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To understand current practice of the treatment of advanced bladder cancer in the United Kingdom, and in particular, the use of second-line chemotherapy.To gain insight into uro-oncologists' use of first-line chemotherapy, imaging following first-line chemotherapy, use of second-line chemotherapy, and the role of the multidisciplinary tumor board (MTB) in making decisions about second-line chemotherapy.From August 2011 to September 2011 uro-oncologists from UK cancer centers were surveyed regarding treatment of advanced bladder cancer.Delegates at the British Uro-oncology Group conference were invited to fill out an electronic survey.Uro-oncologists from 28 of 42 UK cancer centers (67%) participated, who treated, on average 45 patients per year with advanced bladder cancer.Fifteen "always" reimage after first-line chemotherapy, mostly "after 2-4 cycles." Symptomatic patients with progressive distant disease on imaging were most likely to trigger second-line chemotherapy (P = 0.004).Twenty-one respondents would interrupt first-line chemotherapy to start second-line chemotherapy for progressive disease and 10 would never do this. Of the patients given first-line chemotherapy, 19% go on to receive second-line chemotherapy.Seven different regimes were specified for second-line chemotherapy with no clear preference. National Institute for Health and Clinical Excellence approval, trial data, inclusion in hospital formulary, clinical trials, or commissioning guidelines, and easy access to imaging help to access second-line chemotherapy (P ≤ 0.001). Constraints to second-line chemotherapy were lack of evidence and patient comorbidities.MTB effectiveness did not improve access to second-line chemotherapy. Of the 33 respondents, 19 do not rediscuss patients at the MTB before starting second-line chemotherapy.The investigation and treatment of patients with advanced bladder cancer following first-line chemotherapy is variable.Optimizing the modality and frequency of imaging and increasing the usefulness of the MTB process may improve care.

The gene encoding the chicken chemokine 9E3/CEF4 was cloned, sequenced, and mapped; 9E3/CEF4 was the first nonmammalian cytokine cDNA to be cloned and has significant amino acid identity with both human IL8 and human GROalpha. These results show that this cytokine is chicken IL8 and not GROalpha. The exon:intron structure of chicken IL8 corresponds almost exactly to that of human IL8 and differs from those of other known mammalian CXC chemokine genes. Analysis of the predicted amino acid sequence suggests that overall protein structure is conserved between human and chicken IL8, but that the receptor binding sites are not. Genetic distance analysis also suggests that this gene encodes chicken IL8. A number of potential regulatory sequences similar to those found in human IL8 have been identified in the promoter. These include (5'-3') a hepatocyte NF-1 binding site, an NF-kappaB binding site, and a TATAAA box. The human AP-1 binding site and CCAT box are poorly conserved in the promoter of the chicken gene, but there are other potential AP-1 binding sites and a potential CCAT box. The human IRF-1 and octamer binding sites seem to be absent. However, the chicken gene promoter contains a GATA motif not present in the promoter of human IL8. Sequence comparisons also identify conserved regions in the promoter that may function as transcription factor binding sites as yet undescribed in the human IL8 promoter. Promoter sequence polymorphisms have been identified in chicken lines C and 61, but neither lie in any of the regulatory regions mentioned above. Chicken IL8 contains nine repeats of the "instability" motif ATTTA in the 3' untranslated region (UTR) in exon 4. A multiple restriction single-stranded conformational polymorphism was identified which enabled chicken IL8 to be genetically mapped to Chromosome (Chr) 4, linked to SPP1 and ALB1, and thus showing conserved synteny with mouse Chr 5 and human Chr 4. This is the first nonmammalian chemokine gene to be genetically mapped.

EDITOR,—I am writing about the BMJ's coverage of the health policies that the two opposition parties launched at their annual conferences.1 Although I announced several concrete and worthy policies at the Liberal Democrats' conference, John Warden chose to mention only our £350m extra from tax perks and gave many more details of the Labour party's policy than of ours, which seems perverse, given the respective detail of our two policies. I would like to remind readers that the Liberal Democrats are also committed …

The online version of the original article can be found at http://dx.doi.org/10.3758/s13428-012-0214-0 .

