Microarray amplification bias: loss of 30% differentially expressed genes due to long probe – poly(A)-tail distances

BACKGROUND: Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe - poly(A)-tail distances. RESULTS: Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe - poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe - poly(A)-tail distance >500 nucleotides appearing only after one round of amplification. CONCLUSION: Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA

Analysis of Released Circulating Tumor Cells During Surgery for Non-Small Cell Lung Cancer:are they what they appear to be?

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P <0.01) and in higher counts ( P <0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach

The 15q24/25 Susceptibility Variant for Lung Cancer and Chronic Obstructive Pulmonary Disease Is Associated with Emphysema

RATIONALE: Genome-wide association studies have identified genetic variants in the nicotinic acetylcholine receptor (nAChR) on chromosome 15q24/25 as a risk for nicotine dependence, lung cancer and chronic obstructive pulmonary disease (COPD). Assessment of bronchial obstruction by spirometry, typically used for diagnosing COPD, fails, however, to detect emphysema. OBJECTIVES: To determine the association of the 15q24/25 locus with emphysema. METHODS: The rs1051730 variant on 15q24/25 was genotyped in two independent Caucasian cohorts of 661 and 456 heavy smokers. Participants underwent pulmonary function tests, computed tomography (CT) of the chest and took questionnaires assessing smoking behaviour and health status. MEASUREMENTS AND MAIN RESULTS: The rs1051730 A-allele correlated with reduced forced expiratory volume in 1 second (FEV1) and with increased susceptibility for bronchial obstruction with a pooled odds ratio (OR) of 1.33 (95% confidence interval [CI]=1.11-1.61; P=0.0026). In both studies a correlation between the rs1051730 A-allele and lung diffusing capacity (DLCO) and diffusing capacity per unit alveolar volume (KCO) was observed. Consistently, the rs1051730 A-allele conferred increased risk for emphysema as assessed by CT (P=0.0097 and P=0.019), with a pooled OR of 1.39 (CI=1.15-1.68; P=0.00051). Visual emphysema scores and scores based on densities quantified on CT were more pronounced in A-allele carriers, indicating that rs1051730 correlates with the severity of emphysema. CONCLUSIONS: The 15q24/25 locus in nAChR is associated with the presence and severity of emphysema. This association was independent of pack-years smoking, suggesting that nAChR is causally involved in alveolar destruction, as a potentially shared pathogenic mechanism in lung cancer and COPD.status: publishe

New Fissure-Attached Nodules in Lung Cancer Screening: A Brief Report From The NELSON Study

INTRODUCTION: In incidence lung cancer screening rounds, new pulmonary nodules are regular findings. They have a higher lung cancer probability than baseline nodules. Previous studies have shown that baseline perifissural nodules (PFNs) represent benign lesions. Whether this is also the case for incident PFNs is unknown. This study evaluated newly detected nodules in the Dutch-Belgian randomized-controlled NELSON study with respect to incidence of fissure-attached nodules, their classification, and lung cancer probability. METHODS: Within the NELSON trial, 7557 participants underwent baseline screening between April 2004 and December 2006. Participants with new nodules detected after baseline were included. Nodules were classified based on location and attachment. Fissure-attached nodules were re-evaluated to be classified as typical, atypical, or non-PFN by two radiologists without knowledge of participant lung cancer status. RESULTS: One thousand four hundred eighty-four new nodules were detected in 949 participants (77.4% male, median age 59 years [interquartile range: 55-63 years]) in the second, third, and final NELSON screening round. Based on 2-year follow-up or pathology, 1393 nodules (93.8%) were benign. In total, 97 (6.5%) were fissure-attached, including 10 malignant nodules. None of the new fissure-attached malignant nodules was classified as typical or atypical PFN. CONCLUSIONS: In the NELSON study, 6.5% of incident lung nodules were fissure-attached. None of the lung cancers that originated from a new fissure-attached nodule in the incidence lung cancer screening rounds was classified as a typical or atypical PFN. Our results suggest that also in the case of a new PFN, it is highly unlikely that these PFNs will be diagnosed as lung cancer.status: publishe