MicroRNAs regulate several aspects of tumorigenesis and cancer progression. Most cancer tissues are archived formalin-fixed and paraffin-embedded (FFPE). While microRNAs are a more stable form of RNA ...thought to withstand FFPE-processing and degradation there is only limited evidence for the latter assumption. We examined whether microRNA profiling can be successfully conducted on FFPE cancer tissues using SOLiD ligation based sequencing. Tissue storage times (2-9 years) appeared to not affect the number of detected microRNAs in FFPE samples compared to matched frozen samples (paired t-test p>0.7). Correlations of microRNA expression values were very high across microRNAs in a given sample (Pearson's r = 0.71-0.95). Higher variance of expression values among samples was associated with higher correlation coefficients between FFPE and frozen tissues. One of the FFPE samples in this study was degraded for unknown reasons with a peak read length of 17 nucleotides compared to 21 in all other samples. The number of detected microRNAs in this sample was within the range of microRNAs detected in all other samples. Ligation-based microRNA deep sequencing on FFPE cancer tissues is feasible and RNA degradation to the degree observed in our study appears to not affect the number of microRNAs that can be quantified.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The use of electronic cigarettes (e-cigs) is increasing rapidly, but their effects on lung toxicity are largely unknown. Smoking is a well-established cause of lung cancer and respiratory disease, in ...part through inflammation. It is plausible that e-cig use might affect similar inflammatory pathways. E-cigs are used by some smokers as an aid for quitting or smoking reduction, and by never smokers (e.g., adolescents and young adults). The relative effects for impacting disease risk may differ for these groups. Cell culture and experimental animal data indicate that e-cigs have the potential for inducing inflammation, albeit much less than smoking. Human studies show that e-cig use in smokers is associated with substantial reductions in blood or urinary biomarkers of tobacco toxicants when completely switching and somewhat for dual use. However, the extent to which these biomarkers are surrogates for potential lung toxicity remains unclear. The FDA now has regulatory authority over e-cigs and can regulate product and e-liquid design features, such as nicotine content and delivery, voltage, e-liquid formulations, and flavors. All of these factors may impact pulmonary toxicity. This review summarizes current data on pulmonary inflammation related to both smoking and e-cig use, with a focus on human lung biomarkers.
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The urinary metabolites "prostaglandin E2 metabolite" (PGE-M) and (Z)-7-1R,2R,3R,5S)-3,5-dihydroxy-2-(E,3S)-3-hydroxyoct-1-enylcyclopentylhept-5-enoic acid (8-iso-PGF2α) are biomarkers of ...inflammation and oxidative damage, respectively, and are elevated in cigarette smokers. Relatively little is known about the effects of smoking cessation on these biomarkers. To investigate this, current cigarette smokers interested in quitting were recruited and invited to participate in a smoking cessation study where varenicline (Chantix) and brief supportive behavioral counseling were offered at each visit after baseline. Subjects returned to the clinic during the 12 week treatment phase for 9 visits post cessation on days 3, 7, 14, 21, 28, 42, 56, 70 and 84. Urine samples were collected at each visit and analyzed by liquid chromatography-tandem mass spectrometry for PGE-M, 8-iso-PGF2α, and cotinine. Cotinine values demonstrated that 15 of 38 subjects quit smoking for the entire 84 day period. Significant decreases in mean levels of PGE-M and 8-iso-PGF2α per milligram creatinine were observed in these subjects, by 44% (p = 0.0014) and 27% (p<0.001), respectively. The results of this study demonstrate that cessation of smoking for 84 days results in modest but significant declines in urinary PGE-M and 8-iso-PGF2α indicating reductions in systemic inflammation and oxidative damage. Given that levels were only modestly decreased, these markers are not specific to tobacco-smoke exposure. The modest declines in these biomarkers should be considered when planning studies with ex-smokers. There is a "hangover" from smoking that lasts at least 3 months.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Electronic cigarette (e-cig) use is continuing to increase, particularly among youth never-smokers, and is used by some smokers to quit. The acute and chronic toxicity of e-cig use is unclear ...generally in the context of increasing reports of inflammatory-type pneumonia in some e-cig users. To assess lung effects of e-cigs without nicotine or flavors, we conducted a pilot study with serial bronchoscopies over 4 weeks in 30 never-smokers, randomized either to a 4-week intervention with the use of e-cigs containing only 50% propylene glycol (PG) and 50% vegetable glycerine or to a no-use control group. Compliance to the e-cig intervention was assessed by participants sending daily puff counts and by urinary PG. Inflammatory cell counts and cytokines were determined in bronchoalveolar lavage (BAL) fluids. Genome-wide expression, miRNA, and mRNA were determined from bronchial epithelial cells. There were no significant differences in changes of BAL inflammatory cell counts or cytokines between baseline and follow-up, comparing the control and e-cig groups. However, in the intervention but not the control group, change in urinary PG as a marker of e-cig use and inhalation was significantly correlated with change in cell counts (cell concentrations, macrophages, and lymphocytes) and cytokines (IL8, IL13, and TNFα), although the absolute magnitude of changes was small. There were no significant changes in mRNA or miRNA gene expression. Although limited by study size and duration, this is the first experimental demonstration of an impact of e-cig use on inflammation in the human lung among never-smokers.
