The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis.

BACKGROUND: Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with a single-or stratified layer of basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish an in vitro- and in vivo model to mimic HC formation in IPF. We (1) optimized conditions to culture and propagate IPF patient-derived alveolar BC, (2) cultured the cells on an air liquid interface (ALI) or in a three dimensional (3D) organoid model, and (3) investigated the cells` behavior after instillation into bleomycin-challenged mice. METHODS: Alveolar BC were cultured from peripheral IPF lung tissue and grown on tissue-culture treated plastic, an ALI, or in a 3D organoid model. Furthermore, cells were instilled into bleomycin-challenged NRG mice. Samples were analyzed by TaqMan RT-PCR, immunoblotting, immunocytochemistry/immunofluorescence (ICC/IF), or immunohistochemistry (IHC)/IF. Mann-Whitney tests were performed using GraphPad Prism software. RESULTS: Cultured alveolar BC showed high expression of canonical basal cell markers (TP63, keratin (KRT)5, KRT14, KRT17), robust proliferation, and wound closure capacity. The cells could be cryopreserved and propagated for up to four passages without a significant loss of basal cell markers. When cultured on an ALI or in a 3D organoid model, alveolar BC differentiated to ciliated- and secretory epithelial cells. When instilled into bleomycin-challenged mice, human alveolar BC cells formed HC-like structures composed of human basal-, and secretory epithelial cells within the mouse parenchyma. CONCLUSION: IPF patient-derived alveolar BC on an ALI, in 3D organoids or after instillation into bleomycin-challenged mice form HC-like structures that closely resemble HC within the IPF lung. These models therefore represent powerful tools to study honeycomb formation, and its potential therapeutic inhibition in IPF.

Bronchial thermoplasty in asthma: an exploratory histopathological evaluation in distinct asthma endotypes/phenotypes.

BACKGROUND: Bronchial thermoplasty regulates structural abnormalities involved in airway narrowing in asthma. In the present study we aimed to investigate the effect of bronchial thermoplasty on histopathological bronchial structures in distinct asthma endotypes/phenotypes. METHODS: Endobronchial biopsies (n = 450) were collected from 30 patients with severe uncontrolled asthma before bronchial thermoplasty and after 3 sequential bronchial thermoplasties. Patients were classified based on blood eosinophils, atopy, allergy and smoke exposure. Tissue sections were assessed for histopathological parameters and expression of heat-shock proteins and glucocorticoid receptor. Proliferating cells were determined by Ki67-staining. RESULTS: In all patients, bronchial thermoplasty improved asthma control (p < 0.001), reduced airway smooth muscle (p = 0.014) and increased proliferative (Ki67 +) epithelial cells (p = 0.014). After bronchial thermoplasty, airway smooth muscle decreased predominantly in patients with T2 high asthma endotype. Epithelial cell proliferation was increased after bronchial thermoplasty in patients with low blood eosinophils (p = 0.016), patients with no allergy (p = 0.028) and patients without smoke exposure (p = 0.034). In all patients, bronchial thermoplasty increased the expression of glucocorticoid receptor in epithelial cells (p = 0.018) and subepithelial mesenchymal cells (p = 0.033) and the translocation of glucocorticoid receptor in the nucleus (p = 0.036). Furthermore, bronchial thermoplasty increased the expression of heat shock protein-70 (p = 0.002) and heat shock protein-90 (p = 0.001) in epithelial cells and decreased the expression of heat shock protein-70 (p = 0.009) and heat shock protein-90 (p = 0.002) in subepithelial mesenchymal cells. The effect of bronchial thermoplasty on the expression of heat shock proteins -70 and -90 was distinctive across different asthma endotypes/phenotypes. CONCLUSIONS: Bronchial thermoplasty leads to a diminishment of airway smooth muscle, to epithelial cell regeneration, increased expression and activation of glucocorticoid receptor in the airways and increased expression of heat shock proteins in the epithelium. Histopathological effects appear to be distinct in different endotypes/phenotypes indicating that the beneficial effects of bronchial thermoplasty are achieved by diverse molecular targets associated with asthma endotypes/phenotypes.

PD-1+ natural killer cells in human non-small cell lung cancer can be activated by PD-1/PD-L1 blockade.

