chemotherapy-naive elderly patients with advanced non-small-cell lung cancer (INVITE): a randomized, phase II study.

PURPOSE: This phase II, open-label, parallel-group study compared gefitinib with vinorelbine in chemotherapy-naïve elderly patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Chemotherapy-naïve patients (age >or= 70 years) were randomly assigned to gefitinib (250 mg/d orally) or vinorelbine (30 mg/m(2) infusion on days 1 and 8 of a 21-day cycle). The primary end point was progression-free survival (PFS). Secondary end points were overall survival (OS), objective response rate (ORR), quality of life (QOL), pulmonary symptom improvement (PSI), and tolerability. Exploratory end points included epidermal growth factor receptor (EGFR) gene copy number by fluorescent in situ hybridization (FISH). RESULTS: Patients were randomly assigned to gefitinib (n = 97) or to vinorelbine (n = 99). Hazard ratios (HR; gefitinib v vinorelbine) were 1.19 (95% CI, 0.85 to 1.65) for PFS and 0.98 (95% CI, 0.66 to 1.47) for OS. ORR and disease control rates were 3.1% (95% CI, 0.6 to 8.8) and 43.3% (for gefitinib) and 5.1% (95% CI, 1.7 to 11.4) and 53.5% (for vinorelbine), respectively. Overall QOL improvement and PSI rates were 24.3% and 36.6% (for gefitinib) and 10.9% and 31.0% (for vinorelbine), respectively. In the 54 patients who were EGFR FISH-positive, HRs were 3.13 (95% CI, 1.45 to 6.76) for PFS and 2.88 (95% CI, 1.21 to 6.83) for OS. There were fewer treatment-related grade 3 to 5 adverse events with gefitinib (12.8%) than with vinorelbine (41.7%). CONCLUSION: There was no statistical difference between gefitinib and vinorelbine in efficacy in chemotherapy-naïve, unselected elderly patients with advanced NSCLC, but there was better tolerability with gefitinib. Individuals who were EGFR FISH-positive benefited more from vinorelbine than from gefitinib; this unexpected finding requires further study.

A phase II study of weekly docetaxel in patients with anthracycline pretreated metastatic breast cancer.

BACKGROUND: Docetaxel has significant activity in metastatic breast cancer and weekly schedules are associated with less myelosuppression than 3-weekly schedules. We evaluated the toxicity and the activity of weekly docetaxel in anthracycline-pretreated patients. PATIENTS AND METHODS: A total of 42 patients were studied. Treatment consisted of docetaxel 35 mg/m2 weekly as a 30-min infusion for 6 weeks followed by a 2-week rest, with dexamethasone 8 mg i.v. pre-medication and 4 mg orally 12-hourly for 48 h following treatment. RESULTS: The median age of the patients was 53 years (range 34-74). Twenty-six (62%) patients had received prior chemotherapy for advanced disease. Patients received a median 10 weeks of treatment (range 1-24). 11 had a partial response (ORR 26%; 95% CI 13-39%), five of whom had relapsed <12 months since the end of previous anthracycline-based chemotherapy. In addition six patients (14%) had stable disease for >16 weeks. Myelosuppression was rare with only 2 patients (5%) experiencing grade 3 neutropenia (no grade 4 neutropenia). Non-haematological grade III toxicities were as follows: fatigue 17%, neuropathy 0%, hyperlacrimation 5%, stomatitis 7%, diarrhoea 14%, and cutaneous toxicity 19%. Skin toxicity consisted of limb/palmar-plantar erythematous reactions, or fixed-plaque erythrodysaesthesia. CONCLUSIONS: Weekly docetaxel has moderate activity in women with anthracycline pre-treated breast cancer. Although the level of myelosuppression is lower than 3-weekly regimens, this weekly regimen cannot be recommended due to the significant non-haematological toxicities associated with the treatment.

The value of routine serum carcino-embryonic antigen measurement and computed tomography in the surveillance of patients after adjuvant chemotherapy for colorectal cancer.

PURPOSE: This analysis aims to evaluate routine carcino-embryonic antigen (CEA) and computed tomography (CT) of thorax, abdomen, and pelvis as part of protocol-specified follow-up policy for colorectal cancer (CRC). PATIENTS AND METHODS: Patients with resected stage II and III CRC were randomly assigned to bolus fluorouracil/leucovorin or protracted venous infusion fluorouracil. Following completion of chemotherapy, patients were seen in clinic at regular intervals for 5 years. CEA was measured at each clinic visit, and CT of thorax, abdomen, and pelvis was performed at 12 and 24 months after commencement of chemotherapy. RESULTS: Between 1993 and 1999, 530 patients were recruited. The median follow-up was 5.6 years. Disease relapses were observed in 154 patients. Relapses were detected by symptoms (n = 65), CEA (n = 45), CT (n = 49), and others (n = 9). Fourteen patients, whose relapses were detected by CT, had a concomitant elevation of CEA and were included in both groups. The CT-detected group had a better survival compared with the symptomatic group from the time of relapse (P =.0046). Thirty-three patients (21%) proceeded to potentially curative surgery for relapse and enjoyed a better survival than those who did not (P <.00001). For patients who underwent hepatic or pulmonary metastatic resection, 13 (26.5%) were in the CT group, eight (17.8%) in the CEA group, and only two (3.1%) in the symptomatic group (CT v symptomatic, P <.001; CEA v symptomatic, P =.015). CONCLUSION: Surveillance CT and CEA are valuable components of postoperative follow-up in stage II and III colorectal cancer.

