Germline genetic variants of the renin-angiotensin system, hypoxia and angiogenesis in non-small cell lung cancer progression: Discovery and validation studies

Introduction: The renin–angiotensin system (RAS) is involved in cell proliferation, immunoinflammatory response, hypoxia and angiogenesis, which are critical biological processes in lung cancer. Our aim was to study the association of putatively functional genetic polymorphisms in genes coding for proteins involved in RAS, hypoxia and angiogenesis with non-small cell lung cancer (NSCLC) prognosis. Methods: Genotyping of 52 germline variants from genes of the RAS and hypoxic/angiogenic factors/receptors was performed using MassARRAY iPLEX Gold in a retrospective cohort (n = 167) of advanced NSCLC patients. Validation of the resulting genetic markers was conducted in an independent group (n = 190), matched by clinicopathological characteristics. Results: Multivariate analysis on the discovery set revealed that MME rs701109 C carriers were protected from disease progression in comparison with homozygous T (hazard ratio (HR) = 0.5, 95% confidence interval (CI) = 0.2–0.8, p = 0.010). Homozygous A and T genotypes for KDR rs1870377 were at increased risk for disease progression and death compared to heterozygous (HR = 1.7, 95% CI = 1.2–2.5, p = 0.005 and HR = 2.1, 95% CI = 1.2–3.4, p = 0.006, respectively). Carriers of homozygous genotypes for ACE2 rs908004 presented increased risk for disease progression, only in the subgroup of patients without tumour actionable driver mutations (HR = 2.9, 95% CI = 1.3–6.3, p = 0.010). Importantly, the association of homozygous genotypes in MME rs701109 with risk for disease progression was confirmed after multivariate analysis in the validation set. Conclusion: This study provides evidence that MME polymorphism, which encodes neprilysin, may modulate progression-free survival in advanced NSCLC. Present genetic variation findings will foster basic, translational, and clinical research on their role in NSCLC.M.J.C. was supported by the Associação de Estudos Respiratórios and the Portuguese Pulmonology Society

Blood-sampling collection prior to surgery may have a significant influence upon biomarker concentrations measured

Abstract Background Biomarkers can be subtle tools to aid the diagnosis, prognosis and monitoring of therapy and disease progression. The validation of biomarkers is a cumbersome process involving many steps. Serum samples from lung cancer patients were collected in the framework of a larger study for evaluation of biomarkers for early detection of lung cancer. The analysis of biomarker levels measured revealed a noticeable difference in certain biomarker values that exhibited a dependence of the time point and setting of the sampling. Biomarker concentrations differed significantly if taken before or after the induction of anesthesia and if sampled via venipuncture or arterial catheter. Methods To investigate this observation, blood samples from 13 patients were drawn 1–2 days prior to surgery (T1), on the same day by venipuncture (T2) and after induction of anesthesia via arterial catheter (T3). The biomarkers Squamous Cell Carcinoma antigen (CanAG SCC EIA, Fujirebio Diagnostics, Malvern, USA), Carcinoembrionic Antigen (CEA), and CYFRA 21-1 (Roche Diagnostics GmbH, Mannheim, Germany) were analyzed. Results SCC showed a very strong effect in relation to the sampling time and procedure. While the first two points in time (T1; T2) were highly comparable (median fold-change: 0.84; p = 0.7354; correlation ρ = 0.883), patients showed a significant increase (median fold-change: 4.96; p = 0.0017; correlation ρ = -0.036) in concentration when comparing T1 with the sample time subsequent to anesthesia induction (T3). A much weaker increase was found for CYFRA 21-1 at T3 (median fold-change: 1.40; p = 0.0479). The concentration of CEA showed a very small, but systematic decrease (median fold-change: 0.72; p = 0.0039). Conclusions In this study we show the unexpectedly marked influence of blood withdrawal timing (before vs. after anesthesia) and procedure (venous versus arterial vessel puncture) has on the concentration of the protein biomarker SCC and to a less extent upon CYFRA21-1. The potential causes for these effects remain to be elucidated in subsequent studies, however these findings highlight the importance of a standardized, controlled blood collection protocol for biomarker detection

A solid‐phase transfection platform for arrayed CRISPR screens [Corrigendum]

Since the publication of this study, it has come to our attention that a citation to the study by Bulkescher et al (2017) was omitted from the Introduction. The following sentence should have been included in the introduction: “A previously reported solid‐phase reverse transfection method for proteins (Bulkescher et al , 2017) was used for the delivery of RNPs for three endogenous genes suggesting the potential of solid‐phase reverse transfection for CRISPR/Cas9‐based gene editing, despite its low efficiency”. We apologise for any inconvenience this omission may have caused

A solid-phase transfection platform for arrayed CRISPR screens

Arrayed CRISPR‐based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid‐phase transfection platform that enables CRISPR‐based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high‐throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies

Genome-Wide DNA Methylation Profiling in Early Stage I Lung Adenocarcinoma Reveals Predictive Aberrant Methylation in the Promoter Region of the Long Noncoding RNA PLUT: An Exploratory Study

