[ERAS Implementation in Thoracic Surgery]. / ERAS-Implementierung in der Thoraxchirurgie.

This manuscript provides an overview of the principles and requirements for implementing the ERAS program in thoracic surgery.The ERAS program optimises perioperative management of elective lung resection procedures and is based on the ERAS Guidelines for Thoracic Surgery of the ERAS Society. The clinical measures are described as in the current literature, with a focus on postoperative outcome. There are currently 45 enhanced recovery items covering four perioperative phases: from the prehospital admission phase (patient education, screening and treatment of potential risk factors such as anaemia, malnutrition, cessation of nicotine or alcohol abuse, prehabilitation, carbohydrate loading) to the immediate preoperative phase (shortened fasting period, non-sedating premedication, prophylaxis of PONV and thromboembolic complications), the intraoperative measures (antibiotic prophylaxis, standardised anaesthesia, normothermia, targeted fluid therapy, minimally invasive surgery, avoidance of catheters and probes) through to the postoperative measures (early mobilisation, early nutrition, removal of a urinary catheter, hyperglycaemia control). Most of these measures are based on scientific studies, with a high level of evidence and aim to reduce general postoperative complications.The ERAS program is an optimised perioperative treatment approach aiming to improve the postoperative recovery in patients after elective lung resection by reducing the overall complication rates and overall morbidity.

From morbidity reduction to cost-effectiveness: Enhanced recovery after surgery (ERAS) society recommendations in minimal invasive liver surgery.

PURPOSE: Minimal-invasive liver surgery (MILS) reduces surgical trauma and is associated with fewer postoperative complications. To amplify these benefits, perioperative multimodal concepts like Enhanced Recovery after Surgery (ERAS), can play a crucial role. We aimed to evaluate the cost-effectiveness for MILS in an ERAS program, considering the necessary additional workforce and associated expenses. METHODS: A prospective observational study comparing surgical approach in patients within an ERAS program compared to standard care from 2018-2022 at the Charité - Universitätsmedizin Berlin. Cost data were provided by the medical controlling office. ERAS items were applied according to the ERAS society recommendations. RESULTS: 537 patients underwent liver surgery (46% laparoscopic, 26% robotic assisted, 28% open surgery) and 487 were managed by the ERAS protocol. Implementation of ERAS reduced overall postoperative complications in the MILS group (18% vs. 32%, p = 0.048). Complications greater than Clavien-Dindo grade II incurred the highest costs (€ 31,093) compared to minor (€ 17,510) and no complications (€13,893; p < 0.001). In the event of major complications, profit margins were reduced by a median of € 6,640. CONCLUSIONS: Embracing the ERAS society recommendations in liver surgery leads to a significant reduction of complications. This outcome justifies the higher cost associated with a well-structured ERAS protocol, as it effectively offsets the expenses of complications.

Impact of complexity in minimally invasive liver surgery on enhanced recovery measures: prospective study.

BACKGROUND: Adherence to enhanced recovery after surgery (ERAS) protocols is crucial for successful liver surgery. The aim of this study was to assess the impact of minimally invasive liver surgery complexity on adherence after implementing an ERAS protocol. METHODS: Between July 2018 and August 2021, a prospective observational study involving minimally invasive liver surgery patients was conducted. Perioperative treatment followed ERAS guidelines and was recorded in the ERAS interactive audit system. Kruskal-Wallis and ANOVA tests were used for analysis, and pairwise comparisons utilized Wilcoxon rank sum and Welch's t-tests, adjusted using Bonferroni correction. RESULTS: A total of 243 patients were enrolled and categorized into four groups based on the Iwate criteria: low (n = 17), intermediate (n = 81), advanced (n = 74) and expert difficulty (n = 71). Complexity correlated with increased overall and major morbidity rate, as well as longer length of stay (all P < 0.001; standardized mean difference = 0.036, 0.451, 0.543 respectively). Adherence to ERAS measures decreased with higher complexity (P < 0.001). Overall adherence was 65.4%. Medical staff-centred adherence was 79.9%, while patient-centred adherence was 38.9% (P < 0.001). Complexity significantly affected patient-centred adherence (P < 0.001; standardized mean difference (SMD) = 0.420), but not medical staff-centred adherence (P = 0.098; SMD = 0.315). Postoperative phase adherence showed major differences among complexity groups (P < 0.001, SMD = 0.376), with mobilization measures adhered to less in higher complexity cases. CONCLUSION: The complexity of minimally invasive liver surgery procedures impacts ERAS protocol adherence for each patient. This can be addressed using complexity-adjusted cut-offs and 'gradual adherence' based on the relative proportion of cut-off values achieved.