Natural killer (NK) cells are critically involved in anti-tumor immunity by targeting tumor cells. In this study, we show that intratumoral NK cells from NSCLC patients expressed elevated levels of the immune checkpoint receptor PD-1 on their cell surface. In contrast to the expression of activating receptors, PD-1+ NK cells co-expressed more inhibitory receptors compared to PD-1- NK cells. Intratumoral NK cells were less functional compared to peripheral NK cells, and this dysfunction correlated with PD-1 expression. Tumor cells expressing PD-L1 inhibited the functionality of PD-1+ NK cells in ex vivo models and induced PD-1 clustering at the immunological synapse between NK cells and tumor cells. Notably, treatment with PD-1 blockade was able to reverse PD-L1-mediated inhibition of PD-1+ NK cells. Our findings highlight the therapeutic potential of PD-1+ NK cells in immune checkpoint blockade and could guide the development of NK cell-stimulating agents in combination with PD-1 blockade.

The Role of Fluorescence In Situ Hybridization for Predicting Recurrence after Adjuvant bacillus Calmette-Guérin in Patients with Intermediate and High Risk Nonmuscle Invasive Bladder Cancer: A Systematic Review and Meta-Analysis of Individual Patient Data.

PURPOSE: The objective of this study was to assess the value of fluorescence in situ hybridization to predict early recurrence in patients with nonmuscle invasive bladder cancer at intermediate and high risk treated with bacillus Calmette-Guérin. MATERIALS AND METHODS: We performed a systematic review using MEDLINE®, Embase® and the Cochrane Library. Individual patient data from prospective observational studies of fluorescence in situ hybridization in patients treated with bacillus Calmette-Guérin were included. A 2-stage individual patient data meta-analysis was done to assess the value of fluorescence in situ hybridization to predict tumor recurrence after bacillus Calmette-Guérin induction therapy. RESULTS: From a total of 4 studies we obtained individual data on 422 patients, of whom 408 with a median followup of 18.8 months were included in the final analysis. When fluorescence in situ hybridization was positive, the recurrence HR was 1.20 (95% CI 0.81-1.79) before bacillus Calmette-Guérin (time 0), 2.23 (95% CI 1.31-3.62) at 6 weeks (time 1), 3.70 (95% CI 2.34-5.83) at 3 months (time 2) and 23.44 (95% CI 5.26-104.49) at 6 months (time 3). CONCLUSIONS: A positive fluorescence in situ hybridization test after bacillus Calmette-Guérin correlated with higher risk of recurrent tumor. Fluorescence in situ hybridization could aid urologists in risk stratifying and counseling patients. Based on the HR and the narrowest CI the preferred timing of fluorescence in situ hybridization is 3 months after transurethral resection of bladder tumor. This is also in time for patients in whom induction therapy fails to enter clinical trials or change the treatment strategy.

Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction.

AIMS: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly evolved into a sweeping pandemic. Its major manifestation is in the respiratory tract, and the general extent of organ involvement and the microscopic changes in the lungs remain insufficiently characterised. Autopsies are essential to elucidate COVID-19-associated organ alterations. METHODS AND RESULTS: This article reports the autopsy findings of 21 COVID-19 patients hospitalised at the University Hospital Basel and at the Cantonal Hospital Baselland, Switzerland. An in-corpore technique was performed to ensure optimal staff safety. The primary cause of death was respiratory failure with exudative diffuse alveolar damage and massive capillary congestion, often accompanied by microthrombi despite anticoagulation. Ten cases showed superimposed bronchopneumonia. Further findings included pulmonary embolism (n = 4), alveolar haemorrhage (n = 3), and vasculitis (n = 1). Pathologies in other organ systems were predominantly attributable to shock; three patients showed signs of generalised and five of pulmonary thrombotic microangiopathy. Six patients were diagnosed with senile cardiac amyloidosis upon autopsy. Most patients suffered from one or more comorbidities (hypertension, obesity, cardiovascular diseases, and diabetes mellitus). Additionally, there was an overall predominance of males and individuals with blood group A (81% and 65%, respectively). All relevant histological slides are linked as open-source scans in supplementary files. CONCLUSIONS: This study provides an overview of postmortem findings in COVID-19 cases, implying that hypertensive, elderly, obese, male individuals with severe cardiovascular comorbidities as well as those with blood group A may have a lower threshold of tolerance for COVID-19. This provides a pathophysiological explanation for higher mortality rates among these patients.

Adrenomedullin mediates pro-angiogenic and pro-inflammatory cytokines in asthma and COPD.

