COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type-2 and PDE4 gene expression in sputum.

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase-4-inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32-day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh  ≥ 3%; eosinophillow  < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty-one genes were differentially expressed in primary samples (p-adjusted for false discovery rate < 0.05); all up-regulated in eosinophilhigh patients and functionally enriched for type-2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type-2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.

Sputum and blood transcriptomics characterisation of the inhaled PDE4 inhibitor CHF6001 on top of triple therapy in patients with chronic bronchitis.

BACKGROUND: Although phosphodiesterase-4 (PDE4) inhibitors have been shown to reduce COPD exacerbation rate, their biological mechanism of action is not completely elucidated at the molecular level. We aimed to characterise the whole genome gene expression profile of the inhaled PDE4-inhibitor CHF6001 on top of triple therapy in sputum cells and whole blood of patients with COPD and chronic bronchitis. METHODS: Whole genome gene expression analysis was carried out by microarray in 54 patients before and after 32 days treatment with CHF6001 800 and 1600 µg and placebo twice daily (BID) in a randomised crossover study. RESULTS: CHF6001 had a strong effect in sputum, with 1471 and 2598 significantly differentially-expressed probe-sets relative to placebo (p-adjusted for False Discovery Rate < 0.05) with 800 and 1600 µg BID, respectively. Functional enrichment analysis showed significant modulation of key inflammatory pathways involved in cytokine activity, pathogen-associated-pattern-recognition activity, oxidative stress and vitamin D with associated inhibition of downstream inflammatory effectors. A large number of pro-inflammatory genes coding for cytokines and matrix-metalloproteinases were significantly differentially expressed for both doses; the majority (> 87%) were downregulated, including macrophage inflammatory protein-1-alpha and 1-beta, interleukin-27-beta, interleukin-12-beta, interleukin-32, tumour necrosis factor-alpha-induced-protein-8, ligand-superfamily-member-15, and matrix-metalloproteinases-7,12 and 14. The effect in blood was not significant. CONCLUSIONS: Inhaled PDE4 inhibition by CHF6001 on top of triple therapy in patients with COPD and chronic bronchitis significantly modulated key inflammatory targets and pathways in the lung but not in blood. Mechanistically these findings support a targeted effect in the lung while minimising unwanted systemic class-effects. TRIAL REGISTRATION: ClinicalTrial.gov, EudraCT, 2015-005550-35. Registered 15 July 2016.

Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial.

Metformin may reduce the incidence of breast cancer and enhance response to neoadjuvant chemotherapy in diabetic women. This trial examined the effects of metformin on Ki67 and gene expression in primary breast cancer. Non-diabetic women with operable invasive breast cancer received pre-operative metformin. A pilot cohort of eight patients had core biopsy of the cancer at presentation, a week later (without treatment; internal control), then following metformin 500-mg o.d. for 1 week increased to 1-g b.d. for a further week continued to surgery. A further 47 patients had core biopsy at diagnosis were randomized to metformin (the same dose regimen) or no drug, and 2 weeks later had core biopsy at surgery. Ki67 immunohistochemistry, transcriptome analysis on formalin-fixed paraffin-embedded cores and serum insulin determination were performed blinded to treatment. Seven patients (7/32, 21.9%) receiving metformin withdrew because of gastrointestinal upset. The mean percentage of cells staining for Ki67 fell significantly following metformin treatment in both the pilot cohort (P = 0.041, paired t-test) and in the metformin arm (P = 0.027, Wilcoxon rank test) but was unchanged in the internal control or metformin control arms. Messenger RNA expression was significantly downregulated by metformin for PDE3B (phosphodiesterase 3B, cGMP-inhibited; a critical regulator of cAMP levels that affect activation of AMP-activated protein kinase, AMPK), confirmed by immunohistochemistry, SSR3, TP53 and CCDC14. By ingenuity pathway analysis, the tumour necrosis factor receptor 1 (TNFR1) signaling pathway was most affected by metformin: TGFB and MEKK were upregulated and cdc42 downregulated; mTOR and AMPK pathways were also affected. Gene set analysis additionally revealed that p53, BRCA1 and cell cycle pathways also had reduced expression following metformin. Mean serum insulin remained stable in patients receiving metformin but rose in control patients. This trial presents biomarker evidence for anti-proliferative effects of metformin in women with breast cancer and provides support for therapeutic trials of metformin.

A Novel Processing-Free Method for RNAseq Analysis of Spontaneous Sputum in Chronic Obstructive Pulmonary Disease.

Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV200) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV200 higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.

Molecular Subgroup of Primary Prostate Cancer Presenting with Metastatic Biology.

