The existence of an in situ phase of malignant mesothelioma has long been postulated but until recently has been impossible to prove. Here we describe ten patients with mesothelioma in situ, defined ...by a single layer of surface mesothelial cells showing loss of BAP1 nuclear immunostaining, no evidence of tumor by imaging and/or by direct examination of the pleura/peritoneum, and no invasive mesothelioma developing for at least 1 year. Nine cases were pleural and one peritoneal. Most patients were biopsied for repeated effusions of unknown etiology; in two patients mesothelioma in situ was found incidentally in lung cancer resections. In addition to surface mesothelium with BAP1 loss, one case had a surface papillary proliferation with BAP1 loss, and two cases had a small (few millimeter) nodule with BAP1 loss. CDKN2A was deleted by FISH in one of eight cases. Methylthioadenosine phosphorylase showed partial loss in the surface mesothelium by immunohistochemistry in three cases. Invasive malignant mesothelioma developed in seven patients with time between biopsy and invasive disease from 12 to 92 (median 60) months. Invasive mesothelioma has not developed in the other three patients at 12, 57, and 120 months, but the latter patient, who has pleural plaques, still has repeated pleural effusions, probably representing a so-called "benign asbestos effusion." We conclude that mesothelioma in situ, as diagnosed using the criteria outlined above, is associated with a high risk of developing invasive mesothelioma, but typically over a relatively protracted time, so that curable interventions maybe possible.
Since the outbreak of the COVID-19 pandemic, much has been learned regarding its clinical course, prognostic inflammatory markers, disease complications, and mechanical ventilation strategy. ...Clinically, three stages have been identified based on viral infection, pulmonary involvement with inflammation, and fibrosis. Moreover, low and high elastance phenotypes can be distinguished in mechanically ventilated patients, based on lung mechanics, ventilation-to-perfusion ratio, and CT scans; these two phenotypes have presumed differences in their underlying pathophysiology. Although essential for therapeutic guidance, the pathophysiology of COVID-19 is poorly understood. Here, we systematically reviewed published case reports and case series in order to increase our understanding of COVID-19 pathophysiology by constructing a timeline and correlating histopathological findings with clinical stages of COVID-19. Using PRISMA-IPD guidelines, 42 articles reporting 198 individual cases were included in our analysis. In lung samples (n = 131 cases), we identified three main histological patterns: epithelial (n = 110, 85%), with reactive epithelial changes and DAD; vascular (n = 76, 59%) with microvascular damage, (micro)thrombi, and acute fibrinous and organizing pneumonia; and fibrotic (n = 28, 22%) with interstitial fibrosis. The epithelial and vascular patterns can present in all stages of symptomatic COVID-19, whereas the fibrotic pattern presents starting at ~3 weeks. Moreover, patients can present with more than one pattern, either simultaneously or consecutively. These findings are consistent with knowledge regarding clinical patterns of viral infection, development of hyperinflammation and hypercoagulability, and fibrosis. Close collaboration among medical staff is necessary in order to translate this knowledge and classification of pathophysiological mechanisms into clinical stages of disease in individual patients. Moreover, further research, including histopathological studies, is warranted in order to develop reliable, clinically relevant biomarkers by correlating these pathological findings with laboratory results and radiological findings, thus, increasing our understanding of COVID-19 and facilitating the move to precision medicine for treating patients.
This meeting report from the XV Banff conference describes the creation of a multiorgan transplant gene panel by the Banff Molecular Diagnostics Working Group (MDWG). This Banff Human Organ ...Transplant (B‐HOT) panel is the culmination of previous work by the MDWG to identify a broadly useful gene panel based on whole transcriptome technology. A data‐driven process distilled a gene list from peer‐reviewed comprehensive microarray studies that discovered and validated their use in kidney, liver, heart, and lung transplant biopsies. These were supplemented by genes that define relevant cellular pathways and cell types plus 12 reference genes used for normalization. The 770 gene B‐HOT panel includes the most pertinent genes related to rejection, tolerance, viral infections, and innate and adaptive immune responses. This commercially available panel uses the NanoString platform, which can quantitate transcripts from formalin‐fixed paraffin‐embedded samples. The B‐HOT panel will facilitate multicenter collaborative clinical research using archival samples and permit the development of an open source large database of standardized analyses, thereby expediting clinical validation studies. The MDWG believes that a pathogenesis and pathway based molecular approach will be valuable for investigators and promote therapeutic decision‐making and clinical trials.