PURPOSE: Adrenomedullin (AM) is a pluripotent peptide hormone with contradictory effects in human health and disease. In chronic inflammatory lung diseases, such as asthma and COPD, AM has been shown to inhibit inflammation and cell proliferation. In the present study, we aimed to investigate the effect of AM on pro-angiogenic and pro-inflammatory cytokines in asthma and COPD. PATIENTS AND METHODS: Serum levels of pro-AM were measured in patients with asthma, COPD and matched controls. The effect of AM on intracellular signaling proteins and cytokine secretion was assessed in primary cultures of epithelial cells (EC) and airway smooth muscle cells (ASMC) established from endo-bronchial biopsies of patients with asthma, COPD and controls. RESULTS: Serum pro-AM was higher in patients with asthma and COPD, compared to controls. AM stimulated cAMP in ASMC but not in EC. In EC, AM decreased Erk1/2 MAPK expression and activation but in ASMC, AM activated Erk1/2. This effect was similar in asthma, COPD and controls. AM stimulated the secretion of pro-angiogenic CXCL1 by EC of controls and CXCL5 by EC of asthma patients. AM did not affect the secretion of IL-6 or IL-8 by EC but stimulated the secretion of IL-6 by ASMC. In EC, AM inhibited the stimulatory effect of TGF-ß and IL-4 on the secretion of IL-6 and IL-8 but had an additive stimulatory effect with TGF-ß in ASMC. CONCLUSIONS: These data suggest that AM mediates the secretion of pro-angiogenic and pro-inflammatory cytokines in a cell-type and/or a disease-specific way, explaining its association with clinical outcomes in COPD.

Metastatic spread in patients with non-small cell lung cancer is associated with a reduced density of tumor-infiltrating T cells.

Tumor-infiltrating lymphocytes play an important role in cell-mediated immune destruction of cancer cells and tumor growth control. We investigated the heterogeneity of immune cell infiltrates between primary non-small cell lung carcinomas (NSCLC) and corresponding metastases. Formalin-fixed, paraffin-embedded primary tumors and corresponding metastases from 34 NSCLC patients were analyzed by immunohistochemistry for CD4, CD8, CD11c, CD68, CD163 and PD-L1. The percentage of positively stained cells within the stroma and tumor cell clusters was recorded and compared between primary tumors and metastases. We found significantly fewer CD4(+) and CD8(+) T cells within tumor cell clusters as compared with the stromal compartment, both in primary tumors and corresponding metastases. CD8(+) T cell counts were significantly lower in metastatic lesions than in the corresponding primary tumors, both in the stroma and the tumor cell islets. Of note, the CD8/CD4 ratio was significantly reduced in metastatic lesions compared with the corresponding primary tumors in tumor cell islets, but not in the stroma. We noted significantly fewer CD11c(+) cells and CD68(+) as well as CD163(+) macrophages in tumor cell islets compared with the tumor stroma, but no difference between primary and metastatic lesions. Furthermore, the CD8/CD68 ratio was higher in primary tumors than in the corresponding metastases. We demonstrate a differential pattern of immune cell infiltration in matched primary and metastatic NSCLC lesions, with a significantly lower density of CD8(+) T cells in metastatic lesions compared with the primary tumors. The lower CD8/CD4 and CD8/CD68 ratios observed in metastases indicate a rather tolerogenic and tumor-promoting microenvironment at the metastatic site.

Acute exacerbations of COPD are associated with significant activation of matrix metalloproteinase 9 irrespectively of airway obstruction, emphysema and infection.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are associated with accelerated aggravation of clinical symptoms and deterioration of pulmonary function. The mechanisms by which exacerbations may contribute to airway remodeling and declined lung function are poorly understood. In this study, we investigated if AE-COPD are associated with differential expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in bronchoalveolar lavage (BAL). METHODS: COPD patients undergoing diagnostic bronchoscopy, with either stable disease (n = 53) or AE-COPD (n = 44), matched for their demographics and lung function parameters were included in this study. Protein levels of MMP-2,-9,-12 and of TIMP-1 and -2 in BAL were measured by ELISA. Enzymatic activity of MMP-2 and -9 was assessed by gelatin zymography. RESULTS: We observed that MMP-9, TIMP-1 and TIMP-2 were significantly increased in BAL during AE-COPD. Furthermore, there was a significant negative correlation of MMP-9, TIMP-1 and TIMP-2 with FEV1% predicted and a significant positive correlation of TIMP-1 and TIMP-2 with RV% predicted in AE-COPD. None of MMPs and TIMPs correlated with DLCO% predicted, indicating that they are associated with airway remodeling leading to obstruction rather than emphysema. In AE-COPD the gelatinolytic activity of MMP-2 was increased and furthermore, MMP-9 activation was significantly up-regulated irrespective of lung function, bacterial or viral infections and smoking. CONCLUSIONS: The results of this study indicate that during AE-COPD increased expression of TIMP-1, TIMP-2, and MMP-9 and activation of MMP-9 may be persistent aggravating factors associated with airway remodeling and obstruction, suggesting a pathway connecting frequent exacerbations to lung function decline.