Do skip-N2 metastases significantly impact overall survival and disease-free interval in N2 non-small-cell lung cancer patients? A multicentre analysis.

OBJECTIVES: Skip-N2 metastasis (N0N2), thus N2 metastasis in the absence of N1 metastasis, occurs in ∼20-30% of non-small-cell lung cancer patients. N0N2 patients have a better prognosis than continuous-N2 metastasis (N1N2) patients following surgery. However, this effect remains controversial. Therefore, we conducted a multicentre study to compare the long-term survival and disease-free interval (DFI) of N1N2- and N0N2 patients. METHODS: One- and 3-year survival rates were measured. Kaplan-Meier curves and a Cox proportional hazards model assessed survival and were used to identify prognostic factors for overall survival. In addition, we performed propensity score matching (PSM) to rule out confounding factors. All patients received adjuvant chemoradiation therapy according to European guidelines. RESULTS: Between January 2010 and December 2020, 218 stage IIIA/B N2 patients were included in our analysis. The Cox regression analysis revealed that N1N2 significantly influenced the overall survival rate. Before PSM, N1N2 patients showed significantly more metastatic lymph nodes (P < 0.001) and significantly larger tumours (P = 0.05). After PSM, baseline characteristics did not differ between groups. Before and after PSM, N0N2 patients showed significantly better 1- (P = 0.01; P = 0.009) and 3-year (P < 0.001) survival rates than N1N2 patients. Furthermore, N0N2 patients showed significantly longer DFI than N1N2 patients before and after PSM (P < 000.1). CONCLUSIONS: Prior and after PSM analysis, N0N2 patients were confirmed to have better survival and DFI than N1N2 patients. Our results demonstrate that stage IIIA/B N2 patients are heterogeneous and would benefit from a more precise subdivision and differential treatment.

Validation of the Enhanced Recovery after Surgery (ERAS) society recommendations for liver surgery: a prospective, observational study.

Background: Twenty-three recommendations were summarized by the Enhanced Recovery After Surgery (ERAS) society for liver surgery. The aim was to validate the protocol especially with regard to adherence and the impact on morbidity. Methods: Using the ERAS Interactive Audit System (EIAS), ERAS items were evaluated in patients undergoing liver resection. Over a period of 26 months, 304 patients were prospectively enrolled in an observational study (DRKS00017229). Of those, 51 patients (non-ERAS) were enrolled before and 253 patients (ERAS) after the implementation of the ERAS protocol. Perioperative adherence and complications were compared between the two groups. Results: Overall adherence increased from 45.2% in the non-ERAS group to 62.7% in the ERAS group (P<0.001). This was associated with significant improvements in the preoperative and postoperative phase (P<0.001), rather than in the outpatient and intraoperative phase (both P>0.05). Overall complications decreased from 41.2% (n=21) in the non-ERAS group to 26.5% (n=67) in the ERAS group (P=0.0423), which was mainly due to the reduction of grade 1-2 complications from 17.6% (n=9) to 7.6% (n=19) (P=0.0322). As for patients undergoing open surgery, implementation of ERAS lead to a reduction of overall complications in patients scheduled for minimally invasive liver surgery (MILS) (P=0.036). Conclusions: Implementation of the ERAS protocol for liver surgery according to the ERAS guidelines of the ERAS Society reduced Clavien-Dindo grade 1-2 complications particularly in patients who underwent MILS. The ERAS guidelines are beneficial for the outcome, while adherence to the various items has not yet been satisfactorily defined.

A machine learning tool to improve prediction of mediastinal lymph node metastases in non-small cell lung cancer using routinely obtainable [18F]FDG-PET/CT parameters.