Phase III study of mitomycin-C with protracted venous infusion or circadian-timed infusion of 5-fluorouracil in advanced colorectal carcinoma.

The combination of protracted venous infusion (PVI) fluorouracil (5-FU) and mitomycin-C has previously been shown to be superior to PVI 5-FU alone in terms of response rate and failure-free survival. This study explores the effect of dose intensification by circadian timing of 5-FU in this combination on response, toxicity, and survival. Patients with advanced colorectal carcinoma were randomized to receive PVI 5-FU 300 mg/m2 daily or circadian-timed infusion (CTI) of 5-FU, beginning at 600 mg/m2 and subsequently reduced to 450 mg/m2, delivered as a flat-rate infusion from 10:15 PM to 9:45 AM. Both groups received mitomycin-C at a dose of 7 mg/m2 given every 6 weeks. From April 1996 to August 1998, 320 patients were randomized, including 263 with metastatic disease and 21 with circumferential margin involvement. The overall response rate for the PVI 5-FU group was 38%, compared with 30.3% for the CTI group (P = 0.176). There was no statistically significant difference in terms of failure-free survival (8.0 months vs. 9.9 months; P = 0.131) or overall survival (15.8 months vs. 16.3 months; P = 0.275) between the treatment groups. There were no differences in global quality of life. Grade 3/4 diarrhea occurred significantly more frequently with CTI 5-FU (6.5% vs. 19.8%; P < 0.001); a nonsignificant trend toward increased incidences of grade 3/4 infection and palmar plantar erythema were observed with CTI 5-FU. This study confirms the high response rate and overall survival figures for the combination of PVI 5-FU and mitomycin-C in colorectal cancer. However, dose intensification of 5-FU using a circadian-timed, flat-rate infusion did not lead to improved response or survival.

Thymidylate synthase expression in advanced colorectal cancer predicts for response to raltitrexed.

PURPOSE: The purpose of this research was to evaluate the predictive value of expression of thymidylate synthase (TS) and other genes for response to raltitrexed (RTX). EXPERIMENTAL DESIGN: Twenty-five patients with metastatic colorectal cancer received RTX 3 mg/m(2) 3-weekly. Pretreatment tumor biopsies were analyzed for TS, dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), folylpolyglutamate synthetase, and reduced folate carrier mRNA expression by real-time reverse transcription-PCR. TS protein expression was evaluated by immunohistochemistry using a polyclonal TS antibody. RESULTS: Twenty patients were evaluable for response and gene expression. Six of 20 (30%) achieved a partial response. Median TS/beta-actin was 5.7 x 10(3) (range, 2.2-42 x 10(3)). Median TS/beta-actin was 3.7 x 10(3) in responding patients and 6.1 x 10(3) in nonresponders (P = 0.048). Five of 6 patients with TS/beta-actin

Patterns of elevation of plasma 2'-deoxyuridine, a surrogate marker of thymidylate synthase (TS) inhibition, after administration of two different schedules of 5-fluorouracil and the specific TS inhibitors raltitrexed (Tomudex) and ZD9331.

5-Fluorouracil (5-FU) exerts cytotoxic effects through inhibition of thymidylate synthase (TS) and incorporation of metabolites into RNA. TS inhibition may be greater for infusional 5-FU, with bolus regimens more likely to cause RNA effects. Elevation of plasma 2'-deoxyuridine (dUrd) is a surrogate marker of TS inhibition. Nineteen patients were treated with continuous infusion (CI) 5-FU 300mg/m(2)/day or bolus 5-FU 425mg/m(2)/day plus leucovorin (LV) 20mg/m(2)/day days 1-5. Pretreatment (day 1) and day 2, 3, 4, 5, 8, 15, 22, and 29 plasma samples were assayed for dUrd by reverse-phase high-performance liquid chromatography. In patients treated with bolus 5-FU/LV, dUrd elevation at 24 and 48 h was 235 +/- 125 and 254 +/- 119%, respectively, falling to 138 +/- 58%, 156 +/- 89%, and 92 +/- 25% on days 8, 15, and 22, respectively. dUrd elevation with CI 5-FU was 229 +/- 86% at 24 h and 239 +/- 86, 240 +/- 98%, and 255 +/- 109% at days 15, 22, and 29, respectively. Duration of dUrd elevation was generally less than 8 days for bolus 5-FU/LV. A single dose of raltitrexed (3 mg/m(2)) gave a similar profile to this regimen. ZD9331 (130 mg/m(2), days 1 and 8) gave dUrd elevation for 14 of 21 days, with some recovery prior to day 8. Thus, both 5-FU regimens inhibit TS, and prolonged TS inhibition is achieved by CI 5-FU without significant toxicity. This suggests that the mechanism of antiproliferative toxicity from bolus 5-FU/LV is partly non-TS mediated. These results clarify underlying pharmacodynamic processes and could guide scheduling of 5-FU and TS inhibitors.