Abstract Lung cancer exhibits sex-biased molecular characteristics and epidemiological trends, suggesting a need for sex-specific approaches to understanding its etiology and treatment. DNA ...methylation alterations play critical roles in lung carcinogenesis and may serve as valuable biomarkers for precision medicine strategies. We employed the Infinium MethylationEPIC array to identify autosomal sex-related differentially methylated CpG sites (DM-CpGs) in lung epithelium of healthy individuals (32 females and 37 males) while controlling for age, BMI, and tobacco use. We correlated DM-CpGs with gene expression in lung epithelium and immune responses in bronchoalveolar lavage. We validated these DM-CpGs in lung tumors and adjacent normal tissue from The Cancer Genome Atlas (TCGA). Among 522 identified DM-CpGs, 61% were hypermethylated in females, predominantly located in promoter regions. These DM genes were implicated in cell-to-cell signaling, cellular function, transport, and lipid metabolism. Correlation analysis revealed sex-specific patterns between DM-CpGs and gene expression. Additionally, several DM-CpGs were correlated significantly with cytokines (IL-1β, IL-4, IL-12p70, and IFN-γ), macrophage, and lymphocyte counts. Also, some DM-CpGs were observed in TCGA lung adenocarcinoma, squamous cell carcinoma, and adjacent normal tissues. Our findings highlight sex-specific DNA methylation patterns in healthy lung epithelium and their associations with lung gene expression and lung immune biomarkers. These findings underscore the potential role of lung sex-related CpGs as epigenetic predispositions influencing sex disparities in lung cancer risk and outcomes, warranting further investigation for personalized lung cancer management strategies.
Integration of transcriptomic and metabolomic data improves functional interpretation of disease-related metabolomic phenotypes, and facilitates discovery of putative metabolite biomarkers and gene ...targets. For this reason, these data are increasingly collected in large (> 100 participants) cohorts, thereby driving a need for the development of user-friendly and open-source methods/tools for their integration. Of note, clinical/translational studies typically provide snapshot (e.g. one time point) gene and metabolite profiles and, oftentimes, most metabolites measured are not identified. Thus, in these types of studies, pathway/network approaches that take into account the complexity of transcript-metabolite relationships may neither be applicable nor readily uncover novel relationships. With this in mind, we propose a simple linear modeling approach to capture disease-(or other phenotype) specific gene-metabolite associations, with the assumption that co-regulation patterns reflect functionally related genes and metabolites.
The proposed linear model, metabolite ~ gene + phenotype + gene:phenotype, specifically evaluates whether gene-metabolite relationships differ by phenotype, by testing whether the relationship in one phenotype is significantly different from the relationship in another phenotype (via a statistical interaction gene:phenotype p-value). Statistical interaction p-values for all possible gene-metabolite pairs are computed and significant pairs are then clustered by the directionality of associations (e.g. strong positive association in one phenotype, strong negative association in another phenotype). We implemented our approach as an R package, IntLIM, which includes a user-friendly R Shiny web interface, thereby making the integrative analyses accessible to non-computational experts. We applied IntLIM to two previously published datasets, collected in the NCI-60 cancer cell lines and in human breast tumor and non-tumor tissue, for which transcriptomic and metabolomic data are available. We demonstrate that IntLIM captures relevant tumor-specific gene-metabolite associations involved in known cancer-related pathways, including glutamine metabolism. Using IntLIM, we also uncover biologically relevant novel relationships that could be further tested experimentally.
