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Supplementary Tables S1-S2 from Transcription Deregulation at the 15q25 Locus in Association with Lung Adenocarcinoma Risk

Purpose: We characterized the candidacy of the six candidate genes mapping in the chromosome 15q25 locus, which was previously reported as associated with lung cancer risk, and confirmed the locus association with lung cancer risk in an Italian population of lung adenocarcinoma patients and controls.Experimental Design: We did a quantitative analysis of mRNA levels of IREB2 (iron-responsive element-binding protein 2), LOC123688, PMSA4 [proteasome (prosome, macropain) subunit α type 4], CHRNB4 (cholinergic receptor nicotinic β 4), CHRNA3 (cholinergic receptor nicotinic α 3), and CHRNA5 (cholinergic receptor nicotinic α 5) genes in paired normal lung and lung adenocarcinoma tissue, and an immunohistochemical localization of CHRNA3- and CHRNA5-encoded proteins. We also examined the association of CHRNA5 D398N polymorphism with lung cancer risk and with CHRNA5 mRNA levels in the normal lung.Results: Expression analysis of the six candidate genes mapping in the lung cancer risk–associated chromosome 15q25 locus revealed a 30-fold up-regulation of the gene encoding the CHRNA5 subunit and a 2-fold down-regulation of the CHRNA3 subunit in lung adenocarcinoma as compared with the normal lung. The expression of the four other candidate genes resulted either unchanged or absent. The carrier status of the 398N allele at the D398N polymorphism of the CHRNA5 gene was associated with lung adenocarcinoma risk (odds ratio, 1.5; 95% confidence interval, 1.2-2.0) in a population-based series of lung adenocarcinoma patients (n = 467) and healthy controls (n = 739). Analysis of a family-based series of nonsmoker lung cancer cases (n = 80) and healthy sib controls (n = 80) indicated a similar trend. In addition, the same D398N variation correlated with CHRNA5 mRNA levels in normal lung of adenocarcinoma patients.Conclusions: Our results point to the candidacy of the CHRNA5 gene for the 15q25 locus.

Transcripts originating from the transcriptional read through of two adjacent, similarly oriented genes have been identified in normal and neoplastic tissues, but their functional role and the mechanisms that regulate their expression are mostly unknown. Here, we investigated whether the expression of read-through transcripts previously identified in the non-involved lung tissue of lung adenocarcinoma patients was genetically regulated. Data on genome-wide single nucleotide variant genotypes and expression levels of 10 read-through transcripts in 201 samples of lung tissue were combined to identify expression quantitative trait loci (eQTLs). Then, to identify genes whose expression levels correlated with the 10 read-through transcripts, we used whole transcriptome profiles available for 154 patients. For 8 read-though transcripts, we identified 60 eQTLs (false discovery rate

Background: In rapidly lung deteriorating patients, urgent lung transplantation (ULT) seems the only definitive therapy. Few publications on this topic report conflicting results, putting a word of caution about ULT programs. Methods: A national ULT program was introduced in 2010: patients on mechanical support may be transplanted with the first available graft. We reviewed the experience of three national center, focusing on post-operative outcomes after ULT. Results: Ten patients (17.5%) died awaiting transplantation, while 47 underwent LT with a median urgent waiting list time of 6 days. Pre-operatively, 4.3% of patients were supported only by mechanical ventilation (MV), 55.3% by extracorporeal membrane oxygenation (ECMO) and the remaining 40.4% by both. The main indication was cystic fibrosis (64%). Median recipient lung allocation score was 72. In-hospital mortality was 19%. MV and ECMO median duration of 7 and 3 days, respectively while intensive care unit (ICU) and hospital stay were 20 and 46 days, respectively. At long-term, 1- and 3-year survival rate were 74% and 70%, respectively. Highly impact risk factors for in-hospital mortality were both presence and duration of preoperative veno-arterial ECMO and pre-transplant C-reactive protein level. Conclusions : ULT program allows transplantation in a significant percentage of patients with acceptable results. Pre-operative recipient selection is mandatory to improve clinical outcomes.

Background: Despite advances in perioperative care and surgical techniques, patients undergoing pulmonary lobectomy are still at high risk for postoperative complications. Among interventions expected to reduce complications, continuous positive airway pressure (CPAP) is a discussed option. This trial aims to test the hypothesis whether prophylactic application of CPAP following pulmonary lobectomy can reduce postoperative complications. Methods: The study was designed as a prospective, randomized, controlled trial. Patients with clinical stage I non-small cell lung cancer scheduled for pulmonary lobectomy were eligible and were trained for the use of CPAP interface. The control group received standard postoperative pain management and physiotherapy; in addition, the study group received CPAP (PEEP 8–12 cmH 2 O, 2 hours thrice daily for three days). Results: After the appropriate selection, 163 patients were considered for the analysis: 82 patients constituted the control group, 81 the study group. The two groups were substantially comparable for preoperative parameters. The rate of postoperative complications was lower in the study group (24.7% vs . 43.9%; P=0.015) as well as the hospital stay (6 vs . 7 days; P=0.031). The stepwise logistic regression model identified: CPAP [odd ratio (OR): 0.3026, CI: 0.1389–0.6591], smoke habits [OR: 2.5835, confidence interval (CI): 1.0331–6.4610] and length of surgery in minutes (OR: 1.0102, CI: 1.0042–1.0163) as regressors on postoperative complications. Conclusions: The present trial demonstrated that prophylactic application of CPAP during the postoperative period after pulmonary lobectomy for stage I non-small cell lung cancer was effective in prevent postoperative complications.