Born and educated in Lancashire, where he excelled at rugby football, Frank Richard Taylor was the archetypal rural general practitioner, always full of bonhomie and generosity. After qualifying in Dublin and doing house jobs in Northampton, he became a partner in Barton le Clay, Bedfordshire, in 1965, remaining so until 2005. The Siegfried Farnon (from All Creatures Great and Small ) of the GP world, the stories of his exploits over his time in practice are legendary in the area. Though never a GP trainer, he …

Purpose: To remove artefacts that occur between adjacent couch positions in 4DCT volumes acquired in Cine mode. Method and Materials: separate B‐spline non‐rigid registration is performed between an artefact free reference volume and the data from each individual couch position in the 4DCT volume. The registration is performed using an extended control point grid that covers all of the couch positions and is common to all of the registrations. Therefore the result of each registration defines a transformation over all couch positions but was only constrained by data from one couch position. The registration results from all couch position are then combined into a single B‐spline transformation. The control point displacements in the combined transformation are a weighted average of the displacements in the individual registrations. The weight for each registration is different for each row in the control point grid, and depends on the contribution that the control points make to the transformation in the region of the couch position that was registered. The combined transformation is continuous across all the couch positions, and when used to deform the reference volume will produce an artefact free prediction of the anatomy in the same respiratory state as the original 4DCT volume. Results: This method has been applied to 4DCT data from five patients that were subject to artefacts between couch positions. In all cases our method produced volumes that resembled the original 4DCT data but were free of artefacts between adjacent couch positions. Conclusion: We have presented a novel method based on B‐spline non‐rigid registration that can remove the artefacts that occur between adjacent couch positions in 4DCT volumes acquired in Cine model, and have successfully demonstrated this method on data from five patients.

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a “representation” of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Single-cell comparative genomic hybridization (SCOMP), described in this protocol, is a form of ligation-mediated PCR that was specifically designed for WGA of extremely limited sources of genomic DNA. The reaction volume is purposely kept to a minimum, and all buffers are optimized to eliminate the need to purify the reaction between steps. In addition, the entire reaction is performed in a single tube. This avoids initial template loss and reduces the risk of PCR contamination. SCOMP begins by converting the genome to a high-complexity representation with a fragment size of

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). GenomePlex WGA, described in this protocol, is a proprietary amplification technology based on nonenzymatic random fragmentation of genomic DNA. The protocol involves conversion of the genome into an in vitro molecular library of DNA fragments, followed by incubation at various temperatures to add adaptor sequences with specific PCR priming sites to both ends of every fragment. The fragment library can then be amplified several 1000-fold to generate milligram quantities of DNA starting with as little as 10-100 ng.

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a “representation” of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Adaptor-ligation PCR of randomly sheared genomic DNA (PRSG), described here, is based on ligation-mediated PCR and was designed to improve genome coverage. Rather than using enzymatically generated fragments, this method uses randomly fragmented DNA as the template. The process involves three steps: (1) the hydrodynamic shearing of genomic DNA to a 0.5-2-kb size range, (2) end filling and adaptor ligation, and (3) high-stringency PCR for faithful replication of the resulting fragments.

18032 Background: The addition of cetuximab can increase the efficacy of chemotherapy for advanced NSCLC. Concomitant cetuximab and RT is superior to RT alone for locally advanced squamous cell head & neck carcinoma. The SCRATCH study (cohort I) is a phase I study to assess the safety of synchronous cetuximab and radical RT in patients with Stage III NSCLC. Methods: Cohort I will contain 12 patients with inoperable stage III NSCLC. Inclusion criteria are performance status 0–1, adequate organ function, and disease encompassable within a radical RT volume. Exclusion criteria are previous malignancy, thoracic RT or treatment with EGFR targeted therapy. Patients receive platinum-based induction chemotherapy, followed by weekly intravenous cetuximab (initial dose 400mg/m2; maintenance dose 250mg/m2) and concomitant RT (64Gy/32fractions/45days). The primary end-point is toxicity. NCI Common Toxicity Criteria (CTC) V3.0 assessments are performed weekly during radiotherapy, and at regular follow-up visits. Results: Data from the first 9 patients is available. 2 patients stopped receiving cetuximab early due to toxicity. 1 experienced grade 3 fatigue following the initial dose, and the other declined further treatment after developing grade 2 skin toxicity. 2 patients have died, 1 from disease progression and 1 from thromboembolic disease. Both deaths occurred between months 2 and 4 post RT and were not attributed to the cetuximab therapy. Of the 7 living patients, 2 have survived 1 year (measured from the first day of induction chemotherapy). The maximum NCI CTC V3.0 scores are summarised in the table below. Conclusions: The results suggest that the early and late toxicities of synchronous cetuximab and radical RT are acceptable. Data on all 12 patients will be available by June 2007. SCRATCH Study cohorts II-IV follow on and will recruit sequentially. They will assess the safety of adding concomitant cisplatin (±vinorelbine) to cetuximab and radical RT. [Table: see text] No significant financial relationships to disclose.

Background Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples. Results To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA. Conclusion Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.