S-adenosyl-L-methionine decarboxylase of Acanthamoeba castellanii (Neff): purification and properties.

S-Adenosyl-L-methionine decarboxylase (AdoMetDC) has been purified to near homogeneity from the Neff strain of Acanthamoeba castellanii. The holoenzyme molecular mass is 88.8 kDa, including two copies each of a 32.8 kDa alpha-subunit and a 10-15 kDa beta-subunit. The alpha-subunit contains the active site. It has an N-terminal pyruvoyl group, and the first 19 amino acids are 63 and 74% identical with comparable sequences from yeast and mammals, respectively. The apparent Km for S-adenosylmethionine (AdoMet) in the presence of 2 mM putrescine was 30.0 microM. The enzyme was stimulated 2-fold by putrescine, but was unaffected by spermidine. It was inhibited by the following anti-metabolites, listed with their Ki values: Berenil (0.17 microM), pentamidine (19.4 microM), propamidine (334 microM), hydroxystilbamidine (357 microM), methylglyoxal bis(guanylhydrazone) (604 microM) and ethidium bromide (1.3 mM). Activity of the enzyme fell to undetectable levels during cell differentiation (encystment).

Molecular aspects of the cell cycle and encystment of Acanthamoeba.

Evidence for subdivision of the cell cycle of Acanthamoeba into ultradian biochemical cycles is accumulating, and a linkage between these cycles and the length of the cell cycle is possible. The DNA replication cycle differs with the method of assay: no G1 phase is found in asynchronous cultures, and a long G1 phase is found in synchronous cultures. Encystment most likely occurs from G2, but whether it is limited to a portion of this phase is not clear. Encystment-enhancing factors are released by Acanthamoeba castellanii and Acanthamoeba palestinensis, and encystment can be induced by monoclonal antibodies to plasma membrane proteins. Likewise, encystment can be induced by inhibitors of polyamine synthesis, especially diamidines that inhibit S-adenosylmethionine decarboxylase, but inhibition of this enzyme is not necessarily responsible for differentiation. Studies on the regulation of gene expression during encystment have focused on actin and the ribosomal RNA transcriptional unit.

Quantitative enumeration of acanthamoeba for evaluation of cyst inactivation in contact lens care solutions.

A simple, quantitative plate assay has been developed for use in the enumeration of Acanthamoeba. The technique uses an agarose overlay and low-nutrient medium to support the growth of amoebae on a bacterial lawn. The authors found that in this assay, individual trophozoites or dormant cysts will cause plaques to form in an Enterobacter aerogenes lawn. With the assay, it is possible to quantitatively assess the effects of various disinfectant compounds on the viability of Acanthamoeba. The authors used this assay to enumerate Acanthamoeba cyst viability after chemical disinfection in contact lens care solutions. The inactivation data indicated major differences among the four test solutions evaluated.

Genes of Acanthamoeba: DNA, RNA and protein sequences (a review).

This review summarizes knowledge about the structure of nuclear genes and mitochondrial DNA in Acanthamoeba. The information about nuclear genes is derived from studies of DNA, RNA and protein sequences. The genes considered are those for 5S, 5.8S and 18S rRNA, actin I, profilins Ia/b and II, myosins IB, IC and II, and calmodulin. All of the sequences show strong similarities to comparable sequences from other organisms. Introns have been found in the actin and myosin genes. The location of the actin intron is unique, but many of the myosin introns occur at the same sites as introns in myosins of other organisms. Sequence comparisons, especially of 5S and 5.8S rRNA and actin, support previous evidence, based primarily on 18S rRNA, that Acanthamoeba genes are at least as closely related to those of higher plants and animals as they are to various other protistan genera. The functional organization of the promoter region for the nuclear rDNA transcription unit has been studied extensively, but there is a need for information about the functional organization of regulatory sequences for other genes. Restriction fragment length profile (RFLP) studies of mitochondrial DNA reveal relatively high levels of overall sequence diversity, but information on the structure and function of individual genes is needed. The RFLP appear to have potential as tools for taxonomic studies of this genus.