Multi-institutional studies of endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) for mediastinal staging in lung cancer are scarce. It is unclear if the high diagnostic performance of EBUS-TBNA reported by experts' guidelines can be generally achieved.This is a retrospective study performed in five tertiary referral centers of thoracic surgery in Italy, to assess the EBUS-TBNA diagnostic performance in patients with non-small cell lung cancer (NSCLC). Patient inclusion criteria were: both genders;18 years old; with suspect/confirmed NSCLC; undergoing EBUS-TBNA for mediastinal node enlargement at computed tomography (size1 cm, ≤3 cm) and/or pathological uptake at positron emission tomography. Altogether we included 485 patients [male, 366; female, 119; median age, 68 years (IQR, 61-74 years)] undergoing mediastinal staging between January 2011 and July 2016. All EBUS-TBNAs were performed by experienced bronchoscopists, without pre-defined quality standards. Depending on usual practice in each center, EBUS-TBNA was done under conscious sedation, with 21- or 22-Gauge (G) needle, and specimen preparation was cell-block, or cytology slides, or core-tissue. Sampling was classified inadequate in absence of lymphocytes, or when sample was insufficient. We analyzed the EBUS-TBNA procedural steps likely to influence the rate of adequate samplings (diagnostic yield).EBUS-TBNA sensitivity, negative predictive value (NPV) and accuracy respectively were 90%, 78% and 93% in the whole cohort. At multivariate analysis, use of 21-G needle was associated with better diagnostic yield (P0.001). Center and specimen processing technique were not independent factors affecting EBUS-TBNA diagnostic yield.In this multicentric study, EBUS-TBNA was a highly sensitive and accurate method for NSCLC mediastinal node staging. Results indicate better performance of EBUS-TBNA with 21-G needle, and suggest that specimen processing technique could be chosen according to the local practice preference.

Introduction Lung transplantation is considered a therapeutic option in selected patients affected by end-stage pulmonary disease. The mortality on the waiting list is mainly attributed to the shortage of the donor pool available for transplantation. There are various strategies to overcome this shortage; one of them is lobar transplantation. Methods The aim of the current study was to analyze the outcome of lobar lung transplantation from deceased donors in our Lung Transplant Center. Overall survival, perioperative mortality and morbidity, problem on bronchial anastomosis, and chronic rejection were prospectively recorded in a 5-year time-frame. Results From November 2010 to October 2015, we performed 100 lung transplantations; 6 of which (6%) were lobar transplantations from deceased donors. Three recipients were on an emergency list due to preoperative extracorporeal support. The causes of lobectomy leading to lobar transplantation were: size mismatch (3), iatrogenic vascular damage (2), and chronic atelectasis (1). One patient died 5 months after surgery for sepsis; and 5 patients were alive at the study end (median follow-up: 17.5 months). Prevalence of grade 3 primary graft dysfunction at 72 hours was 50%. One patient developed bronchial stenosis. No cases of chronic rejection were recorded. Conclusions Lobar transplantation can be considered a valid tool to overcome the donor pool shortage in selected cases; such a technique has proved particularly useful in critically ill patients who were scheduled in an emergency transplant program.

VATS lobectomy was first described as a surgical treatment for lung cancer about 25 years ago (1); after initial scepticism concerning it, VATS technique has progressively spread in many centers worldwide.

Objectives A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. Methods Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. Results Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor–recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-β-lactamase (VIM)-producing Enterobacter aerogenes. Conclusions This study showed a low risk of donor–recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Alterations in the gene expression of organs in contact with the environment may signal exposure to toxins. To identify genes in lung tissue whose expression levels are altered by cigarette smoking, we compared the transcriptomes of lung tissue between 118 ever smokers and 58 never smokers. In all cases, the tissue studied was non-involved lung tissue obtained at lobectomy from patients with lung adenocarcinoma. Of the 17,097 genes analyzed, 357 were differentially expressed between ever smokers and never smokers (FDR MYO1A (FDR = 6.9 × 10−4) while the gene with the largest difference between groups was FGG (fold change = 31.60). Overall, 100 of the genes identified in this study (38.6%) had previously been found to associate with smoking in at least one of four previously reported datasets of non-involved lung tissue. Seven genes (KMO, CD1A, SPINK5, TREM2, CYBB, DNASE2B, FGG) were differentially expressed between ever and never smokers in all five datasets, with concordant higher expression in ever smokers. Smoking-induced up-regulation of six of these genes was also observed in a transcription dataset from lung tissue of non-cancer patients. Among the three most significant gene networks, two are involved in immunity and inflammation and one in cell death. Overall, this study shows that the lung parenchyma transcriptome of smokers has altered gene expression and that these alterations are reproducible in different series of smokers across countries. Moreover, this study identified a seven-gene panel that reflects lung tissue exposure to cigarette smoke.

Lung transplantation (LTx) in advanced stage chronic obstructive pulmonary disease (COPD) patients is associated with significant improvement in lung function and exercise capacity. However, demonstration that the procedure also provides a survival benefit has been more elusive compared to other respiratory conditions. Identification of patients with increased risk of mortality is crucial: a low forced expiratory volume in 1 second (FEV 1 ) is perhaps the most common reason for referral to a lung transplant center, but in itself is insufficient to identify which COPD patients will benefit from LTx. Many variables have to be considered in the selection of candidates, time for listing, and choice of procedure: age, patient comorbidities, secondary pulmonary hypertension, the balance between individual and community benefit. This review will discuss patient selection, transplant listing, potential benefits and critical issues of bilateral (BLTx) and single lung (SLTx) procedure, donor-to-recipient organ size-matching; furthermore, it will describe LTx outcomes and its effects on recipient survival and quality of life.