Calcineurin inhibitors in bronchiolitis obliterans syndrome following stem cell transplantation.

Bronchiolitis obliterans is a complication after allogeneic haematopoietic stem cell transplantation (HSCT). Management of bronchiolitis obliterans comprises intensive immunosuppression, but treatment response is poor. We investigated the effect of cyclosporine A (CsA), tacrolimus (FK506), methylprednisolone (mPRED), mycophenolate mofetil (MMF) and everolimus on the proliferation of primary lung myofibroblasts from HSCT patients with bronchiolitis obliterans syndrome (BOS). Cells were isolated from surgical lung biopsies of eight HSCT patients with BOS. Proliferation was assessed by [(3)H]-thymidine incorporation. Biopsies revealed constrictive bronchiolitis obliterans in three patients and lymphocytic bronchiolitis in five patients. CsA and FK506 significantly induced proliferation of myofibroblasts. mPRED and MMF caused a significant inhibition of proliferation, whereas everolimus had no effect. Costimulation with FK506, mPRED and MMF significantly inhibited proliferation. Serial pulmonary function tests over 12 months after lung biopsy and under triple therapy demonstrated that patients with lymphocytic bronchiolitis had a significant improvement in forced expiratory volume in 1 s (FEV1), whereas FEV1 of patients with bronchiolitis obliterans was unchanged. Our data demonstrate a pro-proliferative effect of calcineurin inhibitors on primary human lung myofibroblasts obtained from patients with BOS after HSCT. In contrast, based on the observed antiproliferative capacity of MMF in vitro, MMF-based calcineurin inhibitor-free treatment strategies should be further evaluated in patients with bronchiolitis obliterans after HSCT.

An unexpected cause of diffuse alveolar hemorrhage in a kidney transplant patient.

Diffuse alveolar hemorrhage (DAH) is a life-threatening condition requiring urgent treatment. There are many different treatment-relevant causes of DAH, making the diagnostic approach to these patients complex and necessitating a multidisciplinary team. We report the case of a kidney transplant recipient in whom all diagnostic efforts did not reveal the cause of DAH, and only autopsy was able to establish an unexpected diagnosis.

Lung histology predicts outcome of bronchiolitis obliterans syndrome after hematopoietic stem cell transplantation.

Bronchiolitis obliterans (BO) is a severe complication after allogeneic hematopoietic stem cell transplantation with an unfavorable prognosis. Lung biopsy remains the gold standard for diagnosis. In this retrospective single-center study, we describe 33 patients who underwent biopsy for suspected BO. Ten patients had constrictive BO (CBO); 9 had lymphocytic bronchiolitis (LB), characterized by lymphocytic infiltration of the bronchioles. Six additional patients (4, CBO; 2, LB) had concomitant infection; 8 had other pathological diagnoses. Seven patients with CBO and 3 with LB met the National Institutes of Health consensus BO syndrome definition criteria. An additional 7 patients with histologically confirmed CBO did not meet the consensus definition, 4 of them because of concomitant airway infection. At diagnosis, there were no significant differences between the CBO and LB groups in clinical presentation; pulmonary function tests (median forced expiratory volume in one second [FEV1] at baseline, 90.4% and 99% predicted, at time of video-assisted thoracoscopic surgery, 55.1% and 60.8% for CBO and LB groups, respectively); and chest scans. Treatment was similar in both groups but outcome was different depending on histological findings. FEV1 significantly improved in LB patients compared with CBO patients. Survivals at 1 and 3 years were 77% ± 12% and 60% ± 14% for patients with CBO and 91% ± 9% for patients with LB (P = .028). Lung biopsy in patients with suspected BO enables better characterization of the pattern of BO syndrome. In contrast to CBO, LB is associated with a good long-term prognosis.

Androgen Deprivation Therapy for Intracranial Metastasis of a Salivary Duct Carcinoma: Case Report.