BACKGROUND: Approximately 4-25% of patients with early prostate cancer develop disease recurrence following radical prostatectomy. OBJECTIVE: To identify a molecular subgroup of prostate cancers with metastatic potential at presentation resulting in a high risk of recurrence following radical prostatectomy. DESIGN, SETTING, AND PARTICIPANTS: Unsupervised hierarchical clustering was performed using gene expression data from 70 primary resections, 31 metastatic lymph nodes, and 25 normal prostate samples. Independent assay validation was performed using 322 radical prostatectomy samples from four sites with a mean follow-up of 50.3 months. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Molecular subgroups were identified using unsupervised hierarchical clustering. A partial least squares approach was used to generate a gene expression assay. Relationships with outcome (time to biochemical and metastatic recurrence) were analysed using multivariable Cox regression and log-rank analysis. RESULTS AND LIMITATIONS: A molecular subgroup of primary prostate cancer with biology similar to metastatic disease was identified. A 70-transcript signature (metastatic assay) was developed and independently validated in the radical prostatectomy samples. Metastatic assay positive patients had increased risk of biochemical recurrence (multivariable hazard ratio [HR] 1.62 [1.13-2.33]; p=0.0092) and metastatic recurrence (multivariable HR=3.20 [1.76-5.80]; p=0.0001). A combined model with Cancer of the Prostate Risk Assessment post surgical (CAPRA-S) identified patients at an increased risk of biochemical and metastatic recurrence superior to either model alone (HR=2.67 [1.90-3.75]; p<0.0001 and HR=7.53 [4.13-13.73]; p<0.0001, respectively). The retrospective nature of the study is acknowledged as a potential limitation. CONCLUSIONS: The metastatic assay may identify a molecular subgroup of primary prostate cancers with metastatic potential. PATIENT SUMMARY: The metastatic assay may improve the ability to detect patients at risk of metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies should be assessed in this higher-risk population.

Gene expression profiling during intensive cardiovascular lifestyle modification: Relationships with vascular function and weight loss.

Heart disease and related sequelae are a leading cause of death and healthcare expenditure throughout the world. Although many patients opt for surgical interventions, lifestyle modification programs focusing on nutrition and exercise have shown substantial health benefits and are becoming increasing popular. We conducted a year-long lifestyle modification program to mediate cardiovascular risk through traditional risk factors and to investigate how molecular changes, if present, may contribute to long-term risk reduction. Here we describe the lifestyle intervention, including clinical and molecular data collected, and provide details of the experimental methods and quality control parameters for the gene expression data generated from participants and non-intervention controls. Our findings suggest successful and sustained modulation of gene expression through healthy lifestyle changes may have beneficial effects on vascular health that cannot be discerned from traditional risk factor profiles. The data are deposited in the Gene Expression Omnibus, series GSE46097 and GSE66175.

Importance of substantial weight loss for altering gene expression during cardiovascular lifestyle modification.

OBJECTIVE: To examine relationships between weight loss through changes in lifestyle and peripheral blood gene expression profiles. METHODS: A prospective nonrandomized trial was conducted over 1 year in participants undergoing intensive lifestyle modification to reverse or stabilize progression of coronary artery disease. Cardiovascular risk factors, inflammatory biomarkers, and gene expression as a function of weight loss were assessed in 89 lifestyle participants and 71 retrospectively matched controls undergoing usual care. RESULTS: Substantial weight loss (-15.2 ± 3.8%) in lifestyle participants (n = 33) was associated with improvement in selected cardiovascular risk factors and significant changes in peripheral blood gene expression from pre- to post-intervention: 132 unique genes showed significant expression changes (false discovery rate corrected P-value <0.05 and fold-change ≥1.4). Altered molecular pathways were related to immune function and inflammatory responses involving endothelial activation. In contrast, participants losing minimal weight (-3.1 ± 2.5%, n = 32) showed only minor changes in cardiovascular risk factors and markers of inflammation and no changes in gene expression compared to non intervention controls after 1 year. CONCLUSIONS: Weight loss (≥10%) during lifestyle modification is associated with down-regulation of genetic pathways governing interactions between circulating immune cells and the vascular endothelium and may be required to successfully reduce CVD risk.

Intensive cardiovascular risk reduction induces sustainable changes in expression of genes and pathways important to vascular function.

BACKGROUND: Healthy lifestyle changes are thought to mediate cardiovascular disease risk through pathways affecting endothelial function and progression of atherosclerosis; however, the extent, persistence, and clinical significance of molecular change during lifestyle modification are not well known. We examined the effect of a rigorous cardiovascular disease risk reduction program on peripheral blood gene expression profiles in 63 participants and 63 matched controls to characterize molecular responses and identify regulatory pathways important to cardiovascular health. METHODS AND RESULTS: Dramatic changes in dietary fat intake (-61%; P<0.001 versus controls) and physical fitness (+34%; P<0.001) led to significant improvements in cardiovascular disease risk factors. Analysis of variance with false discovery rate correction for multiple testing (P<0.05) identified 26 genes after 12 weeks and 143 genes after 52 weeks that were differentially expressed from baseline in participants. Controls showed little change in cardiovascular disease risk factors or gene expression. Quantitative reverse transcription polymerase chain reaction validated differential expression for selected transcripts. Lifestyle modification effectively reduced expression of proinflammatory genes associated with neutrophil activation and molecular pathways important to vascular function, including cytokine production, carbohydrate metabolism, and steroid hormones. Prescription medications did not significantly affect changes in gene expression. CONCLUSIONS: Successful and sustained modulation of gene expression through lifestyle changes may have beneficial effects on the vascular system not apparent from traditional risk factors. Healthy lifestyles may restore homeostasis to the leukocyte transcriptome by downregulating lactoferrin and other genes important in the pathogenesis of atherosclerosis. Clinical Trial Registration- URL: www.clinicaltrials.gov. Unique identifier: NCT01805492.

Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.