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy., Highlights • Transcriptome analysis performed on tumor-resident CD4+ Th1, Th17, and Treg cells • Tumor-infiltrating Treg cells are defined by the expression of signature genes • Treg-specific signature genes correlate with patients’ survival in both CRC and NSCLC, Tumor-infiltrating regulatory T cells can suppress effector T cells specific for tumor antigens. De Simone et al. (2016) demonstrate that tumor-infiltrating Treg cells display specific gene signatures that were also validated at the single-cell level. These data can contribute to dissect the molecular networks underlying the biology of tumor-infiltrating Treg cells. As part of the IHEC consortium, this study integrates genetic, epigenetic, and transcriptomic profiling in three immune cell types from nearly 200 people to characterize the distinct and cooperative contributions of diverse genomic inputs to transcriptional variation. Explore the Cell Press IHEC webportal at www.cell.com/consortium/IHEC.

Ex vivo lung perfusion (EVLP) has become a reality as a technique to evaluate and recondition lungs from marginal donors. We report the first case on the use of EVLP followed by separate transplantation in two different centres. The local organ procurement organization proposed the lungs of a 53-year-old non-smoker donor who died for cerebral haemorrhage. P/F ratio was 294 after lung recruitment manoeuvres. Oto score was 10. Two centres accepted the grafts for two single transplantations under the condition of EVLP evaluation. After usual retrieval, the bi-pulmonary block was transferred to Centre 1 and EVLP was run as previously described. At the end of the procedure the two lungs were evaluated separately and both judged suitable for transplantation. After cooling and storage on ice, the block was separated on the back table. The left lung was transplanted in a patient with pulmonary fibrosis at Centre 1; surgery was complicated by cardiac arrhythmias that required several defibrillations. The right lung was transferred on ice to Centre 2, 250 km away from Centre 1, and transplanted in a patient with idiopathic pulmonary fibrosis. Thirty months after transplantations Patient 1 and Patient 2 are both alive, in good clinical conditions. This is the first report of the separate use of lungs after EVLP for non-urgent recipients in two different centres. This experience opens the door to a new allocation model with great potentials on organ shortage. Actually, we demonstrated that the perspective of a ‘lung repair centre’ is feasible and effective.

The lung transplantation (LT) follow-up required several computed tomography (CT) controls. The radiological risk is disappointing in patients treated with immunosuppressive drugs and with young age. Regional intensity variations between different lung volumes acquired by CT (Aliverti et al, ERJ 2013) and by MRI (Pennati et al, Rad 2014) can be used to estimate the regional ventilation. The aim of this transversal study is to compare maps of CT-density difference (ΔHU) with the corresponding maps of proton-density difference (Δ 1 H-MRI) after LT. CT and 1 H-MRI of the lungs were scheduled on the same day at full inspiration (INSP) and full expiration (EXP). Corresponding slices at six lung levels were selected at EXP, INSP. EXP images were registered onto INSP by optical flow, and maps of ΔHU and Δ 1 H-MRI were obtained with pixel-by-pixel subtraction (Figure1). Difference maps were compared by computing the linear correlation between the median values of ΔHU and Δ 1 H-MRI in six corresponding regions at the six lung levels. Ten consecutive patients were recruited; the ΔHU and Δ 1 H-MRI correlation coefficient was R 2 =0.56, P 1 H-MRI demonstrate good correlation after LT. The multivolume MRI may be a valid non-ionizing imaging alternative to CT to describe the ventilation distribution in the follow-up of lung transplanted patients.

Many single nucleotide polymorphisms (SNPs) have been associated with lung cancer but lack confirmation and functional characterization. We retested the association of 56 candidate SNPs with lung adenocarcinoma risk and overall survival in a cohort of 823 Italian patients and 779 healthy controls, and assessed their function as expression quantitative trait loci (eQTLs). In the replication study, eight SNPs (rs401681, rs3019885, rs732765, rs2568494, rs16969968, rs6495309, rs11634351, and rs4105144) associated with lung adenocarcinoma risk and three (rs9557635, rs4105144, and rs735482) associated with survival. Five of these SNPs acted as cis-eQTLs, being associated with the transcription of IREB2 (rs2568494, rs16969968, rs11634351, rs6495309), PSMA4 (rs6495309) and ERCC1 (rs735482), out of 10,821 genes analyzed in lung. For these three genes, we obtained experimental evidence of differential allelic expression in lung tissue, pointing to the existence of in-cis genomic variants that regulate their transcription. These results suggest that these SNPs exert their effects on cancer risk/outcome through the modulation of mRNA levels of their target genes.

OBJECTIVES The video-assisted thoracic surgery (VATS) approach is encouraged over postero-lateral thoracotomy (PLT) for lobectomy in lung cancer. We compare the ribcage kinematics during exercise before and after both procedures, assuming that VATS, being minimally invasive, could better preserve ribcage expansion. METHODS Thirty-one patients undergoing lobectomy by means of VATS (n = 20) or PLT (n = 11) were compared presurgery, after chest drainage removal (T1) and 2 months post-surgery (T2) during quiet breathing and incremental exercise. Spirometry, chest pain, ventilatory pattern and expansions of the ribcage (ΔVRC) and abdomen were measured. Furthermore, the expansion of the ribcage and abdomen in the operated (ΔVRC-OP and ΔVAB-OP, respectively) and non-operated (ΔVRC-NO and ΔVAB-NO, respectively) sides was also considered. RESULTS At T1, in both groups, spirometry worsened and chest pain increased, being higher after PLT. Tidal volume (VT) decreased after PLT because the ribcage expanded less due to reduced ΔVRC-OP. Contrary to this, in VATS, there were no changes in VT and ΔVRC, although ΔVRC-OP was lower, because ΔVRC-NO increased at high level of exercise. At T2, ΔVRC-OP was completely restored after VATS. At high levels of exercise following PLT, although patients still showed reduced ΔVRC and ΔVRC-OP, VT was restored owing to increased ΔVAB-NO. CONCLUSIONS We demonstrate VATS to have a reduced impact on ribcage kinematics while PLT induced restriction more markedly during exercise and still present 2 months after surgery. Patients adopt 2 different compensatory mechanisms, by shifting the expansion toward the contralateral ribcage after VATS and toward the abdomen after PLT. Our study justifies thoracoscopic lobectomy prompt recovery. Clinical trial registration clinicaltrials.gov (NCT02910453).

