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OBJECTIVE Spread through air spaces (STAS) is a pattern of invasion present in some adenocarcinomas (ADC). The goal of this study was to assess the impact of STAS in patients treated with different types of surgical resections and on the clinical outcome in patients with ADC of different diameters and with different degrees of nodal involvement. METHODS A total of 109 patients were reviewed. Complete surgical resection with systematic nodal dissection was achieved in all patients. The median follow-up was 65 months (3–90 months). RESULTS STAS was observed in 70 cases (64.2%); 13 patients (18.5%) had lymph node involvement (N1 and N2). Overall survival and progression-free survival were higher in patients without STAS (P = 0.042; P = 0.027). The presence of STAS in tumours ≤2 cm was a predictor of worse progression-free survival following sublobar resection compared to major resections (P = 0.011). Sublobar resection of N0 STAS-positive tumours was associated with worse long-term survival compared to a major resection (P = 0.04). Statistical analyses showed that age >70 years and recurrence were independent variables for survival; smoking pack-years >20, sublobar resection and nodal involvement were independent variables for recurrence; and smoking pack-years >20 were independent variables for a history of cancer and pleural invasion for local recurrence. CONCLUSIONS STAS seems to play a role in long-term survival, particularly for patients with N0 and tumours smaller than 2 cm. Further studies are necessary to validate this hypothesis.

Background Hypertrophic cardiomyopathy (HCM) is characterized by severe alterations of cardiac architecture and function involving cardiomyocytes (CM) and coronary microvessels (MV). Coronary microvascular dysfunction, cardiomyocyte hypertrophy and disarray, sarcomeric alterations and interstitial fibrosis are HCM features. The transcriptome profile associated with coronary MV and CM in HCM patients is presently unknown. Purpose Aim of this study was to improve knowledge of the molecular and biological pahways involved in HCM. To this purpose, the gene expression profile of coronary MV and CM was investigated. Methods Interventricular septum myectomies from patients with obstructive HCM and donors' hearts (CTR) were collected. Coronary MV (HCM=20, CTR=6) and CM (HCM=10, CTR=5) were laser capture microdissected. RNA-seq was performed by Illumina Nextseq 500, with 76 nt long single-reads. Adapter trimming and quality filtering of the sequenced reads were performed before alignment to the human reference genome. Univocally mapped reads estimated gene expression/sample. Normalized expressed gene levels were quantified. Statistical tests compared HCM and CTR to identify differentially expressed genes (DEG), i.e. up- and down-expressed genes in CM and MV samples. Functional enrichment analysis was performed. Biological categories, i.e. KEGG and Reactome pathways, Gene Ontology terms, protein domains in InterPro database, putative interactors collected in the Intact database and protein annotations in UniProt were considered for inter group comparison of DEGs. Results Transcriptome analysis identified 392 genes significantly up-regulated and 514 down-regulated in CM samples of HCM vs. CTR, while in MV 681 genes were up-regulated and 815 down-regulated. Although some DEGs were shared between MV and CM (26 and 146 are up- and down-expressed in both sample types), the majority of DEGs displayed a sample-specific pattern. A comparative functional analysis of DEGs highlighted some statistically enriched biological categories including an enrichment of phosphoproteins, with down-expressed genes both in CM (490) and MV (314). Other biological categories annotated as “ubiquitin-like protein conjugation” or “acetylation” in Uniprot database were enriched in down-regulated genes, both in MV and CM. Interestingly, “ribosomal protein” and “ribonucleoprotein” categories resulted as enriched up-regulated DEGs in MV. Conversely, the “citrullination” category was specifically present in annotations associated to down-regulated DEGs in MV from HCM compared to CTR. Conclusions Our preliminary results support the suitability of RNA-seq analysis to assess: i. the transcriptome profiles and pathways associated to coronary MV and CM; ii. the possible relationship/interplay of MV and CM profiles and HCM disease. The enrichment functional analysis provides preliminary data on candidate DEGs and target proteins for in vitro studies on HCM-related mechanisms. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Ministry of Health

Background Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy, heterogeneous in phenotype and clinical course. The genotype-phenotype relationship and associated molecular mechanisms are still incompletely understood. In the HCM milieu, increased energy cost of force production, impairing performance and mitochondrial function, may be associated to patients' genotype and/or phenotype. Purpose To determine abnormalities in mitochondrial energetics in HCM and their possible relationship with genotype and clinical phenotype of patients. Methods Septal myectomies from characterized patients with obstructive HCM (HOCM, n=18) and donor hearts discarded from transplantation (CTRL, n=8) have been compared. HOCM patients were screened and demographic, clinical and instrumental data (routine ECG, echocardiography and cardiac magnetic resonance-CMR) were collected. Genetic analysis was performed on total DNA extracted from myectomies by NGS on MiSeq platform using the TruSight Cardio Sequencing kit and testing a panel of 26 genes. Activity and amount of mitochondrial enzymes generating/scavenging reactive oxygen species were investigated. Results Within the HOCM cohort 30% of patients was bearing mutations in sarcomeric genes associated with HCM. The amount and/or activity of mitochondrial Complex I NADH dehydrogenase, of SOD2 and (m)-aconitase were upregulated in HOCM vs. CTRL. NADH dehydrogenase level was inversely correlated with the degree of mitral valve regurgitation and mitral valve backward volume by CMR (Spearman R=−0.5 and −0.8, respectively). The Complex V enzyme ATP synthase activity decreased, whilst its amount was comparable in HOCM vs. CTRL. Analogously the SOD1 activity was similar in HOCM and CTRL. No difference in mitochondrial DNA (mtDNA) copy number was found. Results were unrelated to HCM-associated mutations. Conclusions HOCM hearts are characterized by mitochondrial hyperactivity aimed at quenching reactive oxygen species, but decreased ATP synthase activity. Overall, these data suggest an abnormal mitochondrial activity in the myocardium of HOCM patients independent of the presence of HCM-associated mutations. Moreover, our results underpin the markedly abnormal cellular energetics of HOCM, identifying potential therapeutic targets. Acknowledgement/Funding NET-2011-02347173, Italian Minister of Health

We describe a rare case of Dirofilaria repens infection presenting as peripheral lung nodules and mimicking a metastatic focus from a previously diagnosed cutaneous melanoma. To avoid invasive investigations before arriving at the correct diagnosis, dirofilariasis should be included as a part of the diagnostic process in subjects with lung nodules who live in (or have traveled to) endemic regions.

Aim Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. Methods and results We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. controls. Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. Conclusions Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies.

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(-/-)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(-/-) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration.

Mitochondrial disease refers to a heterogenous group of genetic disorders that result from dysfunction of the final common pathway of energy metabolism. Mitochondrial DNA mutations affect key components of the respiratory chain and account for the majority of mitochondrial disease in adults. Owing to critical dependence of the heart on oxidative metabolism, cardiac involvement in mitochondrial disease is common and may occur as the principal clinical manifestation or part of multisystem disease. Recent advances in our understanding of the clinical spectrum and genetic aetiology of cardiac involvement in mitochondrial DNA disease have important implications for cardiologists in terms of the investigation and multi-disciplinary management of patients.

In the March, 2009, edition of Europace (volume 11, number 3), Ejima et al. 1 reported the case of a young woman in whom a diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) was obtained with of a novel endomyocardial biopsy (EMB) approach, guided by electroanatomic mapping of the right ventricle (RV). In order to increase the diagnostic accuracy of EMB, Ejima et al. 1 performed …