This Banff meeting report summarizes the progress of the Banff Molecular Diagnostics Working Group, which generated consensus on a transplant‐specific discovery gene panel and a potential roadmap for its validation for diagnostic application.
Abstract
Lower respiratory tract (LRT) disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can deteriorate to acute respiratory distress syndrome (ARDS). Because the ...release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill patients with COVID-19. Plasma NET levels peaked early after intensive care unit admission and were correlated with the SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma samples. The baseline plasma NET quantity was correlated with disease severity but was not associated with soluble markers of thrombosis or with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from deceased patient with COVID-19. Thus, NETs are produced and retained in the LRT of critically ill patients with COVID-19 and could contribute to SARS-CoV-2–induced ARDS disease.
Aberrant neutrophil extracellular trap (NET) formation is implicated in acute respiratory distress syndrome (ARDS) pathogenesis. We demonstrate that NETs are present in blood and persist at high levels in the lower respiratory tract of critically ill patients with coronavirus disease 2019 and ARDS.
Aims
Immunohistochemical programmed death‐ligand 1 (PD‐L1) staining to predict responsiveness to immunotherapy in patients with advanced non‐small cell lung cancer (NSCLC) has several drawbacks: a ...robust gold standard is lacking, and there is substantial interobserver and intraobserver variance, with up to 20% discordance around cutoff points. The aim of this study was to develop a new deep learning‐based PD‐L1 tumour proportion score (TPS) algorithm, trained and validated on a routine diagnostic dataset of digitised PD‐L1 (22C3, laboratory‐developed test)‐stained samples.
Methods and results
We designed a fully supervised deep learning algorithm for whole‐slide PD‐L1 assessment, consisting of four sequential convolutional neural networks (CNNs), using aiforia create software. We included 199 whole slide images (WSIs) of ‘routine diagnostic’ histology samples from stage IV NSCLC patients, and trained the algorithm by using a training set of 60 representative cases. We validated the algorithm by comparing the algorithm TPS with the reference score in a held‐out validation set. The algorithm had similar concordance with the reference score (79%) as the pathologists had with one another (75%). The intraclass coefficient was 0.96 and Cohen’s κ coefficient was 0.69 for the algorithm. Around the 1% and 50% cutoff points, concordance was also similar between pathologists and the algorithm.
Conclusions
We designed a new, deep learning‐based PD‐L1 TPS algorithm that is similarly able to assess PD‐L1 expression in daily routine diagnostic cases as pathologists. Successful validation on routine diagnostic WSIs and detailed visual feedback show that this algorithm meets the requirements for functioning as a ‘scoring assistant’.
Lymph node micrometastases could be one of the reasons for the high recurrence rate after complete surgical resection in stage I–IIIA non‐small cell lung cancer (NSCLC). The standard evaluation of a ...single haematoxylin and eosin (H&E) slide of a paraffin‐embedded section of a lymph node is insufficient for the detection of micrometastases, and there is a need for additional histopathological evaluation. The association of lymph node micrometastases with survival remains as yet unresolved. The aim of this systematic review and meta‐analysis is to investigate if lymph node micrometastases and isolated tumour cells in patients with stage I–IIIA NSCLC, detected with multiple sectioning and/or immunohistochemistry (IHC) and/or reverse transcriptase polymerase chain reaction (RT‐PCR), are associated with overall survival (OS) and disease‐free survival (DFS) after surgical resection. We performed a meta‐analysis of time‐to‐event outcomes based on 15 articles using ancillary techniques to detect micrometastases. We extracted the OS and DFS every 3–6 months after surgery, for patients with and without occult lymph node micrometastasis, from the survival curves published in each article. These data were used to reconstruct OS and DFS for ‘micrometastasis’ and ‘no micrometastasis’ groups. Based on all included studies that used IHC, serial sectioning, or RT‐PCR, we found a 5‐year OS of 55% (micrometastasis) vs. 75% (no micrometastasis), and a 5‐year DFS of 53% (micrometastasis) vs. 75% (no micrometastasis). Patients with stage I–IIIA NSCLC with lymph node micrometastases detected by ancillary histopathological and molecular techniques have a significantly poorer OS and DFS compared to patients without lymph node micrometastases.