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p

This paper describes the initial clinical experience of ex vivo lung perfusion (EVLP) at the Fondazione Ca' Granda in Milan between January 2011 and May 2013. EVLP was considered if donor PaO2 /FiO2 was below 300 mmHg or if lung function was doubtful. Donors with massive lung contusion, aspiration, purulent secretions, pneumonia, or sepsis were excluded. EVLP was run with a low-flow, open atrium and low hematocrit technique. Thirty-five lung transplants from brain death donors were performed, seven of which after EVLP. EVLP donors were older (54 ± 9 years vs. 40 ± 15 years, EVLP versus Standard, P < 0.05), had lower PaO2 /FiO2 (264 ± 78 mmHg vs. 453 ± 119 mmHg, P < 0.05), and more chest X-ray abnormalities (P < 0.05). EVLP recipients were more often admitted to intensive care unit as urgent cases (57% vs. 18%, P = 0.05); lung allocation score at transplantation was higher (79 [40-84] vs. 39 [36-46], P < 0.05). After transplantation, primary graft dysfunction (PGD72 grade 3, 32% vs. 28%, EVLP versus Standard, P = 1), mortality at 30 days (0% vs. 0%, P = 1), and overall survival (71% vs. 86%, EVLP versus Standard P = 0.27) were not different between groups. EVLP enabled a 20% increase in available donor organs and resulted in successful transplants with lungs that would have otherwise been rejected (ClinicalTrials.gov number: NCT01967953).

BackgroundSex and age strongly influence the pathophysiology of human lungs, but scarce information is available about their effects on pulmonary gene expression.MethodsWe followed a discovery-validation strategy to identify sex-and age-related transcriptional differences in lung.ResultsWe identified transcriptional profiles significantly associated with sex (215 genes; FDR ConclusionsGene expression in non-tumor lung tissue is modulated by both sex and age. These findings represent a validated starting point for research on the molecular mechanisms underlying the observed differences in the course of lung diseases among men and women of different ages.

Background The use of extracorporeal membrane oxygenation (ECMO) as a bridge to lung transplant (LTX) is still being debated. Methods We performed a retrospective two-center analysis of the relationship between ECMO bridging duration and survival in 25 patients. Further survival analysis was obtained by dividing the patients according to waiting time on ECMO: up to 14 days (Early group) or longer (Late group). We also analyzed the impact of the ventilation strategy during ECMO bridging (ie, spontaneous breathing and noninvasive ventilation [NIV] or intubation and invasive mechanical ventilation [IMV]). Results Seventeen of 25 patients underwent a transplant (with a 76% 1-year survival), whereas eight patients died during bridging. In the 17 patients who underwent a transplant, mortality was positively related to waiting days until LTX (hazard ratio [HR], 1.12 per day; 95% CI, 1.02-1.23; P = .02), and the Early group showed better Kaplan-Meier curves (P = .02), higher 1-year survival rates (100% vs 50%, P = .03), and lower morbidity (days on IMV and length of stay in ICU and hospital). During the bridge to transplant, mortality increased steadily with time. Considering the overall outcome of the bridging program (25 patients), bridge duration adversely affected survival (HR, 1.06 per day; 95% CI, 1.01-1.11; P = .015) and 1-year survival (Early, 82% vs Late, 29%; P = .015). Morbidity indexes were lower in patients treated with NIV during the bridge. Conclusions The duration of the ECMO bridge is a relevant cofactor in the mortality and morbidity of critically ill patients awaiting organ allocation. The NIV strategy was associated with a less complicated clinical course after LTX.

// Giulia Pintarelli 1, * , Alice Dassano 1, * , Chiara E. Cotroneo 1, 6 , Antonella Galvan 7 , Sara Noci 1 , Rocco Piazza 2, 3 , Alessandra Pirola 2 , Roberta Spinelli 8 , Matteo Incarbone 4 , Alessandro Palleschi 5 , Lorenzo Rosso 5 , Luigi Santambrogio 5 , Tommaso A. Dragani 1 , Francesca Colombo 1 1 Department of Predictive and Prevention Medicine, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Health Sciences, University of Milano-Bicocca, Monza, Italy 3 Hematology and Clinical Research Unit, San Gerardo Hospital, Monza, Italy 4 Department of Surgery, San Giuseppe Hospital, Multimedica, Milan, Italy 5 Department of Surgery, IRCCS Fondazione Ca Granda Ospedale Maggiore Policlinico, Universita degli Studi di Milano, Milan, Italy 6 Present Address: UCD School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland 7 Formerly, Department of Predictive and Prevention Medicine, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy 8 Formerly, Department of Health Sciences, University of Milano-Bicocca, Monza, Italy * These authors have contributed equally to this work Correspondence to: Tommaso A. Dragani, e-mail: tommaso.dragani@istitutotumori.mi.it Keywords: conjoined genes, gene fusion, lung adenocarcinoma, RNA-Seq, read-through transcripts Received: November 17, 2015 Accepted: February 18, 2016 Published: April 02, 2016 ABSTRACT Read-through transcripts result from the continuous transcription of adjacent, similarly oriented genes, with the splicing out of the intergenic region. They have been found in several neoplastic and normal tissues, but their pathophysiological significance is unclear. We used high-throughput sequencing of cDNA fragments (RNA-Seq) to identify read-through transcripts in the non-involved lung tissue of 64 surgically treated lung adenocarcinoma patients. A total of 52 distinct read-through species was identified, with 24 patients having at least one read-through event, up to a maximum of 17 such transcripts in one patient. Sanger sequencing validated 28 of these transcripts and identified an additional 15, for a total of 43 distinct read-through events involving 35 gene pairs. Expression levels of 10 validated read-through transcripts were measured by quantitative PCR in pairs of matched non-involved lung tissue and lung adenocarcinoma tissue from 45 patients. Higher expression levels were observed in normal lung tissue than in the tumor counterpart, with median relative quantification ratios between normal and tumor varying from 1.90 to 7.78; the difference was statistically significant ( P < 0.001, Wilcoxon’s signed-rank test for paired samples) for eight transcripts: ELAVL1–TIMM44, FAM162B–ZUFSP, IFNAR2–IL10RB, INMT–FAM188B, KIAA1841–C2orf74, NFATC3–PLA2G15, SIRPB1–SIRPD , and SHANK3–ACR . This report documents the presence of read-through transcripts in apparently normal lung tissue, with inter-individual differences in patterns and abundance. It also shows their down-regulation in tumors, suggesting that these chimeric transcripts may function as tumor suppressors in lung tissue.