IntLIM provides a user-friendly, reproducible framework to integrate transcriptomic and metabolomic data and help interpret metabolomic data and uncover novel gene-metabolite relationships. The IntLIM R package is publicly available in GitHub ( https://github.com/mathelab/IntLIM ) and includes a user-friendly web application, vignettes, sample data and data/code to reproduce results.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The bovine paranasal sinuses are a group of complex cavernous air-filled spaces, lined by respiratory epithelium, the exact function of which is unclear. While lesions affecting these sinuses are ...occasionally reported in cattle, their microbial flora has not been defined. Furthermore, given that the various bacterial and viral pathogens causing bovine respiratory disease (BRD) persist within herds, we speculated that the paranasal sinuses may serve as a refuge for such infectious agents. The paranasal sinuses of clinically normal cattle (n = 99) and of cattle submitted for post-mortem examination (PME: n = 34) were examined by microbial culture, PCR and serology to include bacterial and viral pathogens typically associated with BRD: Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica and Pasteurella multocida, bovine respiratory syncytial virus (BRSV) and bovine parainfluenza-3 virus (BPIV-3). Overall, the paranasal sinuses were either predominantly sterile or did not contain detectable microbes (83.5%: 94.9% of clinically normal and 50.0% of cattle submitted for PME). Bacteria, including BRD causing pathogens, were identified in relatively small numbers of cattle (<10%). While serology indicated widespread exposure of both clinically normal and cattle submitted for PME to BPIV-3 and BRSV (seroprevalences of 91.6% and 84.7%, respectively), PCR identified BPIV-3 in only one animal. To further explore these findings we investigated the potential role of the antimicrobial molecule nitric oxide (NO) within paranasal sinus epithelium using immunohistochemistry. Expression of the enzyme responsible for NO synthesis, inducible nitric oxide synthase (iNOS), was detected to varying degrees in 76.5% of a sub-sample of animals suggesting production of this compound plays a similar protective role in the bovine sinus as it does in humans.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lung cancer remains the number one cause of cancer-related mortality worldwide, but less known is that lung cancer patients are among the most psychologically disabled of all cancer groups. Patients ...with stage IV non-small cell lung cancer (NSCLC) were studied to test the hypothesis that trajectories of depression and/or anxiety symptoms after diagnosis would show an adverse relationship with survival, beyond relevant controls.
Patients with stage IV NSCLC (n = 157) were enrolled (ClinicalTrials.gov Identifier: NCT03199651) at diagnosis and completed validated measures for depressive symptoms (Patient Health Questionnaire-9) and anxiety symptoms (Generalized Anxiety Disorder-7). Patients were reassessed every 1 to 2 months through 24 months (16 assessments; 80% average completion rate) and survival monitored. Joint statistical models provided simultaneous modeling of longitudinal (psychological) and time-to-event (survival) processes. Control variables were age, sex, marital status, education, smoking status, cancer type, and treatment received.
Depression and anxiety symptoms significantly decreased with time since diagnosis. The 2-year trajectory of depressive symptoms was significantly associated with cancer survival after adjustment for covariates (hazard ratio = 1.09 per unit increase in the Patient Health Questionnaire-9, 95% confidence interval = 1.03-1.15, p = .002). Anxiety was marginally significant in the unadjusted (p = .053) but not the adjusted (p = .39) model.
For the first time, joint model analyses test the interaction of a longitudinal trajectory of psychological symptoms, assessed from diagnosis to 24 months, and cancer survival. New data show the continuation of depressive and anxiety symptoms through treatment and thereafter. Immunotherapy and targeted therapies have dramatically improved survival for patients with advanced NSCLC; however, novel data suggest their benefit may be constrained by depressive symptoms.
Lynch syndrome is the most common cause of inherited endometrial cancer, attributable to germline pathogenic variants (PV) in mismatch repair (MMR) genes. Tumor microsatellite instability (MSI-high) ...and MMR IHC abnormalities are characteristics of Lynch syndrome. Double somatic MMR gene PV also cause MSI-high endometrial cancers. The aim of this study was to determine the relative frequency of Lynch syndrome and double somatic MMR PV.
341 endometrial cancer patients enrolled in the Ohio Colorectal Cancer Prevention Initiative at The Ohio State University Comprehensive Cancer Center from 1/1/13–12/31/16. All tumors underwent immunohistochemical (IHC) staining for the four MMR proteins, MSI testing, and MLH1 methylation testing if the tumor was MMR-deficient (dMMR). Germline genetic testing for Lynch syndrome was undertaken for all cases with dMMR tumors lacking MLH1 methylation. Tumor sequencing followed if a germline MMR gene PV was not identified.
Twenty-seven percent (91/341) of tumors were either MSI-high or had abnormal IHC indicating dMMR. As expected, most dMMR tumors had MLH1 methylation; (69, 75.8% of the dMMR cases; 20.2% of total). Among the 22 (6.5%) cases with dMMR not explained by methylation, 10 (2.9% of total) were found to have Lynch syndrome (6 MSH6, 3 MSH2, 1 PMS2). Double somatic MMR PV accounted for the remaining 12 dMMR cases (3.5% of total).
Since double somatic MMR gene PV are as common as Lynch syndrome among endometrial cancer patients, paired tumor and germline testing for patients with non-methylated dMMR tumor may be the most efficient approach for LS screening.
•2.9% of endometrial cancers (EC) are due to Lynch syndrome.•Double somatic mismatch repair gene mutations (3.5%) are as common as Lynch syndrome (2.9%) among EC patients.•EC with absence of MSH6, PMS2, or microsatellite instability with normal IHC are more likely due to Lynch syndrome.•EC with absence of MLH1 & PMS2 and no MLH1 methylation are more likely due to double somatic mismatch repair mutations.•Endometrial cancers with absence of MSH2 & MSH6 are more likely due to double somatic mismatch repair gene mutations.
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