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Lung transplantation offers a lifesaving option for patients with end-stage lung disease, but it is marred by a high risk of post-transplant infections, particularly involving multidrug-resistant bacteria, Cytomegalovirus, and fungal pathogens. This elevated infection rate, the highest among solid organ transplants, poses a significant challenge for clinicians, particularly within the first year post-transplantation, where infections are the leading cause of mortality. The direct exposure of lung allografts to the external environment exacerbates this vulnerability leading to constant immune stimulation and consequently to an elevated risk of triggering alloimmune responses to the lung allograft. The necessity of prolonged immunosuppression to prevent allograft rejection further complicates patient management by increasing susceptibility to infections and neoplasms, and complicating the differentiation between rejection and infection, which require diametrically opposed management strategies. This review explores the intricate balance between preventing allograft rejection and managing the heightened infection risk in lung transplant recipients.

Mutual interactions between the diaphragm and lung transplantation (LTx) are known to exist. Before LTx, many factors can exert notable impact on the diaphragmatic function, such as the underlying respiratory disease, the comorbidities, and the chronic treatments of the patient. In the post-LTx setting, even the surgical procedure itself can cause a stressful trauma to the diaphragm, potentially leading to morphological and functional alterations. Conversely, the diaphragm can significantly influence various aspects of the LTx process, ranging from graft-to-chest cavity size matching to the long-term postoperative respiratory performance of the recipient. Despite this, there are still no standard criteria for evaluating, defining, and managing diaphragmatic dysfunction in the context of LTx to date. This deficiency hampers the accurate assessment of those factors which affect the diaphragm and its reciprocal influence on LTx outcomes. The objective of this narrative review is to delve into the complex role the diaphragm plays in the different stages of LTx and into the modifications of this muscle following surgery.

Introduction: Telemedicine has been successfully employed in a wide range of conditions, such as such as chronic lung disease and COVID-19. This study evaluate the role of telemonitoring for the early diagnosis of acute lung allograft dysfunction in cystic fibrosis adults who underwent lung transplant (LuTx). Quality of life and functional level achieved during a 12 months follow up were assessed. Methods: Patients were randomized into two groups; control group received traditional hospital-based follow-up, whereas patients in the intervention group received, on top of standard care, a telemonitoring device, with a pulse oximeter and a spirometer integrated. Telemonitoring data were digitally transmitted to our centre. Results: Sixteen patients were enrolled in each group. No statistically significant difference was found between the two groups in terms of incidence of allograft dysfunction, time from onset of symptoms to diagnosis and time of occurrence from LuTx. Moreover, both groups achieved similar quality of life and functional level. With reference to the telemonitoring group: 1) hospital reported data were consistent with those being remotely registered; 2) adherence to telemonitoring decreased during the follow up; 3) the majority of patients reported a high degree of satisfaction. Conclusion: The COVID19 pandemic highlighted the necessity to investigate alternative practices to treat chronically ill individuals. Telemonitoring is a valuable tool to improve quality care to LuTx recipients.

Background: Unilateral proximal interruption of the pulmonary artery (UPIPA) is a rare congenital disease, and its optimal management remains undefined in the existing literature. The occasional necessity for pneumonectomy is still supported by limited evidence. Methods: A systematic review of the literature was conducted using the PubMed search engine, focusing on UPIPA cases that received pneumonectomy. Thirty-one pertinent articles were selected and included in the analysis. A case reported from our institution was included in the analysis. Results: We found 25 adults and seven children affected by UPIPA who received an indication for pneumonectomy, plus an additional case that was reported by our institution. Among adult patients, the predominant indication was hemoptysis (57%), followed by suspected or confirmed lung cancer (23%). Approximately 46% of surgical procedures were classified as urgent or emergent. Postoperative complications were observed in 36% of cases, with no recorded mortality. In pediatric cases, pneumonectomy was primarily a life-saving intervention, performed urgently or emergently in 75% of instances. A possible late complication in pediatric patients involves a mediastinal shift leading to respiratory distress, which may be mitigated using an inflatable prosthesis. Conclusions: Pneumonectomy achieves complete resolution of UPIPA symptoms. In the adult population, its primary indication is hemoptysis, with procedures conducted in both elective and urgent/emergent settings. Despite a mortality rate of zero, a notable proportion of patients may experience postoperative complications. In pediatric cases, the clinical presentation varies more extensively, and pneumonectomy is typically reserved for life-threatening situations, emphasizing the need for careful patient selection.