Posterior reversible encephalopathy syndrome is a neurological problem characterized by headache, altered mental status, focal neurological deficits, visual disorders, and seizures. The disorder is related to a number of diseases including calcineurin inhibitor therapy in solid organ transplantation. The incidence of posterior reversible encephalopathy syndrome in lung transplantation patients is unclear; probably the majority of the cases are unreported. The authors have described a case series constituted of four patients presenting posterior reversible encephalopathy syndrome after bilateral lung transplantation. The cases had in common complicated surgery and a posttransplant course characterized by hypertension, hypomagnesemia and acidosis. Invasive mechanical ventilation, calcineurin inhibitor discontinuation, aggressive antihypertensive therapy, and electrolyte regulation led to near complete recovery of symptoms.

Tumors successfully adapt to constantly changing intra- and extra-cellular environments, but the wirings of this process are still largely elusive. Here, we show that Heat Shock Protein 90 (HSP90)-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase (AMPK), inhibition of rapamycin-sensitive mTOR complex 1 (mTORC1), induction of autophagy, and expression of an endoplasmic reticulum (ER) unfolded protein response (UPR). This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial HSP90s are adaptive regulators of tumor bioenergetics, and tractable targets for cancer therapy.

Ex vivo lung perfusion (EVLP) has been validated as a valuable technique to increase the pool of organs available for lung transplantation.After a preclinical experience, we obtained permission from the Ethics Committee of our institution to transplant lungs after EVLP reconditioning. ABO compatibility, size match, and donor arterial oxygen pressure (PaO(2))/fraction of inspired oxygen (FiO(2)) ≤ 300 mm Hg were considered to be inclusion criteria, whereas the presence of chest trauma and lung contusion, evidence of gastric content aspiration, pneumonia, sepsis, or systemic disease were exclusion criteria. We only considered subjects on an extra corporeal membrane oxygenation (ECMO) bridge to transplantation with rapid functional deterioration. Using Steen solution with packed red blood cells oxygenated with 21% O(2), 5% to 7% CO(2) was delivered, targeted with a blood flow of approximately 40% predicted cardiac output. Once normothermic, the lungs were ventilated with a tidal volume of 7 mL/kg a PEEP of 5 cmH(2)O and a respiratory rate of 7 bpm. Lungs were considered to be suitable for transplantation if well oxygenated [P(v-a) O(2)350 mm Hg on FiO(2) 100%], in the absence of deterioration of pulmonary vascular resistance and lung mechanics over the perfusion time.From March to September 2011, six lung transplantations were performed, including two with EVLP. The functional outcomes were similar between groups: at T72 posttransplantation, the median PaO(2)/FiO(2) were 306 mm Hg (range, 282 to 331 mm Hg) and 323 mm Hg (range, 270 to 396 mm Hg) (P = 1, EVLP versus conventional). Intensive care unit ICU and hospital length of stay were similar (P = .533 and P = .663, respectively) with no mortality at 60 days in both groups. EVLP donors were older (49 ± 6 y versus 21 ± 7 y, P.05), less well oxygenated (184 ± 6 mm Hg versus 570 ± 30, P.05), displaying higher Oto scores (9.5 ± 0.7 versus 1.7 ± 1.5, P.05).The first 6 months of the EVLP program allowed us to increase the number of organs available for transplantation with short-term outcomes comparable to conventional transplantations.

The majority of stage I lung cancer patients undergo a complete resection of their tumor; however, they still harbor a considerable risk of mortality due to recurrences. A correlation between the presence of lymph node micrometastases and poor prognosis has been observed. The aim of this study was to correlate the lymph node molecular staging with the 5-year survival and disease-free interval following pulmonary lobectomy for non-small cell lung cancer (NSCLC). A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) mRNA was performed on primary lung tumors and regional lymph nodes from 55 surgically resected NSCLC patients classified as clinical stage I. CEA mRNA was found to be present in all the primary tumors. RT-PCR revealed the presence of cancer cells in the lymph nodes of 20 patients (36.3%) and routine staining detected lymph node metastases in 11 patients. Significant differences in survival and disease-free intervals were observed in patients with lymph node micrometastases versus patients with negative lymph nodes (P=0.0026 and P=0.0044, respectively). Multivariate analyses confirmed that micrometastases were an independent predictor for worse prognosis (P=0.0098) and a short disease-free interval (P=0.0137). This study demonstrated strong correlations between the molecular detection of lymph node micrometastases and 5-year survival rates and disease-free interval in patients who underwent pulmonary lobectomy for early-stage lung cancer.

