Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

<p>Abstract</p> <p>Background</p> <p>The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven.</p> <p>Results</p> <p>We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares <it>GeNorm </it>and <it>NormFinder</it>, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.</p> <p>Conclusion</p> <p>Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.</p

Elevated Levels of the Complement Activation Product C4d in Bronchial Fluids for the Diagnosis of Lung Cancer

Molecular markers in bronchial fluids may contribute to the diagnosis of lung cancer. We previously observed a significant increase of C4d-containing complement degradation fragments in bronchoalveolar lavage (BAL) supernatants from lung cancer patients in a cohort of 50 cases and 22 controls (CUN cohort). The present study was designed to determine the diagnostic performance of these complement fragments (hereinafter jointly referred as C4d) in bronchial fluids. C4d levels were determined in BAL supernatants from two independent cohorts: the CU cohort (25 cases and 26 controls) and the HUVR cohort (60 cases and 98 controls). A series of spontaneous sputum samples from 68 patients with lung cancer and 10 controls was also used (LCCCIO cohort). Total protein content, complement C4, complement C5a, and CYFRA 21-1 were also measured in all cohorts. C4d levels were significantly increased in BAL samples from lung cancer patients. The area under the ROC curve was 0.82 (95% CI = 0.71-0.94) and 0.67 (95% CI = 0.58-0.76) for the CU and HUVR cohorts, respectively. In addition, unlike the other markers, C4d levels in BAL samples were highly consistent across the CUN, CU and HUVR cohorts. Interestingly, C4d test markedly increased the sensitivity of bronchoscopy in the two cohorts in which cytological data were available (CUN and HUVR cohorts). Finally, in the LCCCIO cohort, C4d levels were higher in sputum supernatants from patients with lung cancer (area under the ROC curve: 0.7; 95% CI = 0.56-0.83). In conclusion, C4d is consistently elevated in bronchial fluids from lung cancer patients and may be used to improve the diagnosis of the disease

A novel epigenetic signature for early diagnosis in lung cancer

Purpose: lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies. Experimental design: the identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts. Results: we identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes: BCAT1, CDO1, TRIM58, and ZNF177. Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis. Conclusions: minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients

Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced lung cancer

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n=3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (p(combined)=5.66x10(-5); ORcombined=2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (p(combined)=1.02x10(-4); ORcombined=2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10D mRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p=0.01 and p<0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p=0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC

Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model

<p>Abstract</p> <p>Background</p> <p>There is strong evidence demonstrating that activation of epidermal growth factor receptors (EGFRs) leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown to be relevant drugs for lung cancer treatment. Recent studies demonstrate that lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, is clinically effective against HER-2-overexpressing metastatic breast cancer. In this report, we investigated the activity of lapatinib against non-small cell lung cancer (NSCLC).</p> <p>Methods</p> <p>We selected the lung cancer cell line A549, which harbors genomic amplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenic assays, and signaling cascade analyses (by western blot) were performed <it>in vitro</it>. <it>In vivo </it>experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy) were also carried out.</p> <p>Results</p> <p>Lapatinib dramatically reduced cell proliferation (<it>P </it>< 0.0001), DNA synthesis (<it>P </it>< 0.006), and colony formation capacity (<it>P </it>< 0.0001) in A549 cells <it>in vitro</it>. Furthermore, lapatinib induced G1 cell cycle arrest (<it>P </it>< 0.0001) and apoptotic cell death (<it>P </it>< 0.0006) and reduced cyclin A and B1 levels, which are regulators of S and G2/M cell cycle stages, respectively. Stimulation of apoptosis in lapatinib-treated A549 cells was correlated with increased cleaved PARP, active caspase-3, and proapoptotic Bak-1 levels, and reduction in the antiapoptic IAP-2 and Bcl-xL protein levels. We also demonstrate that lapatinib altered EGFR/HER-2 signaling pathways reducing p-EGFR, p-HER-2, p-ERK1/2, p-AKT, c-Myc and PCNA levels. <it>In vivo </it>experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PET analysis) (<it>P </it>< 0.04) and smaller in size than controls. In addition, tumors from lapatinib-treated mice showed a dramatic reduction in angiogenesis (<it>P </it>< 0.0001).</p> <p>Conclusion</p> <p>Overall, these data suggest that lapatinib may be a clinically useful agent for the treatment of lung cancer.</p

Effectiveness and Safety of the Sequential Use of a Second and Third Anti-TNF Agent in Patients With Inflammatory Bowel Disease: Results From the Eneida Registry

