RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

Functional and quality of life outcomes of localised prostate cancer treatments (prostate testing for cancer and treatment [ProtecT] study)

Objective To investigate the functional and quality of life (QoL) outcomes of treatments for localised prostate cancer and inform treatment decision-making. Patients and Methods Men aged 50–69 years diagnosed with localised prostate cancer by prostate-specific antigen testing and biopsies at nine UK centres in the Prostate Testing for Cancer and Treatment (ProtecT) trial were randomised to, or chose one of, three treatments. Of 2565 participants, 1135 men received active monitoring (AM), 750 a radical prostatectomy (RP), 603 external-beam radiotherapy (EBRT) with concurrent androgen-deprivation therapy (ADT) and 77 low-dose-rate brachytherapy (BT, not a randomised treatment). Patient-reported outcome measures (PROMs) completed annually for 6 years were analysed by initial treatment and censored for subsequent treatments. Mixed effects models were adjusted for baseline characteristics using propensity scores. Results Treatment-received analyses revealed different impacts of treatments over 6 years. Men remaining on AM experienced gradual declines in sexual and urinary function with age (e.g., increases in erectile dysfunction from 35% of men at baseline to 53% at 6 years and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6 years. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6 years, and after EBRT this was reported by 69% and 74%, respectively (P < 0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1 pad/day, persisting for 20% at 6 years, compared with no change in men receiving EBRT or AM (P < 0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6 years and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (P < 0.001) with lesser effects after BT. No treatment affected mental or physical QoL. Conclusion Treatment decision-making for localised prostate cancer can be informed by these 6-year functional and QoL outcomes

A História da Alimentação: balizas historiográficas

Os M. pretenderam traçar um quadro da História da Alimentação, não como um novo ramo epistemológico da disciplina, mas como um campo em desenvolvimento de práticas e atividades especializadas, incluindo pesquisa, formação, publicações, associações, encontros acadêmicos, etc. Um breve relato das condições em que tal campo se assentou faz-se preceder de um panorama dos estudos de alimentação e temas correia tos, em geral, segundo cinco abardagens Ia biológica, a econômica, a social, a cultural e a filosófica!, assim como da identificação das contribuições mais relevantes da Antropologia, Arqueologia, Sociologia e Geografia. A fim de comentar a multiforme e volumosa bibliografia histórica, foi ela organizada segundo critérios morfológicos. A seguir, alguns tópicos importantes mereceram tratamento à parte: a fome, o alimento e o domínio religioso, as descobertas européias e a difusão mundial de alimentos, gosto e gastronomia. O artigo se encerra com um rápido balanço crítico da historiografia brasileira sobre o tema

RNA-Seq differentiates the tumour (human) transcriptional response to cediranib from the host (mouse).

<p>Gene Set Enrichment Analysis (GSEA) reveals significant enrichment of (A) hypoxia and (B) cell cycle associated signatures in tumour genes differentially regulated in response to cediranib dosed at 6 mg/kg once daily for 4 days (** indicates gene sets enriched with <i>p</i><0.001, FDR <i>q</i><0.05 and FWER <i>p</i><0.1; * indicates gene sets enriched with <i>p</i><0.001 and FDR <i>q</i><0.05). GSEA-defined “Leading Edge” genes most frequently included in (C) hypoxia-associated gene sets and up-regulated in response to cediranib include CA9, HK2 and VEGFA. Leading edge cell cycle associated genes are given in (D). (E) Ingenuity Pathway Analysis (IPA) highlights functions significantly enriched (<i>p</i><1×10⁻⁵) amongst host genes differentially regulated in response to cediranib. For both GSEA and IPA, genes achieving a log₂ fold change magnitude>1 and <i>p</i><0.1 were defined as differentially regulated. <i>n</i> = 2 in treated and control groups; a positive fold change indicates genes up-regulated in response to cediranib, and <i>vice versa</i>.</p

RT-qPCR validation of mouse gene expression changes in response to cediranib identified by RNA-Seq.

<p>RT-qPCR validation of mouse gene expression changes in response to cediranib identified by RNA-Seq.</p

The phenotypic effects of cediranib observed by immunohistochemical analysis of supporting vasculature in cediranib treated tumours.

<p>Calu-6 xenografts were established and dosed for 4 days with cediranib at 6mg/kg once daily and fixed in formalin. Micro Vessel Density was then quantified by histological staining for CD31 as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066003#pone.0066003-Smith2" target="_blank">[32]</a>. Images are representative for CD31 staining in control and cediranib treated tumours, and arrows indicate blood vessels. Graph depicts the significant (<i>p</i>-value<0.001; student t-test) reduction of the supporting vasculature in cediranib treated tumours; 8 control tumours and 6 cediranib tumours, error bars are standard error of the mean.</p

Schematic of the species-specific mapping workflow applicable to RNA-Seq data from xenografts.

<p>(1) Human tumour cells originating from the Calu-6 non-small cell lung carcinoma cell line were grown in mouse, and RNA extracted and sequenced using standard protocols. (2) Sequenced reads were then mapped to human and mouse genomes separately, and reads mapping to both species discarded. The remaining reads were used to quantify and delineate tumour (human) from host (mouse) gene expression.</p

RNA-Seq versus microarray gene expression correspondence.

<p>Comparison between RNA-Seq and microarray platforms across 8621 human and 5467 mouse genes from a Calu-6 human xenograft sample (Control_2) and detected on both platforms. Correlation in gene expression in (A) human (<i>r²</i> = 0.50) and (B) mouse (<i>r²</i> = 0.35). <i>r</i>² values are calculated only from genes detected on both platforms. The transcript mapped by the highest number of reads was chosen to represent the expression of its parent gene. Any gene containing at least one mappable read was classed as detected in RNA-Seq. A gene was called “present” on the array if the signals of all probesets assigned unambiguously to that gene were separable from the general background with a <i>p</i>-value<0.01. Correlation increases between (C) human and (D) mouse gene expression values across both samples when probes on the array at high risk of cross-species hybridization are removed. Only genes detected on both platforms were considered. A mean of ∼8400 human and ∼5300 mouse genes were used in the comparison of which ∼6800 and ∼4400 genes were deemed at high risk respectively. Genes at high risk were defined as those corresponding to a probe with up to three mismatches to the transcriptome of the alternative species.</p

RNA-Seq gene quantification and detection closely corresponds to values obtained by species-specific RT-qPCR.

<p>Comparison between RNA-Seq and RT-qPCR across 170 human and 174 mouse genes from a Calu-6 human xenograft sample (Control_2). Correlation in gene expression in (A) human (<i>r²</i> = 0.72) and (B) mouse (<i>r²</i> = 0.60). <i>r²</i> values are calculated only from genes detected on both platforms. Overlap in number of (C) human and (D) mouse genes detected in both RNA-Seq and RT-qPCR, and genes detected on one platform only. The transcript mapped to by the highest number of reads was chosen to represent the expression of its parent gene. Any gene containing at least one mappable read was classed as detected in RNA-Seq. The genes included in this comparison are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066003#pone.0066003.s009" target="_blank">Table S5</a>. Details of gene expression and detection correspondence across both Control_1 and Control_2 samples are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066003#pone.0066003.s012" target="_blank">Table S8</a>.</p