Associations between clinical outcome of cancer patients and the gene expression signature in primary tumors at time of diagnosis have been reported. To test whether gene expression patterns in noninvolved lung tissue might correlate with clinical stage in lung adenocarcinoma (ADCA) patients, we compared the transcriptome of noninvolved lung samples from 60 ADCA smoker patients of clinical stage I versus 60 patients with stage >I. Quantitative PCR of 10 genes with the most significant differential expression confirmed the statistical association with clinical stage in eight genes, six of which were downregulated in high-stage patients. Five of these six genes were also downregulated in lung ADCA tissue as compared to noninvolved tissue. Studies in vitro indicated that four of the genes (SLC14A1, SMAD6, TMEM100 and TXNIP) inhibited colony formation of lung cancer cell lines transfected to overexpress the genes, suggesting their potential tumor-suppressor activity. Our findings suggest that individual variations in the transcriptional profile of noninvolved lung tissue may reflect the lung ADCA patient's predisposition to tumor aggressiveness.

This study was planned to observe prospectively the effects of standard open pulmonary lobectomy on leukocyte subsets and their connection with oncological outcome. Leukocyte subsets from 200 patients undergoing pulmonary lobectomy were analyzed: 151 patients had non-small-cell lung cancer, and 49 had non-malignant diseases. Blood samples were taken for leukocyte flow cytometry before and five, 30 and 60 days after operation. The end points were: observation of postoperative leukocyte subsets that are dynamic in patients with lung cancer vs. patients without malignant disease; correlations between leukocyte subsets trend and disease-free interval or survival; and identification of prognostic factors related to preoperative leukocyte subsets. Lymphocyte counts significantly decreased at five days after lobectomy while monocyte counts increased, and complete recovery of the preoperative leukocyte setting was documented at 30 and 60 days. The patients with lung cancer showed a significant low percentage of human leukocyte antigens on their monocytes before surgery (P=0.0017), followed by a peculiar disarrangement of leukocytes subsets compared with patients without malignant disease at the five-day control point. There was no correlation between leukocyte subset dynamics and disease free interval or survival. This study proves reductions of T-, B and natural killer cells, and the expression of DR on T-lymphocyte after pulmonary lobectomy; oncological patients were significantly less affected by surgery than non-cancer patients.

Introduction. Ex vivo lung perfusion (EVLP) has been recently proposed to recondition organs before transplantation from donors with marginal or unacceptable features. The aim of our investigation was to explore glucose consumption during EVLP. Materials and Methods. We investigated 8 domestic pigs (mean weight, 21 ± 0.8 kg). After perfusion with Perfadex, retrieval, and back table surgery, we initiated EVLP. The lungs were perfused with Steen solution with added methylprednisolone, cefazoline, and heparin. The blood flow was gradually increased with a target of 40% of the estimated cardiac output (or less if the pulmonary artery pressure was >15 mm Hg), while keeping the left atrial pressure between 3 and 5 mm Hg. The temperature of the perfusate was increased from 25°C to 37°C. Once the temperature of the lung outflow was >32°C, we began gas flow (4 L/min, 5%-8% CO 2 in air) and mechanical ventilation. EVLP parameters and blood gases were measured throughout the experiment; glucose consumption was calculated as (glucose initial-glucose final)/time. The wet to dry ratio was also calculated as an index of lung edema. Results. When stratified by median glucose consumption (0.237 mg/min), high glucose consumers (0.588 ± 0.17) were characterized by worse lung function, as assessed by oxygenation (partial pressure of oxygen/inspiratory fraction of oxygen [PaO 2 /FiO 2 ] 326 ± 63 mm Hg vs 218 ± 84; P = .083 low vs high, respectively), and lung edema (wet/dry ratio 6.5 ± 0.7 vs 8.6 ± 0.9; P = .012). Glucose consumption correlated with wet to dry ratio (R 2 = 0.663; P = .014). Conclusions. We found that the worse the lung function, the greater the consumption of glucose during EVLP. This observation suggests the need to explore lung metabolism during EVLP to possibly obtain metrics for evaluation.

Purpose: The main prognostic factor of lung cancer patient outcome is clinical stage, a parameter of tumor aggressiveness. Our study was conducted to test whether germ line variations modulate individual differences in clinical stage. Experimental Design: We conducted a case-only genome-wide association study (GWAS) using a 620,901 single-nucleotide polymorphism (SNP) array in a first series of 600 lung adenocarcinoma (ADCA) patients and in a replication series of 317 lung ADCA patients. Results: GWAS identified 54 putatively associated SNPs, 3 of which were confirmed in the replication series. Joint analysis of the two series pointed to 22 statistically associated (P < 0.01) genetic variants that together explained about 20% of the phenotypic variation in clinical staging (P < 2 × 10−16) and showed a statistically significant difference in overall survival (P = 8.0 × 10−8). The strongest statistical association was observed at rs10278557 (P = 1.1 × 10−5), located in the mesenchyme homeobox 2 (MEOX2) gene. Conclusion: These data point to the role of germ line variations involving multiple loci in modulating clinical stage and, therefore, prognosis in lung ADCA patients. Clin Cancer Res; 17(8); 2410–6. ©2011 AACR.

Among patients with respiratory insufficiency awaiting lung transplantation, small adult patients have a lower opportunity of receiving size-matched pulmonary grafts, because of the shortage of donors, particularly those of small size. Reducing the size of an oversized graft is one of the methods to increase the donor pool; similarly, ex vivo lung perfusion is an emerging technique aimed toward the same purpose. We describe how we combined the 2 techniques (lobar transplantation plus contralateral nonanatomic graft reduction during ex vivo lung perfusion) to overcome graft shortage in a clinical case. For the 1st time, this case report demonstrates that surgical manipulation during ex vivo lung perfusion does not affect the functional improvement in a lung previously judged to be not suitable for transplantation. The 6-month follow-up results are similar to those of standard bilateral lung transplantation.