Background: The effectiveness of the switch to another anti-tumor necrosis factor (anti-TNF) agent is not known. The aim of this study was to analyze the effectiveness and safety of treatment with a second and third anti-TNF drug after intolerance to or failure of a previous anti-TNF agent in inflammatory bowel disease (IBD) patients. Methods: We included patients diagnosed with IBD from the ENEIDA registry who received another anti-TNF after intolerance to or failure of a prior anti-TNF agent. Results: A total of 1122 patients were included. In the short term, remission was achieved in 55% of the patients with the second anti-TNF. The incidence of loss of response was 19% per patient-year with the second anti-TNF. Combination therapy (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.8-3; P < 0.0001) and ulcerative colitis vs Crohn's disease (HR, 1.6; 95% CI, 1.1-2.1; P = 0.005) were associated with a higher probability of loss of response. Fifteen percent of the patients had adverse events, and 10% had to discontinue the second anti-TNF. Of the 71 patients who received a third anti-TNF, 55% achieved remission. The incidence of loss of response was 22% per patient-year with a third anti-TNF. Adverse events occurred in 7 patients (11%), but only 1 stopped the drug. Conclusions: Approximately half of the patients who received a second anti-TNF achieved remission; nevertheless, a significant proportion of them subsequently lost response. Combination therapy and type of IBD were associated with loss of response. Remission was achieved in almost 50% of patients who received a third anti-TNF; nevertheless, a significant proportion of them subsequently lost response

Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776260/[EN] Background There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. Methods We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test. Results Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 +/- 0.87 vs 0.16 +/- 0.11 mu g/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 +/- 2.02 vs 1.86 +/- 0.95 mu g/mL, P < 0.001; 3.18 +/- 3.20 vs 1.13 +/- 0.69 mu g/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). Conclusions Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.This work was supported by UTE project CIMA; the Spanish Government (grant numbers ISCIII-RTICC RD06/0020/0066, RD06/0020/1024, RD12/0036/0025, RD12/0036/0040, RD12/0036/0062, PI08/0923, PI10/01652, PI10/00166, and PI11/00618); the European Regional Development Fund; the European Community’s Seventh Framework Programme (HEALTH-F2-2010-258677- CURELUNG); and the Early Detection Research Network from the National Cancer Institute (grant number U01 CA152662). This work was supported (in part) by a grant (RD12/0036/XXXX) from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness & European Regional Development Fund “Una manera de hacer Europa”.Jantus Lewintre, E. (2013). Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer. JNCI: Journal of the National Cancer Institute. 105:1385-1393. https://doi.org/10.1093/jnci/djt205S1385139310

Risk Factors for COVID-19 in Inflammatory Bowel Disease: A National, ENEIDA-Based Case–Control Study (COVID-19-EII)

(1) Scant information is available concerning the characteristics that may favour the acquisition of COVID-19 in patients with inflammatory bowel disease (IBD). Therefore, the aim of this study was to assess these differences between infected and noninfected patients with IBD. (2) This nationwide case-control study evaluated patients with inflammatory bowel disease with COVID-19 (cases) and without COVID-19 (controls) during the period March-July 2020 included in the ENEIDA of GETECCU. (3) A total of 496 cases and 964 controls from 73 Spanish centres were included. No differences were found in the basal characteristics between cases and controls. Cases had higher comorbidity Charlson scores (24% vs. 19%; p = 0.02) and occupational risk (28% vs. 10.5%; p < 0.0001) more frequently than did controls. Lockdown was the only protective measure against COVID-19 (50% vs. 70%; p < 0.0001). No differences were found in the use of systemic steroids, immunosuppressants or biologics between cases and controls. Cases were more often treated with 5-aminosalicylates (42% vs. 34%; p = 0.003). Having a moderate Charlson score (OR: 2.7; 95%CI: 1.3-5.9), occupational risk (OR: 2.9; 95%CI: 1.8-4.4) and the use of 5-aminosalicylates (OR: 1.7; 95%CI: 1.2-2.5) were factors for COVID-19. The strict lockdown was the only protective factor (OR: 0.1; 95%CI: 0.09-0.2). (4) Comorbidities and occupational exposure are the most relevant factors for COVID-19 in patients with IBD. The risk of COVID-19 seems not to be increased by immunosuppressants or biologics, with a potential effect of 5-aminosalicylates, which should be investigated further and interpreted with caution