ObjectiveWe report our experience of transition to robotic-assisted thoracic surgery (RATS) for lung resections with the da Vinci Xi surgical system, exposing short-term results.Materials and methodsThis is a single-center, retrospective analysis of RATS lung resections performed between April 2021 and September 2022 during our new robotic program. The surgical approach evolved over time, starting from a four-arm approach with four incisions. Alternative RATS approaches were subsequently evaluated, such as uniportal and biportal.ResultsDuring a 17-month period, 29 lung resections were performed. Of them, 16 were lobectomies, 7 were segmentectomies, and 6 were wedge resections. The most common indication for anatomical lung resection was non-small cell lung cancer. A uniportal approach was used for two simple segmentectomies and a biportal RATS was performed in five lobectomies and two segmentectomies. A mean number of 8.1 lymph nodes and a mean of 2.6 N2 and 1.9 N1 stations were resected during surgery, and no nodal upstaging was observed. Negative resection margins were 100%. There were two (7%) conversions, one to open surgery and one to video-assisted thoracic surgery (VATS). Eight (28%) patients experienced complications with no 30-day mortality.DiscussionHigh-ergonomic and high-quality views were immediately observed. After some procedures, we abandoned uniportal RATS because of the possibility of arm collisions and the necessity of a VATS-skilled surgeon at the operating table.ConclusionRATS for lung resections was safe and effective, and from the surgeon's standpoint, several practical advantages over VATS were observed. Further analysis on outcomes will help better understand the value of this technology.

Abstract Pulmonary lobectomy, which consists of the partial or complete resection of a lung lobe, is the gold standard intervention for lung cancer removal. The removal of functional tissue during the surgery and the re-adaptation of the remaining thoracic structures decrease the patient's post-operative pulmonary function. Residual functionality is evaluated through pulmonary function tests, which account for the number of resected segments without considering local structural alterations and provide an average at-the-mouth estimation. Computational Fluid Dynamics (CFD) has been demonstrated to provide patient-specific, quantitative, and local information about airways airflow dynamics. A CFD investigation was performed on image-based airway trees reconstructed before and after the surgery for twelve patients who underwent lobectomy at different lobes. The geometrical alterations and the variations in fluid dynamics parameters and in lobar ventilation between the pre and post-operative conditions were evaluated. The post-operative function was estimated and compared with current clinical algorithms and with actual clinical data. The post-operative configuration revealed a high intersubject variability: regardless of the lobectomy site, an increment of global velocity, wall pressure, and wall shear stress was observed. Local flow disturbances also emerged at, and downstream of, the resection site. The analysis of lobar ventilation showed severe variations in the volume flow rate distribution, highlighting the compensatory effects in the contralateral lung with an increment of inflow. The estimation of post-operative function through CFD was comparable with the current clinical algorithm and the actual spirometric measurements. The results confirmed that CFD could provide additional information to support the current clinical approaches both in the operability assessment and in the prescription of personalized respiratory rehabilitation.

Nowadays, lung transplantation is a clinical reality for the treatment of benign end-stage respiratory diseases [...]

Abstract Background Video-assisted thoracoscopic surgery (VATS) lobectomy is currently the recommended approach for treating early-stage non-small cell lung cancer (NSCLC). Different VATS approaches have been proposed so far, and the actual advantages of one technique over the other are still under debate. The aim of our study is to compare postoperative pain and analgesic drug consumption in uniportal VATS and triportal VATS for pulmonary lobectomy in early-stage lung cancer patients. Methods This study is a single-center, prospective, two-arm, parallel-group, randomized controlled trial. It is designed to compare uniportal video-assisted thoracic surgery (u-VATS) and three-port video-assisted thoracic surgery (t-VATS) in terms of postoperative pain. The trial will enroll 120 patients with a 1:1 randomization. The primary outcome is the assessment of analgesic drug consumption. Secondary outcomes are postoperative pain measurement, evaluation of postoperative pulmonary function, and metabolic recovery after pulmonary lobectomy. Discussion The choice of which VATS approach to adopt for treating patients undergoing pulmonary resection mostly depends on the surgeon’s preferences; therefore, it is hard to prove whether one VATS technique is superior to the other. Moreover, postoperative analgesic protocols vary consistently among different centers. To date, only a few studies have evaluated the effects of the most popular VATS techniques. There is no evidence about the difference between multiport VATS and u-VATS in terms of postoperative pain. We hope that the results of our trial will provide valuable information on the outcomes of these different surgical approaches. Trial registration ClinicalTrials.gov NCT03240250 . Registered on 07 August 2017; retrospectively registered.