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p0.01). We then genotyped these candidate SNPs in a validation series of 748 patients from Italy that resulted genetically compatible with the UK series based on principal component analysis. In a Cox proportional hazard model adjusted for age, sex and clinical stage, four SNPs were confirmed on the basis of their having a hazard ratio (HR) indicating the same direction of effect in the two series and p0.05. The strongest association was provided by rs2107561, an intronic SNP of PTPRG, protein tyrosine phosphatase, receptor type, G; the C allele was associated with poorer survival in both patient series (pooled analysis loge HR = 0.31; 95% CI: 0.15-0.46, p = 8.5 × 10(-5) ). PTPRG mRNA levels in 43 samples of lung adenocarcinoma were 40% of those observed in noninvolved lung tissue from the same patients. PTPRG overexpression significantly inhibited the clonogenicity of A549 lung carcinoma cells and the anchorage-independent growth of the NCI-H460 large cell lung cancer line. These four germline variants represent promising candidates that, with further study, may help predict clinical outcome. In addition, the PTPRG locus may have a role in tumor progression.

Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca2+-activated Cl- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca2+-dependent Cl- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways.

Survivin is an inhibitor of apoptosis protein, overexpressed in most human malignancies and implicated in mitosis regulation and preservation of cell viability. In order to investigate the prevalence and clinical significance of survivin in early-stage non-small cell lung carcinoma (NSCLC), survivin mRNA levels and protein expression were evaluated, using quantitative real-time RT-PCR and immunohistochemistry, respectively, in a series of 83 patients with stage I (IA and IB) surgically resected NSCLC. Detectable survivin mRNA levels could be demonstrated in all non-neoplastic lung tissue samples and in the tumours analysed. Survivin mRNA levels were elevated in 80 carcinomas (96%) compared to normal lung (p = 0.008). Among all tumours, survivin transcripts were present at a higher level in squamous cell carcinomas (p = 0.0022). Cytoplasmic and nuclear immunoreactivity was found in 70% and 80% of tumours, respectively and both were present in 54%. Cytoplasmic immunoreactivity correlated with tumour stage (p = 0.019). Survivin expression levels did not correlate with patient survival. In one specimen, cytoplasmic and focal nuclear immunostaining was observed in dysplastic bronchial squamous metaplasia. These results document that survivin overexpression is almost always present in early-stage NSCLC, suggesting that this protein may play a role in lung tumourigenesis. This ubiquitous expression makes survivin an appealing new target for novel therapies in lung cancer. In addition, this study also documents that survivin overexpression could be exploited for diagnostic purposes and that quantitative real-time RT-PCR can be a useful tool for evaluating survivin activation in NSCLC.

Preliminary studies have shown that (99m)Tc-hexakis-2-methoxyisobutylisonitrile (MIBI) is an interesting tracer for various tumors. The aim of this study was to determine the feasibility of using (99m)Tc-MIBI as a diagnostic and staging procedure for lung cancer.We prospectively compared the results of biopsy with (99m)Tc-MIBI imaging in patients with potentially resectable lung lesions (stages IIIA or lower). In the patients with radiopharmaceutical uptake, the staging provided by CT was compared with that obtained with (99m)Tc-MIBI.Ninety-nine of the 116 patients examined had lung cancer. For the diagnosis of malignancy, the specificity of (99m)Tc-MIBI was 100%, sensitivity was 89.8%, positive predictive value was 100%, negative predictive value was 62.9%, and accuracy was 91.4%. In the 87 patients with radiopharmaceutical uptake in their lung cancer, the values for the specificity and sensitivity of (99m)Tc-MIBI in the detection of mediastinal lymph node metastases were 100% and 54.5%, respectively. The corresponding values for CT in the same patients were 87.6% and 63.3%, respectively. The difference in specificity is statistically significant (p = 0.011).This study demonstrates that (99m)Tc-MIBI provides significant diagnostic and staging information in patients with lung lesions. The high specificity and positive predictive value of (99m)Tc-MIBI suggest that this radiopharmaceutical could be a very useful tool for the diagnosis of lung cancer, especially in consideration of its low costs and wide availability.

Objectives Preventive analgesia is defined as a treatment that is commenced before the surgical procedure in order to diminish the physiological consequences of afferent nociceptive transmission caused by the procedure and prevent central sensitization. The analysis of randomized studies of preventive analgesia is controversial. The aim of this study was to check the analgesic efficacy of preoperative administration of dextromethorphan associated with intercostal nerve block with levobupivacaine in thoracotomy patients who refused or had a contraindication to epidural analgesia. Methods This study was a four-arm, double-blinded, randomized placebo-controlled trial. Patients were allocated following close block randomization into four arms: 'Group A' preoperative dextromethorphan and preoperative intercostal block (IB), 'Group B' preoperative placebo and preoperative IB, 'Group C' preoperative dextromethorphan and postoperative IB, 'Group D' preoperative placebo and postoperative block. The primary end-point was the cumulative morphine consumption (CMC) within the first 14 days after surgery. Results A total of 400 patients were enrolled and 395 completed the study. There were no statistical differences among the groups in terms of demographic and surgical data; in contrast, preoperative quality-of-life scores were heterogeneous. The mean CMCs were as follows: Group A 111.4 mg, Group B 121.5 mg, Group C 126.8 mg, Group D 138.3 mg. Group A mean was lower than the maximum (P = 0.0001). The CMC value did not correlate with age, sex, body mass index, education, type of surgery, length or width of the incision and rib fracture. Postoperative functional data and post-thoracotomy syndrome prevalence were homogeneous; female gender resulted predictive for post-thoracotomy syndrome. Conclusions Results indicate that preoperative administration of dextromethorphan associated with preoperative IB with levobupivacaine provided preventive analgesia, decreasing analgesic administration during the early postoperative period compared with placebo and/or postoperative IB. This study failed in detecting any effect of preventive analgesia on functional items and post-thoracotomy syndrome.