BACKGROUND: In patients with non-small cell lung cancer (NSCLC), accuracy of [18F]FDG-PET/CT for pretherapeutic lymph node (LN) staging is limited by false positive findings. Our aim was to evaluate machine learning with routinely obtainable variables to improve accuracy over standard visual image assessment. METHODS: Monocentric retrospective analysis of pretherapeutic [18F]FDG-PET/CT in 491 consecutive patients with NSCLC using an analog PET/CT scanner (training + test cohort, n = 385) or digital scanner (validation, n = 106). Forty clinical variables, tumor characteristics, and image variables (e.g., primary tumor and LN SUVmax and size) were collected. Different combinations of machine learning methods for feature selection and classification of N0/1 vs. N2/3 disease were compared. Ten-fold nested cross-validation was used to derive the mean area under the ROC curve of the ten test folds ("test AUC") and AUC in the validation cohort. Reference standard was the final N stage from interdisciplinary consensus (histological results for N2/3 LNs in 96%). RESULTS: N2/3 disease was present in 190 patients (39%; training + test, 37%; validation, 46%; p = 0.09). A gradient boosting classifier (GBM) with 10 features was selected as the final model based on test AUC of 0.91 (95% confidence interval, 0.87-0.94). Validation AUC was 0.94 (0.89-0.98). At a target sensitivity of approx. 90%, test/validation accuracy of the GBM was 0.78/0.87. This was significantly higher than the accuracy based on "mediastinal LN uptake > mediastinum" (0.7/0.75; each p < 0.05) or combined PET/CT criteria (PET positive and/or LN short axis diameter > 10 mm; 0.68/0.75; each p < 0.001). Harmonization of PET images between the two scanners affected SUVmax and visual assessment of the LNs but did not diminish the AUC of the GBM. CONCLUSIONS: A machine learning model based on routinely available variables from [18F]FDG-PET/CT improved accuracy in mediastinal LN staging compared to established visual assessment criteria. A web application implementing this model was made available.

[Treatment of Persistent Parenchymal Lung Injuries in Thoracic Trauma: Lung Laceration, Pleural Fistula and Pneumothorax]. / Behandlung von persistierenden Lungenparenchymverletzungen beim Thoraxtrauma: Lungenlazeration, pleurale Fistel und Pneumothorax.

Thoracic trauma is a frequent injury pattern with high patient morbidity and mortality. Preclinical and clinical emergency treatment is consented in a national S3-guideline. Following emergency therapy one third of patients may develop lung lacerations, pleural fistulation and persisting pneumothorax. An interdisciplinary working group of the German Society for Thoracic Surgery and the German Society for Traumatology reviewed the published medical literature on treatment of those injuries and assessed the existing evidence according to consensus recommendations. An inconsistent classification of those subsequent lung injuries was found. Evidence for diagnostic and therapeutic recommendations is small.

[VATS in Thorax Trauma]. / Videoassistierte Thorakoskopie (VATS) beim Thoraxtrauma.

Since the early 1990s, video-assisted thoracoscopy (VATS) has been increasingly established for a variety of indications in the treatment of patients with thoracic trauma. During this time, one premise for the use of thoracoscopy has not changed. Its use is consistently recommended only for trauma patients with stable circulation and respiration. To define the indications of VATS for use in thoracic trauma, the Pulmonary Injury Group - as part of the Working Committee for Thoracic Trauma of the German Society for Thoracic Surgery (DGT) and the German Society for Trauma Surgery (DGU) - has developed treatment recommendations based on a current literature review (based on the PRISMA Checklist/here: MEDLINE via PubMed from 1993 to 2022). In the present study, after reviewing the available literature, the indications for VATS in the care of thoracic trauma were identified, in order to formulate clinical recommendations for the use of VATS in thoracic trauma. The analysis of 1679 references identified a total of 4 randomised controlled trials (RCTs), 4 clinical trials, and 5 meta-analyses or systematic reviews and 39 reviews, which do not allow a higher level of recommendation than consensual recommendations, due to the low evidence of the available literature. Over the past 30 years, stabilisation options in the care of trauma patients have improved significantly, allowing expansion of indications for the use of VATS. Moreover, the recommendation for more than 50 years to thoracotomise trauma patients in case of an initial blood loss ≥ 1500 ml via the inserted chest drainage or in case of continuous blood loss ≥ 250 ml/h over 4 h is now only relative with today's better stabilisation measures. For unstable/non-stabilisable patients with a thoracic injury requiring emergency treatment, thoracotomy remains the method of choice, while VATS is recommended for a wide range of indications in the diagnosis and treatment of stable patients with a penetrating or blunt thoracic trauma. The indications for VATS are persistent haemothorax, treatment of injuries and haemorrhages to the lung, diaphragm, thoracic wall and other organ injuries, and in the secondary phase, treatment of thoracic sequelae of injury (empyema, persistent pulmonary fistula, infected atelectasis, etc.).

Transcriptomic comparison of primary human lung cells with lung tissue samples and the human A549 lung cell line highlights cell type specific responses during infections with influenza A virus.