In this paper, we study the trajectory tracking problem using iterative learning control for continuous-time nonlinear systems with a generic fixed relative degree in the presence of disturbances. This class of controllers iteratively refine the control input relying on the tracking error of the previous trials and some properly tuned learning gains. Sufficient conditions on these gains guarantee the monotonic convergence of the iterative process. However, the choice of the gains is heuristically hand-tuned given an approximated system model and no information on the disturbances. Thus, in the cases of inaccurate knowledge of the model or iteration-varying measurement errors, external disturbances, and delays, the convergence condition is unlikely to be verified at every iteration. To overcome this issue, we propose a robust convergence condition, which ensures the applicability of the pure feedforward control even if other classical conditions are not fulfilled for some trials due to the presence of disturbances. Furthermore, we quantify the upper bound of the nonrepetitive disturbance that the iterative algorithm is able to handle. Finally, we validate the convergence condition simulating the dynamics of a two degrees of freedom underactuated arm with elastic joints, where one is active, and the other is passive, and a Franka Emika Panda manipulator.

Abstract Despite the promising results achieved so far in long-term survival after lung transplantation (LuTx), airway complications (ACs) still arise in the post-operative period. Early diagnosis and prompt treatment of ACs play a critical role in preventing their onset. Specifically, large bronchi ischemia has been recognized as a triggering factor for ACs. Autofluorescence bronchoscopy, which was first introduced for early cancer diagnosis, displays ischemic mucosae as red spots, while normal vascularized mucosae appear in green. The aim of this study is to investigate whether a significant correlation exists between ACs and the red/green (RG) ratio detected on scheduled autofluorescence bronchoscopy up to 1 year after LuTx. This prospective, observational, single-center cohort study initially considered patients who underwent LuTx between July 2014 and February 2016. All patients underwent concomitant white-light and autofluorescence bronchoscopy at baseline (immediately after LuTx), on POD7, POD14, POD21, POD28, POD45, 3 months, 6 months, and 1 year after LuTx. An autofluorescence image of the first bronchial carina distal to the anastomosis was captured and analyzed using histograms for red and green pixels; the R/G ratio was then recorded. Potential ACs were classified according according to the presence of a white-light following the MDS (macroscopic aspect, diameter and suture) criteria. The authors assessed the association between the R/G ratio and the ACs occurrence using a generalized estimating equations model. Thirty-one patients met the inclusion criteria and were therefore selected. Out of a total of 53 bronchial anastomoses, 8 developed complications (late bronchial stenosis), with an average onset time of 201 days after LuTx. ACs showed a similar baseline covariate value when compared to anastomoses that involved no complication. Generalized estimating equations regression indicated a clear association over time between the R/G ratio and the rise of complications (p = 0.023). The authors observed a significant correlation between post-anastomotic stenosis and the delayed decrease of the R/G ratio. Preliminary outcomes suggest that autofluorescence bronchoscopy may be an effective and manageable diagnostic tool, proving complementary to other instruments for early diagnosis of ACs after LuTx. Further research is needed to confirm and detail preliminary findings.

Abstract Background Phosphaturic mesenchymal tumors are rare neoplasms, frequently presenting with osteomalacia. These neoplasms usually grow at a slow rate and are associated with unspecific symptoms. Case In this study, we present the case of a 70‐year‐old woman who had been suffering from musculoskeletal pain, hypophosphatemia, and spontaneous fractures. Positron emission tomography with Gallium showed increase uptake in a subpleural lesion. Conclusion The patient underwent surgical excision of the subpleural lesion with a non‐intubated uniportal video‐assisted thoracoscopic surgery approach.

This study describes the case of an 18-years-old male affected by severe COVID-19, who was receiving bilateral lung transplantation (LT), after 71 days of mechanical ventilation and 55 days of veno-venous extracorporeal membrane oxygenation. From post-operative day 2, early mobilization and physiotherapy treatments were performed. Weaning from mechanical ventilation, the use of non-invasive ventilation and tracheostomy management were included in the treatment. Forty-five days after LT the patient was discharged at home, showing improvements in terms of functional and respiratory parameters, quality of life and mood. While evidences about physiotherapy treatments in lung transplantation post severe COVID-19 remain limited, early approach and a multidisciplinary team may be considered key elements for functional recovery of these subjects.