Salivary duct carcinoma (SDC) is a rare subtype of salivary cancers associated with androgen receptor and human epidermal growth factor receptor 2 (HER2/neu) overexpression. It shows a high propensity to give rise to distant metastases mainly to the lung, the bone, and the liver. Intracranial metastases are rare. We report the case of a 61-year-old male patient with SDC developing intracranial metastases. Unresponsive to radiotherapy and anti-HER/neu targeted therapy the intracranial metastases showed a very good partial remission to androgen deprivation therapy with goserelin acetate. This case demonstrates the potential of a highly targeted therapy with a relatively cheap and well-known drug in a patient with a rare disease without other good therapeutic options, which is a good example of modern, personalized medicine.

Steroids and ß2-agonists regulate hyaluronan metabolism in asthmatic airway smooth muscle cells.

Glycosaminoglycans (GAGs), especially hyaluronic acid (HA), regulate tissue flexibility, cell motility, and inflammation. Airway smooth muscle cells (ASMCs) of patients with asthma exhibit abnormal HA metabolism, which contributes to inflammation and remodeling. Here, we investigated the effects of glucocorticoids and long-acting ß(2)-agonists (LABAs) on GAG synthesis and HA metabolism by human primary ASMCs. ASMCs were isolated from airway specimens of 10 patients without asthma and 11 patients with asthma. ASMCs were incubated with glucocorticoids, LABAs, or their combination, as well as with their specific receptor antagonists. Secreted and deposited total GAGs were measured by [(3)H]-glucosamine incorporation. The expression of specific GAGs was determined by ELISA and electrophoresis. The expression of HA synthases (HAS), of hyaluronidases (HYALs), and of the HA receptor CD44 was determined by RT-PCR, immunoblotting in cell cultures, and immunohistochemistry in tissue sections of asthmatic lungs. In serum-activated asthmatic ASMCs, glucocorticoids and LABAs significantly inhibited the increased secretion and deposition of total GAGs, but they stimulated secreted and deposited HA of high molecular mass. This effect was attributed to increased mRNA and protein expression of HAS-1 and to the reduced expression of HYAL-1. Furthermore, drug treatment stimulated the expression of CD44 receptors in asthmatic ASMCs. These effects of the drugs were eliminated by their respective receptor inhibitors. Our findings indicate that the combination of glucocorticoids with LABAs counteracts the pathologic degradation of HA, and thereby may reduce the proinflammatory potential of asthmatic ASMCs.

Role of fluorescence in situ hybridization in lung cancer cytology.

There are several challenges in lung cytology including equivocal cytological findings, lung cancer subclassification, and predictive marker analysis. Fluorescence in situ hybridization (FISH) detects chromosomal alterations that underlie the development and progression of cancer, and pinpoints targets of new anticancer drugs. Detection of such targets by FISH can be of diagnostic utility in morphologically difficult cases. Multitarget FISH with four different chromosomal probes improves the sensitivity of cytology in the diagnosis of lung cancer and clarifies equivocal cytological findings. FISH is becoming increasingly important in the field of predictive marker analysis. FISH is the gold standard to identify ALK rearrangements for treatment with the ALK inhibitor crizotinib (Xalkori). EGFR mutation analysis is the method of choice for selecting patients for therapy with EGFR tyrosine kinase inhibitors (TKIs), whereas the EGFR gene copy number has not been confirmed as a reliable predictive marker. MET amplification is an important mechanism of secondary resistance to EGFR TKIs and a candidate predictive marker for targeted second-line treatment of TKI-resistant non-squamous non-small cell lung cancer. FGFR1 amplification is a promising new marker in squamous cell carcinoma that may predict the response to FGFR1 inhibitors. In conclusion, there are an increasing number of clinically relevant FISH applications in lung cytology. We provide an overview on the current role of FISH in respiratory cytology.

Basal-Like Cell-Conditioned Medium Exerts Anti-Fibrotic Effects In Vitro and In Vivo.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

TargetPlex FFPE-Direct DNA Library Preparation Kit for SiRe NGS panel: an international performance evaluation study.

AIM: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow. METHODS: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols. RESULTS: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants. CONCLUSION: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.

Gene profiling of clinical routine biopsies and prediction of survival in non-small cell lung cancer.