Objectives
A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of ...tumor mutational load (TML), CD8
+
T cell infiltration, HLA class-I and PD-L1 expression in the tumor.
Materials and methods
Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8
+
T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan–Meier methodology.
Results
30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (
p
= 0.004) and OS (
p
= 0.025). Interaction analyses revealed that patients with both high TML and high total CD8
+
T cell infiltrate (
p
= 0.023) or no loss of HLA class-I (
p
= 0.026), patients with high total CD8
+
T cell infiltrate and no loss of HLA class-I (
p
= 0.041) or patients with both high PD-L1 and high TML (
p
= 0.003) or no loss of HLA class-I (
p
= 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (
p
= 0.007).
Conclusion
This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8
+
T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.
Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role ...of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice.
B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis.
More IgA
memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA
germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88).
Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•The sequence of genomic events that lead to the development of mesothelioma in situ are unknown.•Mesothelioma in situ development is associated with BAP1 somatic mutations/deletions.•BAP1 ...mutation/deletion is an early event in the development of some malignant mesotheliomas.
We have recently described the first cases of mesothelioma in situ, identified as a pure surface population of mesothelial cells that have lost BAP1 nuclear staining, in the setting of no clinically/radiologically demonstrable mesothelial tumor. These cases have a high propensity to develop invasive mesothelioma. The genetic events that lead to the development of mesothelioma in situ are unknown, nor is it known whether mesothelioma in situ cases carry somatic or germline mutations.
Whole exome sequencing (WES) was performed on two cases of mesothelioma in situ (1 pleural, 1 peritoneal) and paired formalin fixed paraffin embedded normal tissue to characterize driver mutations and copy number alterations.
The analysis demonstrated somatic alterations in the BAP1 gene only. The pleural mesothelioma in situ showed copy number loss and LOH in the BAP1 locus on chromosome 3. The peritoneal mesothelioma in situ showed both a BAP1 somatic splice site mutation involving intron 5-exon 6 boundary (A126_splice) with an allelic fraction of 10%, and BAP1 copy number loss. No other driver point mutations, indels or somatic DNA copy number alterations reported to occur in invasive mesothelioma, or novel genetic alterations, were identified.
Whole exome sequencing confirms that mesothelioma in situ development is associated with BAP1 somatic mutations/deletions, and suggests that BAP1 mutation/deletion represents a very early event in the development of malignant mesothelioma. Whether BAP1 mutation/deletion alone is sufficient to lead to invasive mesothelioma or whether additional genetic alterations are required remains to be determined.
Aims
Lung tissue from COVID‐19 patients shares similar histomorphological features with chronic lung allograft disease, also suggesting activation of autoimmune‐related pathways in COVID‐19. To more ...clearly understand the underlying spectrum of pathophysiology in COVID‐19 pneumonia, we analysed mRNA expression of autoimmune‐related genes in post‐mortem lung tissue from COVID‐19 patients.
Methods and results
Formalin‐fixed, paraffin‐embedded lung tissue samples of 18 COVID‐19 patients and eight influenza patients were used for targeted gene expression profiling using NanoString technology. Multiplex immunofluorescence for tryptase and chymase was applied for validation. Genes related to mast cells were significantly increased in COVID‐19. This finding was strengthened by multiplex immunofluorescence also showing a significant increase of tryptase‐ and chymase‐positive cells in COVID‐19. Furthermore, receptors for advanced glycation end‐products (RAGE) and pro‐platelet basic protein (PPBP) were up‐regulated in COVID‐19 compared to influenza. Genes associated with Type I interferon signalling showed a significant correlation to detected SARS‐CoV2 pathway‐related genes. The comparison of lung tissue samples from both groups based on the presence of histomorphological features indicative of acute respiratory distress syndrome did not result in finding any specific gene or pathways.
Conclusion
Two separate means of measuring show a significant increase of mast cells in SARS‐CoV‐2‐infected lung tissue compared to influenza. Additionally, several genes involved in fibrosis and thrombosis, among which are RAGE and PPBP, are up‐regulated in COVID‐19. As mast cells are able to induce thrombosis and fibrosis, they may play an important role in the pathogenesis of COVID‐19.
This study depicts the role of mast cells in COVID‐19 pneumonia.
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