After normothermic ex vivo lung perfusion (EVLP), pulmonary grafts are usually flush-cooled and stored on ice until implantation although evidence for this practice lacks. We compared outcomes between 2 post-EVLP preservation strategies in a porcine left single-lung transplantation model.After cold flush and 2-h EVLP, donor lungs were prepared and split. In [C], (n = 5) lungs cooled on device to 15°C were preserved in ice-water; in [W] (n = 5), lungs were disconnected from EVLP at 37°C and kept at room temperature. The left lung was transplanted in a recipient animal. Posttransplant, 6 h-monitoring included hourly assessment of pulmonary vascular resistance, pulmonary artery pressure, plateau airway pressure, compliance, and oxygenation before and after exclusion of the right lung. Lung biopsies and bronchoscopy with bronchoalveolar lavage (BAL) were performed at retrieval, at the end of EVLP (R lung), and 1 and 6 h after reperfusion (L lung).Lungs in [W] showed the highest compliance (P 0.05) and the lowest plateau airway pressure (not statistically significant) throughout the whole reperfusion period. Oxygenation and pulmonary artery pressure were similar between groups. Pulmonary vascular resistance was stable in [C], but rose after reperfusion in [W]. Histologic signs of lung injury and BAL neutrophilia were more pronounced in [C] at 1 h (not statistically significant and P 0.05, respectively). BAL cytokine levels and lung tissue expression of intercellular adhesion molecule 1 did not differ between groups.Normothermic preparation after EVLP results in similar graft performances compared with lung cooling after EVLP.

Background: Surgery with pleurectomy/decortication (P/D) or extrapleural pneumonectomy (EPP) can be an option for selected patients with resectable malignant pleural mesothelioma (MPM). The aim of this study was to investigate the impact of surgical treatment on the outcome of patients with MPM. Methods: We retrospectively reviewed data from 1365 consecutive patients with histologically proven MPM, treated from 1982 to 2012 in six Institutions. Patients received chemotherapy alone (n = 172), best supportive care (n = 690), or surgical treatment (n = 503), by either P/D (n = 202) or EPP (n = 301) with or without chemotherapy. Results: After a median follow-up of 6.7 years (range, 1.1–14.8), 230 patients (16.8%) were alive; median survival for patients who received palliative treatment or chemotherapy alone, P/D, and EPP were 11.7 (95% CI, 10.5–12.5), 20.5 (95% CI, 18.2–23.1), and 18.8 (95% CI, 17.2–20.9) months, respectively. The 30-day mortality was 2.6% after P/D and 4.1% after EPP ( p = 0.401). According to multivariate analysis (n = 1227), age less than 70, epithelial histology, and chemotherapy were independent favorable prognostic factors. In the subset of 313 patients (25.5%) with all favorable prognostic factors, median survival was 18.6 months after medical therapy alone, 24.6 months after P/D, and 20.9 months after EPP ( p = 0.596). Conclusions: Our data suggest that patients with good prognostic factors had a similar survival whether they received medical therapy only, P/D, or EPP. The modest benefit observed after surgery during medical treatment requires further investigation, and a large multicenter, randomized trial, testing P/D after induction chemotherapy versus chemotherapy alone in MPM patients with good prognostic factors, is needed.

Lung adenocarcinoma patients of similar clinical stage and undergoing the same treatments often have marked interindividual variations in prognosis. These clinical discrepancies may be due to the genetic background modulating an individual's predisposition to fighting cancer. Herein, we hypothesized that the lung microenvironment, as reflected by its expression profile, may affect lung adenocarcinoma patients' survival. The transcriptome of non-involved lung tissue, excised from a discovery series of 204 lung adenocarcinoma patients, was evaluated using whole-genome expression microarrays (with probes corresponding to 28 688 well-annotated coding sequences). Genes associated with survival status at 60 months were identified by Cox regression analysis (adjusted for gender, age and clinical stage) and retested in a validation series of 78 additional cases. RNA-Seq analysis from non-involved lung tissue of 12 patients was performed to characterize the different isoforms of candidate genes. Ten genes for which the loge-transformed hazard ratios expressed the same direction of effect in the discovery (P < 1.0 × 10(-3)) and validation series comprised the gene expression signature associated with survival: CNTNAP1, PKNOX1, FAM156A, FRMD8, GALNTL1, TXNDC12, SNTB1, PPP3R1, SNX10 and SERPINH1. RNA sequencing highlighted the complex expression pattern of these genes in non-involved lung tissue from different patients and permitted the detection of a read-through gene fusion between PPP3R1 and the flanking gene (CNRIP1) as well as a novel isoform of CNTNAP1. Our findings support the hypothesis that individual genetic characteristics, evidenced by the expression pattern of non-involved tissue, influence the outcome of lung adenocarcinoma patients.

Background Lung function after lung transplantation (LTx) has been widely studied. On the contrary, the thoracoabdominal volume rearrangement after LTx has yet to be investigated. Methods Patients with cystic fibrosis and listed for double LTx at our institution were enrolled for the prospective study to explore the effects of LTx on the rearrangement of respiratory volumes in patients affected by cystic fibrosis, by utilizing the opto-electronic plethysmography (OEP), a noninvasive method to study the volume and motion of the human trunk. Rib cage and abdominal volumes were tested with OEP (OEP system, BTS, Milano, Italy). Results Eight patients were enrolled (male-to-female ratio: 1:3; mean age 29.3 ± 7.8 years). After LTx the volume changes analyzed with OEP revealed a significant decrease of the total lung capacity (TLC) as well as the functional residual capacity and residual volume when the chest wall volume was considered. Dividing the whole respiratory volume in the three compartments showed different trends. Conclusions We consider OEP a particularly useful device in patients with severe respiratory disease, in that it allows a noninvasive estimate of the volume change of the chest wall. This study demonstrates a significant reduction of thoracoabdominal volumes in patients affected by cystic fibrosis treated with bilateral LTx. Abdomen and upper rib cage were congruent with the volume reduction, while the lower rib cage showed an opposite tendency.