Introduction: Surgical procedure is the treatment of choice in early stage I lung adenocarcinoma. However, a considerable number of patients experience recurrence within the first 2 years after complete resection. Suitable prognostic biomarkers that identify patients at high risk of recurrence (who may probably benefit from adjuvant treatment) are still not available. This study aimed at identifying methylation markers for early recurrence that may become important tools for the development of new treatment modalities. Methods: Genome-wide DNA methylation profiling was performed on 30 stage I lung adenocarcinomas, comparing 14 patients with early metastatic recurrence with 16 patients with a long-term relapse-free survival period using methylated-CpG-immunoprecipitation followed by high-throughput next-generation sequencing. The differentially methylated regions between the two subgroups were validated for their prognostic value in two independent cohorts using the MassCLEAVE assay, a high-resolution quantitative methylation analysis. Results: Unsupervised clustering of patients in the discovery cohort on the basis of differentially methylated regions identified patients with shorter relapse-free survival (hazard ratio: 2.23; 95% confidence interval: 0.66-7.53; p = 0.03). In two validation cohorts, promoter hypermethylation of the long noncoding RNA PLUT was significantly associated with shorter relapse-free survival (hazard ratio: 0.54; 95% confidence interval: 0.31-0.93; p < 0.026) and could be reported as an independent prognostic factor in the multivariate Cox regression analysis. Conclusions: Promoter hypermethylation of the long noncoding RNA PLUT is predictive in patients with early stage I adenocarcinoma at high risk for early recurrence. Further studies are needed to validate its role in carcinogenesis and its use as a biomarker to facilitate patient selection and risk stratification

Modern microwave methods in solid state inorganic materials chemistry: from fundamentals to manufacturing

No abstract available

Loss of aquaporin-4 expression and putative function in non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Aquaporins (AQPs) have been recognized to promote tumor progression, invasion, and metastasis and are therefore recognized as promising targets for novel anti-cancer therapies. Potentially relevant AQPs in distinct cancer entities can be determined by a comprehensive expression analysis of the 13 human AQPs.</p> <p>Methods</p> <p>We analyzed the presence of all AQP transcripts in 576 different normal lung and non-small cell lung cancer (NSCLC) samples using microarray data and validated our findings by qRT-PCR and immunohistochemistry.</p> <p>Results</p> <p>Variable expression of several AQPs (AQP1, -3, -4, and -5) was found in NSCLC and normal lung tissues. Furthermore, we identified remarkable differences between NSCLC subtypes in regard to AQP1, -3 and -4 expression. Higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favourable prognosis. Beyond water transport, data mining of co-expressed genes indicated an involvement of AQP4 in cell-cell signalling, cellular movement and lipid metabolism, and underlined the association of AQP4 to important physiological functions in benign lung tissue.</p> <p>Conclusions</p> <p>Our findings accentuate the need to identify functional differences and redundancies of active AQPs in normal and tumor cells in order to assess their value as promising drug targets.</p

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients

Gene–gene interaction of AhRwith and within the Wntcascade affects susceptibility to lung cancer

Background Aberrant Wnt signalling, regulating cell development and stemness, influences the development of many cancer types. The Aryl hydrocarbon receptor (AhR) mediates tumorigenesis of environmental pollutants. Complex interaction patterns of genes assigned to AhR/Wnt-signalling were recently associated with lung cancer susceptibility. Aim To assess the association and predictive ability of AhR/Wnt-genes with lung cancer in cases and controls of European descent. Methods Odds ratios (OR) were estimated for genomic variants assigned to the Wnt agonist and the antagonistic genes DKK2, DKK3, DKK4, FRZB, SFRP4 and Axin2. Logistic regression models with variable selection were trained, validated and tested to predict lung cancer, at which other previously identified SNPs that have been robustly associated with lung cancer risk could also enter the model. Furthermore, decision trees were created to investigate variant x variant interaction. All analyses were performed for overall lung cancer and for subgroups. Results No genome-wide significant association of AhR/Wnt-genes with overall lung cancer was observed, but within the subgroups of ever smokers (e.g., maker rs2722278 SFRP4; OR = 1.20; 95% CI 1.13-1.27; p = 5.6 x 10(-10)) and never smokers (e.g., maker rs1133683 Axin2; OR = 1.27; 95% CI 1.19-1.35; p = 1.0 x 10(-12)). Although predictability is poor, AhR/Wnt-variants are unexpectedly overrepresented in optimized prediction scores for overall lung cancer and for small cell lung cancer. Remarkably, the score for never-smokers contained solely two AhR/Wnt-variants. The optimal decision tree for never smokers consists of 7 AhR/Wnt-variants and only two lung cancer variants. Conclusions The role of variants belonging to Wnt/AhR-pathways in lung cancer susceptibility may be underrated in main-effects association analysis. Complex interaction patterns in individuals of European descent have moderate predictive capacity for lung cancer or subgroups thereof, especially in never smokers