BackgroundDespite significant improvement in screening programs for cancers of the respiratory district, especially in at-risk subjects, early disease detection is still a major issue. In this scenario, new molecular and non-invasive biomarkers are needed to improve early disease diagnosis.MethodsWe profiled the miRNome in exhaled breath condensate (EBC) and plasma samples from fourteen patients affected by lung AdCa, nine healthy subjects. miRNA signatures were then analyzed in another neoplasia of the respiratory district, i.e. pleural mesothelioma (n = 23) and subjects previously exposed to asbestos were used as controls for this cohort (n = 19). Selected miRNAs were analyzed in purified pulmonary neoplastic or normal epithelial and stromal cell subpopulation from AdCa patients. Finally, the plasmatic miRNA signature was analyzed in a publicly available cohort of NSCLC patients for data validation and in silico analysis was performed with predicted miRNA targets using the multiMiR tool and STRING database.ResultsmiR-597-5p and miR-1260a are significantly over-expressed in EBC from lung AdCa and are associated with AdCa. Similarly, miR-1260a is also up-regulated in the plasma of AdCa patients together with miR-518f-3p and correlates with presence of lung cancer, whereas let-7f-5p is under-expressed. Analysis of these circulating miRNAs in pleural mesothelioma cases confirmed that up-regulation of miR-518f-3p, -597-5p and -1260a, is specific for lung AdCa. Lastly, quantification of the miRNAs in laser-assisted microdissected lung tissues revealed that miR-518f-3p, 597-5p and miR-1260a are predominantly expressed in tumor epithelial cells. Validation analysis confirmed miR-518f-3p as a possible circulating biomarker of NSCLC. In silico analysis of the potentially modulated biological processes by these three miRNAs, shows that tumor bioenergetics are the most affected pathways.ConclusionsOverall, our data suggest a 3-miRNAs signature as a non-invasive and accurate biomarker of lung AdCa. This approach could supplement the current screening approaches for early lung cancer diagnosis.

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs’ GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

Background: The Anaplastic Lymphoma Kinase (ALK) gene is known to be affected by several genetic alterations, such as rearrangement, amplification and point mutation. The main goal of this study was to comprehensively analyze ALK amplification (ALK-A) and ALK gene copy number gain (ALK-CNG) in a large cohort of non-small-cell lung cancer (NSCLC) patients in order to evaluate the effects on mRNA and protein expression. Methods: ALK locus number status was evaluated in 578 NSCLC cases by fluorescence in situ hybridization (FISH). In addition, ALK immunohistochemistry and ALK mRNA in situ hybridization were performed. Results: Out of 578 cases, 17 cases showed ALK-A. In addition, 14 cases presented ALK-CNG and 72 cases presented chromosome 2 polyploidy. None of those carrying ALK-A and -CNG showed either ALK immunohistochemical expression or ALK mRNA expression through in situ hybridization. We observed a high frequency of extra copies of the ALK gene. Conclusions: Our findings demonstrated that ALK-A is not involved in mRNA production and consequently is not involved in protein production; these findings support the hypothesis that ALK-A might not play a role in the pathogenesis of NSCLC, underlining the absence of a specific clinical application.

Malignant Pleural Mesothelioma (MPM) is an aggressive cancer mainly caused by asbestos exposure and refractory to current therapies. Specific diagnostic markers for early MPM diagnosis are needed. Changes in miRNA expression have been implicated in several diseases and cancers, including MPM. We examined if a specific miRNA signature in plasmatic extracellular vesicles (EV) may help to discriminate between malignant pleural mesothelioma patients (MPM) and subjects with Past Asbestos Exposure (PAE).We investigated 23 MPM patients and 19 cancer-free subjects with past asbestos exposure (PAE). We screened 754 miRNAs in plasmatic EVs by OpenArray and found 55 differential miRNAs using logistic regression models adjusted for age, sex, BMI, and smoking. Among the top-20 differential miRNAs chosen for validation by Real time PCR, 16 were confirmed. Using receiver operating characteristic (ROC) curve analysis, the most discriminating miRNA combination was given by miR-103a-3p + miR-30e-3p, which generated an AUC of 0.942 (95% CI 0.87-1.00), with a sensitivity of 95.5% and a specificity of 80.0%. Using multivariate Cox regression analysis including gender, age, BMI and smoking we found a Hazard Ratio for miR-103a-3p above the median of 0.37 (95%CI 0.13-1.13) and of 0.51 (95%CI 0.17-1.52) for miR-30e-3p.This study suggests EV-associated miR-103a-3p and miR-30e-3p are able to discriminate MPM from PAE subjects. Larger and prospective studies are needed to confirm these two-miRNA signature alone or in combination with other biomarkers as diagnostic tools for MPM.