Influenza A virus (IAV) causes pandemics and annual epidemics of severe respiratory infections. A better understanding of the molecular regulation in tissue and cells upon IAV infection is needed to thoroughly understand pathogenesis. We analyzed IAV replication and gene expression induced by IAV strain H3N2 Panama in isolated primary human alveolar epithelial type II cells (AECIIs), the permanent A549 adenocarcinoma cell line, alveolar macrophages (AMs) and explanted human lung tissue by bulk RNA sequencing. Primary AECII exhibit in comparison to AM a broad set of strongly induced genes related to RIG-I and interferon (IFN) signaling. The response of AECII was partly mirrored in A549 cells. In human lung tissue, we observed induction of genes unlike in isolated cells. Viral RNA was used to correlate host cell gene expression changes with viral burden. While relative induction of key genes was similar, gene abundance was highest in AECII cells and AM, while weaker in the human lung (due to less IAV replication) and A549 cells (pointing to their limited suitability as a model). Correlation of host gene induction with viral burden allows a better understanding of the cell-type specific induction of pathways and a possible role of cellular crosstalk requiring intact tissue.

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but virus-induced expansion of inflammatory macrophages.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

Reducing Immunosuppression in Patients with De Novo Lung Carcinoma after Liver Transplantation Could Significantly Prolong Survival.

(1) Background: Liver transplantation (LT) is an established treatment for selected patients with end-stage liver disease resulting in a subsequent need for long-term immunosuppressive therapy. With cumulative exposure to immunosuppression (IS), the risk for the development of de novo lung carcinoma increases. Due to limited therapy options and prognosis after diagnosis of lung cancer, the question of the mode and extent of IS in this particular situation is raised. (2) Methods: All patients diagnosed with de novo lung cancer in the follow-up after LT were identified from the institution's register of liver allograft recipients (Charité-Universitätsmedizin Berlin, Germany) transplanted between 1988 and 2021. Survival analysis was performed based on the IS therapy following diagnosis of lung cancer and the oncological treatment approach. (3) Results: Among 3207 adult LTs performed in 2644 patients at our institution, 62 patients (2.3%) developed de novo lung carcinoma following LT. Lung cancer was diagnosed at a median interval of 9.7 years after LT (range 0.7-27.0 years). Median survival after diagnosis of lung carcinoma was 13.2 months (range 0-196 months). Surgical approach with curative intent significantly prolonged survival rates compared to palliative treatment (median 67.4 months vs. 6.4 months). Reduction of IS facilitated a significant improvement in survival (median 38.6 months vs. 6.7 months). In six patients (9.7%) complete IS weaning was achieved with unimpaired liver allograft function. (4) Conclusion: Reduction of IS therapy after the diagnosis of de novo lung cancer in LT patients is associated with prolonged survival. The risk of acute rejection does not appear to be increased with restrictive IS management. Therefore, strict reduction of IS should be an early intervention following diagnosis. In addition, surgical resection should be attempted, if technically feasible and oncologically meaningful.

The single-cell transcriptional landscape of lung carcinoid tumors.

Lung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown. To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGFß and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data. Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the noninflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

[Current State of ERAS Implementation in Thoracic Surgery in Germany]. / Aktuelle ERAS-Implementierung in der Thoraxchirurgie an deutschen Kliniken.

BACKGROUND, OBJECTIVES: In recent years, ERAS treatment pathways have found their way into many surgical fields, as they reduce complications and accelerate postoperative recovery. For thoracic surgery, the first ERAS guidelines were published by the ERAS Society and the European Society of Thoracic Surgeons (ESTS) in 2019. We have now evaluated how ERAS-items are implemented in clinical practice by using an online survey. MATERIAL AND METHODS: An online survey was conducted from 12/5/2021 until 1/6/2021. The survey consisted of 22 questions focusing on the key elements of an ERAS program according to the published ERAS guidelines. Results were summarised, descriptively analysed and put into context with the current literature. RESULTS: Of 155 thoracic surgeons, 32 responded to the survey. In 28.1% (n = 9) of the hospitals, an ERAS core unit was established, and a database to record the ERAS items existed in 15.6% (n = 5). Only 3.1% (n = 1) kept an ERAS-diary preoperatively. A so-called Carboloading was conducted at 15.6% (n = 5) of surgeons. Standard PONV prophylaxis was administered to 59.4% (n = 19) of the patients. In most cases (84.4%, n = 29), a single drain was inserted into the pleural cavity during anatomic resections. In 3% (n = 1) of the centres two drains, in 12.5% (n = 4) no drainage was placed. The most commonly applied initial suction was -10 cmH2O (75%, n = 24). Suction ≤ 2 cmH2O was used by only two of those interviewed. Drainage removal took place in 50% (n = 16) of cases between the 1st or 2nd POD, in 34.4% of cases (n = 11) between the 3rd and 4th POD and in 9.4% (n = 3) the drain remained longer than the 4th POD. The first postoperative mobilisation took place in 71.9% (n = 23) of the centres on the day of the operation. CONCLUSIONS: The implementation of ERAS guidelines varies in Germany between centres. Certain perioperative processes are covered sufficiently, but the implementation of key features of ERAS is yet to be fully established in clinical practice. The first steps in this direction have already been taken and lay the foundation for cooperation across centres.

Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma.

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.

Thoracic Surgery in the COVID-19 Pandemic: A Novel Approach to Reach Guideline Consensus.

The COVID-19 pandemic challenges international and national healthcare systems. In the field of thoracic surgery, procedures may be deferred due to mandatory constraints of the access to diagnostics, staff and follow-up facilities. There is a lack of prospective data on the management of benign and malignant thoracic conditions in the pandemic. Therefore, we derived recommendations from 14 thoracic societies to address key questions on the topic of COVID-19 in the field of thoracic surgery. Respective recommendations were extracted and the degree of consensus among different organizations was calculated. A high degree of consensus was found to temporarily suspend non-critical elective procedures or procedures for benign conditions and to prioritize patients with symptomatic or advanced cancer. Prior to hospitalization, patients should be screened for respiratory symptoms indicating possible COVID-19 infection and most societies recommended to screen all patients for COVID-19 prior to admission. There was a weak consensus on the usage of serology tests and CT scans for COVID-19 diagnostics. Nearly all societies suggested to postpone elective procedures in patients with suspected or confirmed COVID-19 and recommended constant reevaluation of these patients. Additionally, we summarized recommendations focusing on precautions in the theater and the management of chest drains. This study provides a novel approach to informed guidance for thoracic surgeons during the COVID-19 pandemic in the absence of scientific evidence-based data.

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories.

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts.

FDG-PET/CT for pretherapeutic lymph node staging in non-small cell lung cancer: A tailored approach to the ESTS/ESMO guideline workflow.

OBJECTIVES: In patients with NSCLC, current ESTS and ESMO guidelines recommend invasive lymph node (LN) staging with EBUS-TBNA even if FDG-PET/CT is negative for mediastinal LNs if at least one of three risk factors is present (cN1, non-peripheral primary or primary >3 cm). Modified workflows to avoid unnecessary invasive procedures were evaluated. MATERIALS AND METHODS: Monocentric retrospective analysis of pretherapeutic FDG-PET/CT in 247 patients with NSCLC (62 % male; age, 68 [43-88] years) using an analog or digital PET/CT scanner. PET windowing was standardized. LNs were positive if 'LN uptake > mediastinal blood pool' or short axis >10 mm. Surgery or EBUS-TBNA served as reference for diagnostic accuracy per LN station. In all patients with negative mediastinal LNs by PET/CT, LN histology from surgery was available. RESULTS: Among 700 L N stations analyzed, 180 were malignant. Sensitivity and specificity of PET/CT per LN station were 93 % and 71 %. Following current guidelines, 76 patients with mediastinal negative PET/CT required confirmatory invasive staging. Only 5/76 patients had unexpected pN2 (all had adenocarcinoma). In a modified approach, confirmatory invasive staging was confined to patients with mediastinal negative PET/CT who showed all three risk factors. Using this modification, EBUS-TBNA could have been omitted in 62 (82 %) of the 76 patients who required EBUS-TBNA based on current recommendation. Among these 62 patients, only one patient had unsuspected pN2 (single-level) while the remaining 61 of 62 omitted EBUS-TBNA were deemed unnecessary because mediastinal LNs were confirmed to be negative. No multi-level pN2 would have been missed. CONCLUSION: In the current analysis, 82 % of EBUS-TBNA procedures in patients with mediastinal negative PET/CT could have been omitted by modifying the current guideline workflow as proposed (i.e., restricting EBUS-TBNA in patients with cN0/1 to those with all three risk factors). This was consistent with different PET/CT scanners. Prospective confirmation is required.

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.