The challenges faced in the design, conduct and analysis of surgical randomised controlled trials

Randomised evaluations of surgical interventions are rare; some interventions have been widely adopted without rigorous evaluation. Unlike other medical areas, the randomised controlled trial (RCT) design has not become the default study design for the evaluation of surgical interventions. Surgical trials are difficult to successfully undertake and pose particular practical and methodological challenges. However, RCTs have played a role in the assessment of surgical innovations and there is scope and need for greater use. This article will consider the design, conduct and analysis of an RCT of a surgical intervention. The issues will be reviewed under three headings: the timing of the evaluation, defining the research question and trial design issues. Recommendations on the conduct of future surgical RCTs are made. Collaboration between research and surgical communities is needed to address the distinct issues raised by the assessmentof surgical interventions and enable the conduct of appropriate and well-designed trials.The Health Services Research Unit is funded by the Scottish Government Health DirectoratesPeer reviewedPublisher PD

Receptor and secreted targets of Wnt-1/β-catenin signalling in mouse mammary epithelial cells

BACKGROUND: Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells. METHODS: Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway. RESULTS: The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue. CONCLUSIONS: HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo11C-acetate and PET-CT scanning to show that colonic acetate crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression. Furthermore, we demonstrate through 13C high-resolution magic-angle-spinning that 13C acetate from fermentation of 13C-labelled carbohydrate in the colon increases hypothalamic 13C acetate above baseline levels. Hypothalamic 13C acetate regionally increases the 13C labelling of the glutamate–glutamine and GABA neuroglial cycles, with hypothalamic 13C lactate reaching higher levels than the ‘remaining brain’. These observations suggest that acetate has a direct role in central appetite regulation

Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

The yeast prion [PSI+] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI+] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI+] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI+]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI+]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth

Cyclin A is a prognostic indicator in early stage breast cancer with and without tamoxifen treatment

Overexpression of G1-S regulators cyclin D1 or cyclin A is frequently observed in breast cancer and is also to result in ligand-independent activation of oestrogen receptor in vitro. This might therefore, provide a mechanism for failure of tamoxifen treatment. We examined by immunohistochemical staining the effect of deregulation of these, and other cell cycle regulators on tamoxifen treatment in a group of 394 patients with early stage breast cancer. In univariate analysis, expression of cyclin A, Neu, Ki-67 index, and lack of OR expression were significantly associated with worse prognosis. When adjusted by the clinical model (for lymph node status, age, performance status, T-classification, grade, prior surgery, oestrogen receptor status and tamoxifen use), only overexpression of cyclin A and Neu were significantly associated with worse prognosis with hazard ratios of, respectively, 1.709 (P=0.0195) and 1.884 (P=0.0151). Overexpression of cyclin A was found in 86 out of the 201 OR-positive cases treated with tamoxifen, and was the only independent marker associated with worse prognosis (hazard ratio 2.024, P=0.0462). In conclusion, cyclin A is an independent predictor of recurrence of early stage breast cancer and is as such a marker for response in patients treated with tamoxifen

A Densely Interconnected Genome-Wide Network of MicroRNAs and Oncogenic Pathways Revealed Using Gene Expression Signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA–pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA–pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA–pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes (“hubs”), most nodes in the miRNA–pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA–pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

Abstract Introduction Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines. Methods Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response. Results Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade. Conclusions These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991

Non-essential role for TLR2 and its signaling adaptor Mal/TIRAP in preserving normal lung architecture in mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr42/2 mice by 6 months of age, the lungs of Tlr22/2 mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr42/2 mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal2/2 mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal2/2 mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr42/2 mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema

Reprogramming of hepatic fat accumulation and 'browning' of adipose tissue by the short-chain fatty acid acetate

Background/Objectives: Short-chain fatty acids, produced by microbiome fermentation of carbohydrates, have been linked to a reduction in appetite, body weight and adiposity. However, determining the contribution of central and peripheral mechanisms to these effects has not been possible. Subjects/Methods:C57BL/6 mice fed with either normal or high-fat diet were treated with nanoparticle-delivered acetate, and the effects on metabolism were investigated. Results:In the liver, acetate decreased lipid accumulation and improved hepatic function, as well as increasing mitochondrial efficiency. In white adipose tissue, it inhibited lipolysis and induced 'browning', increasing thermogenic capacity that led to a reduction in body adiposity. Conclusions:This study provides novel insights into the peripheral mechanism of action of acetate, independent of central action, including ‘browning’ and enhancement of hepatic mitochondrial function

Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays

The CCND1 gene, a key cell-cycle regulator, is often altered in breast cancer, but the mechanisms underlying CCND1 dysregulation and the clinical significance of CCND1 status are unclear. We used real-time quantitative PCR and RT–PCR assays based on fluorescent TaqMan methodology to quantify CCND1 gene amplification and expression in a large series of breast tumours. CCND1 overexpression was observed in 44 (32.8%) of 134 breast tumour RNAs, ranging from 3.3 to 43.7 times the level in normal breast tissues, and correlated significantly with positive oestrogen receptor status (P=0.0003). CCND1 overexpression requires oestrogen receptor integrity and is exacerbated by amplification at 11q13 (the site of the CCND1 gene), owing to an additional gene dosage effect. Our results challenge CCND1 gene as the main 11q13 amplicon selector. The relapse-free survival time of patients with CCND1-amplified tumours was shorter than that of patients without CCND1 alterations, while that of patients with CCND1-unamplified-overexpressed tumours was longer (P=0.011). Only the good prognostic significance of CCND1-unamplified-overexpression status persisted in Cox multivariate regression analysis. This study confirms that CCND1 is an ER-responsive or ER-coactivator gene in breast cancer, and points to the CCND1 gene as a putative molecular marker predictive of hormone responsiveness in breast cancer. Moreover, CCND1 amplification status dichotomizes the CCND1-overexpressing tumors into two groups with opposite outcomes