BACKGROUND:Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and has a high risk of distant metastasis at every disease stage. We aimed to characterize the genomic landscape of LUAD and identify mutation signatures associated with tumor progression. METHODS AND FINDINGS:We performed an integrative genomic analysis, incorporating whole exome sequencing (WES), determination of DNA copy number and DNA methylation, and transcriptome sequencing for 101 LUAD samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We detected driver genes by testing whether the nonsynonymous mutation rate was significantly higher than the background mutation rate and replicated our findings in public datasets with 724 samples. We performed subclonality analysis for mutations based on mutant allele data and copy number alteration data. We also tested the association between mutation signatures and clinical outcomes, including distant metastasis, survival, and tumor grade. We identified and replicated two novel candidate driver genes, POU class 4 homeobox 2 (POU4F2) (mutated in 9 [8.9%] samples) and ZKSCAN1 (mutated in 6 [5.9%] samples), and characterized their major deleterious mutations. ZKSCAN1 was part of a mutually exclusive gene set that included the RTK/RAS/RAF pathway genes BRAF, EGFR, KRAS, MET, and NF1, indicating an important driver role for this gene. Moreover, we observed strong associations between methylation in specific genomic regions and somatic mutation patterns. In the tumor evolution analysis, four driver genes had a significantly lower fraction of subclonal mutations (FSM), including TP53 (p = 0.007), KEAP1 (p = 0.012), STK11 (p = 0.0076), and EGFR (p = 0.0078), suggesting a tumor initiation role for these genes. Subclonal mutations were significantly enriched in APOBEC-related signatures (p < 2.5×10-50). The total number of somatic mutations (p = 0.0039) and the fraction of transitions (p = 5.5×10-4) were associated with increased risk of distant metastasis. Our study's limitations include a small number of LUAD patients for subgroup analyses and a single-sample design for investigation of subclonality. CONCLUSIONS:These data provide a genomic characterization of LUAD pathogenesis and progression. The distinct clonal and subclonal mutation signatures suggest possible diverse carcinogenesis pathways for endogenous and exogenous exposures, and may serve as a foundation for more effective treatments for this lethal disease. LUAD's high heterogeneity emphasizes the need to further study this tumor type and to associate genomic findings with clinical outcomes.

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

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.

Lung cancer encompasses a constellation of malignancies with no validated prognostic markers. p16Ink4A expression has been reported in different subtypes of lung cancers; however, its prognostic value is controversial. Here, we sought to investigate the clinical significance of p16Ink4A immunoexpression according to specific staining patterns and its operational implications. A total of 502 tumors, including 277 adenocarcinomas, 84 squamous cell carcinomas, 22 large cell carcinomas, 47 typical carcinoids, 12 atypical carcinoids, 28 large cell neuroendocrine carcinomas, and 32 small cell carcinomas were reviewed and subjected to immunohistochemical analysis for p16Ink4A and Ki67. The spectrum of p16Ink4A expression was annotated for each case as negative, sporadic, focal, or diffuse. Expression at immunohistochemical level showed intra-tumor homogeneity, regardless tumor histotype. Enrichments in cells expressing p16Ink4A were observed from lower- to higher-grade neuroendocrine malignancies, whereas a decrease was seen in poorly and undifferentiated non-neuroendocrine carcinomas. Tumor proliferation indices were higher in neuroendocrine tumors expressing p16Ink4A while non-neuroendocrine malignancies immunoreactive for p16Ink4A showed a decrease in Ki67-positive cells. Quantitative statistical analyses including each histotype and the p16Ink4A status confirmed the independent prognostic role of p16Ink4A expression, being a high-risk indicator in neuroendocrine tumors and a marker of good prognosis in non-neuroendocrine lung malignancies. In this study, we provide circumstantial evidence to suggest that the routinary assessment of p16Ink4A expression using a three-tiered scoring algorithm, even in a small biopsy, may constitute a reliable, reproducible, and cost-effective substrate for a more accurate risk stratification of each individual patient.