Volcanology is a rapidly evolving science that has recently received a high media profile through Hollywood movies and television coverage of Montserrat in the West Indies and the Mexican volcanoes of Colima and Popocatepetl. One of the movies and all three of the real-life eruptions involve volcanoes that are subject to sudden, explosive eruptions that can cause many hazards. In the region of the volcanic vent, high altitude eruption columns that resemble the mushroom cloud of an atomic blast can repeatedly disrupt regional air traffic. Within several kilometers of the volcanic edifice, fallout from the eruption can destroy roofs and hinder access while hot and ash-laden pyroclastic density currents can lay to waste any population centers that are encountered in their paths. Such events have claimed thousands of lives in this century alone. Over time, the potential also exists for economic disruption and, on a global scale, climatic change.

Rates have been measured of sulphonyl-activated eliminative ring fissions of a series of six cyclopropanes in which the leaving group is stabilised by alkoxycarbonyl, cyano, or sulphonyl groups. The measurements allow assignment of ranks (nucleofugalities) to carbon leaving groups in systems in which the connection to the leaving group is strained by incorporation in a cyclopropane ring. The values obtained are compared with those obtained for a unstrained (acyclic) analogues. Rank enhancements of about 9 (log) units are obtained; these enhancements suggest that free energies of activation for leaving-group expulsion are reduced by about 53 kJ mol–1, or about 46% of the excess of enthalpy of the strained ring, notwithstanding the small degree of ring fission in the transition structure. The effect of phenyl substitution at the leaving group suggests that cleavage of the ring is very little advanced in the transition structure, although this is variable with the nature of the leaving-group stabilisation. This is the first direct determination of the effect of strain on nucleofugality.

Background Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion. Methods Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided. Results Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04). Conclusion This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.

BACKGROUND: Urothelial cancers (UC) are the fourth most common tumours worldwide after prostate (or breast), lung and colorectal cancer. Despite recent improvements in their management, UC remain an aggressive disease associated with a poor outcome. Following disease progression on first-line platinum-based chemotherapy, very few effective treatment options are available and none of them have shown significant improvement in overall survival. Alterations of the fibroblast growth factor receptor (FGFR) pathway including amplification, mutations and overexpression are common in UC. Pre-clinical data suggest that the presence of such dysregulations may confer sensitivity to FGFR inhibitors. MATERIALS AND METHODS: We present here the case of a patient with a metastatic UC of the renal pelvis with lymph node metastases treated with the selective FGFR inhibitor AZD4547. RESULTS: To date, the patient has been on a study drug for 32 months with acceptable tolerance and maintained radiological partial response as per RECIST 1.1 criteria. Exploratory biomarker analysis showed FGFR3, FGFR1, FGF-ligand and fibroblast growth factor receptor substrate 2 (FRS2) expression in the patient's tumour, together with the presence of a germ-line mutation in the FGFR3 extracellular binding domain. This is not a known hotspot mutation, and the functional significance remains unclear. CONCLUSIONS: The FGFR inhibitor AZD4547 exhibits antitumour activity in a metastatic urothelial cancer displaying FGFR1, FGFR3, FGF-ligand and FRS2 expression. This lends support to the further exploration of FGFR inhibitors in urothelial cancer. Further studies are required to determinate the most effective way to select those patients most likely to respond.

Rates have been measured of sulphonyl-activated eliminative ring fissions of a series of six cyclopropanes in which the leaving group is stabilised by alkoxycarbonyl, cyano, or sulphonyl groups. The measurements allow assignment of ranks (nucleofugalities) to carbon leaving groups in systems in which the connection to the leaving group is strained by incorporation in a cyclopropane ring. The values obtained are compared with those obtained for a unstrained (acyclic) analogues. Rank enhancements of about 9 (log) units are obtained; these enhancements suggest that free energies of activation for leaving-group expulsion are reduced by about 53 kJ mol–1, or about 46% of the excess of enthalpy of the strained ring, notwithstanding the small degree of ring fission in the transition structure. The effect of phenyl substitution at the leaving group suggests that cleavage of the ring is very little advanced in the transition structure, although this is variable with the nature of the leaving-group stabilisation. This is the first direct determination of the effect of strain on nucleofugality.

In eliminative ring fission of cyclopropanes, the extent of ring fission in the transition state is sensitive to the function stabilizing the leaving group; it is small when the stabilizing group is phenylsuphonyl, but substantial when it is cyano.

Comparison of eliminative ring fission in a cyclopropane and elimination in an acyclic analogue allows determination of acceleration of elimination by ring strain; a factor of a least 1011.7 has been found, the largest for any heterolytic reaction.