RATIONALE: Global gene expression analysis provides a comprehensive molecular characterization of non-small cell lung cancer (NSCLC). OBJECTIVES: To evaluate the feasibility of integrating expression profiling into routine clinical work-up by including both surgical and minute bronchoscopic biopsies and to develop a robust prognostic gene expression signature. METHODS: Tissue samples from 41 chemotherapy-naive patients with NSCLC and 15 control patients with inflammatory lung diseases were obtained during routine clinical work-up and gene expression profiles were gained using an oligonucleotide array platform (NovaChip; 34'207 transcripts). Gene expression signatures were analyzed for correlation with histological and clinical parameters and validated on independent published data sets and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Diagnostic signatures for adenocarcinoma and squamous cell carcinoma reached a sensitivity of 80%/80% and a specificity of 83%/94%, respectively, dependent on the proportion of tumor cells. Sixty-seven of the 100 most discriminating genes were validated with independent observations from the literature. A 13-gene metagene refined on four external data sets was built and validated on an independent data set. The metagene was a strong predictor of survival in our data set (hazard ratio = 7.7, 95% CI [2.8-21.2]) and in the independent data set (hazard ratio = 1.6, 95% CI [1.2-2.2]) and in both cases independent of the International Union against Cancer staging. Vascular endothelial growth factor-beta, one of the key prognostic genes, was further validated by immunohistochemistry on 508 independent tumor samples. CONCLUSIONS: Integration of functional genomics from small bronchoscopic biopsies allows molecular tumor classification and prediction of survival in NSCLC and might become a powerful adjunct for the daily clinical practice.

The Paris System for reporting urinary cytology in daily practice with emphasis on ancillary testing by multiprobe FISH.

AIMS: The Paris System (TPS) was introduced in the diagnostic routine with the goal to simplify and standardise diagnostic reporting of urinary cytology. The diagnostic categories of TPS are based on defined cytological criteria, with a focus on high-grade urothelial carcinoma (HGUC). While the categories 'negative for HGUC (NHGUC)' and 'HGUC' are straightforward, the categories 'atypical urothelial cells (AUC)' and 'suspicious of HGUC (SHGUC)' remain inconclusive. In this study, we evaluated the feasibility of TPS in daily practice with special emphasis on ancillary fluorescence in situ hybridisation (FISH) testing in the setting of TPS categories. METHODS: In a 19-month period, TPS was prospectively applied in the routine diagnostic setting on 3900 urinary cytology cases comprising bladder and upper urinary tract washings and voided urine specimens. Additionally, we analysed the results of the FISH assay UroVysion prospectively performed on a cohort of 128 cases enriched for AUC and SHGUC categories. RESULTS: The most frequently reported category was NHGUC (n=3496, 89.7%), followed by AUC (n=178, 4.6%), HGUC (n=155, 4%), SHGUC (n=61, 1.6%), low-grade urothelial neoplasia (n=6, 0.1%) and other malignancies (n=4, 0.1%). In the FISH cohort, 40/90 (44%) cases within the AUC category were FISH positive, consistent with urothelial neoplasia. In the SHGUC category, 16/21 (76%) cases were FISH positive. CONCLUSIONS: When prospectively applying TPS in urinary cytology, inconclusive atypia accounts only for a small subset of cases. FISH additionally improves the stratification between reactive and malignant cells in the indeterminate AUC and SHGUC categories.

Narrative review of molecular pathways of kinase fusions and diagnostic approaches for their detection in non-small cell lung carcinomas.

The discovery of actionable oncogenic driver alterations has significantly improved treatment options for patients with advanced non-small cell lung cancer (NSCLC). In lung adenocarcinoma (LUAD), approved drugs or drugs in clinical development can target more than half of these altered oncogenic driver genes. In particular, several gene fusions have been discovered in LUAD, including ALK, ROS1, NTRK, RET, NRG1 and FGFR. All these fusions involve tyrosine kinases (TK), which are activated due to structural rearrangements on the DNA level. Although the overall prevalence of these fusions in LUAD is rare, their detection is extremely important, as they are linked to an excellent response to TK inhibitors. Therefore, reliable screening methods applicable to small tumor samples (biopsies and cytology specimens) are required in the diagnostic workup of advanced NSCLC. Several methods are at disposal in a routine laboratory to demonstrate, directly or indirectly, the presence of a gene fusion. These methods include immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR), multiplex digital color-coded barcode technology or next-generation sequencing (NGS) either on DNA or RNA level. In our review, we will summarize the increasing number of relevant fusion genes in NSCLC, point out their underlining molecular mechanisms and discuss different methods for the detection of fusion genes.