Molecular Testing in Lung Cancer: Recommendations and Update.

Adoption of molecular testing in lung cancer is increasing. Molecular testing for staging and prediction of response for targeted therapy remain the main indications, and although utilization of blood-based testing for tumor is growing, the use of the diagnostic cytology and tissue specimens is equally important. The pathologist needs to optimize reflex testing, incorporate stage-based algorithms, and understand types of tests for timely and complete assessment in the majority of cases. When tissue is limited, testing should capture the most frequent alterations to maximize the yield of what are largely mutually exclusive alterations, avoiding the need for repeat biopsy.

Pathology of COVID-19 Lung Disease.

The pathology of severe COVID-19 lung injury is predominantly diffuse alveolar damage, with other reported patterns including acute fibrinous organizing pneumonia, organizing pneumonia, and bronchiolitis. Lung injury was caused by primary viral injury, exaggerated immune responses, and superinfection with bacteria and fungi. Although fatality rates have decreased from the early phases of the pandemic, persistent pulmonary dysfunction occurs and its pathogenesis remains to be fully elucidated.

A signature of enhanced proliferation associated with response and survival to anti-PD-L1 therapy in early-stage non-small cell lung cancer.

In early-stage non-small cell lung cancer, the combination of neoadjuvant anti-PD-L1 and subablative stereotactic body radiation therapy (SBRT) is associated with higher rates of major pathologic response compared to anti-PD-L1 alone. Here, we identify a 140-gene set, enriched in genes characteristic of highly proliferating cells, associated with response to the dual therapy. Analysis of on-treatment transcriptome data indicate roles for T and B cells in response. The 140-gene set is associated with disease-free survival when applied to the combined trial arms. This 140-gene set identifies a subclass of tumors in all 7 of The Cancer Genome Atlas tumor types examined. Worse survival is associated with the 140-gene signature in 5 of these tumor types. Collectively, our data support that this 140-gene set, discovered in association with response to combined anti-PD-L1 and SBRT, identifies a clinically aggressive subclass of solid tumors that may be more likely to respond to immunotherapies.

Pathogenesis of Pulmonary Long COVID-19.

COVID-19 is characterized by an acute respiratory illness that, in some patients, progresses to respiratory failure, largely demonstrating a pattern of acute respiratory distress syndrome. Excluding fatal cases, the outcome of this severe illness ranges from complete resolution to persistent respiratory dysfunction. This subacute-to-chronic respiratory illness has different manifestations and is collectively termed as "long COVID." The pathogenesis of organ dysfunction in acute injury stems from exaggerated innate immune response, complement activation, and monocyte influx, with a shift toward an organ injury state with abnormalities in cellular maturation. Although the increased rate of thrombosis observed in acute COVID-19 does not appear to persist, interestingly, ongoing symptomatic COVID-19 and post-COVID pathogeneses appear to reflect the persistence of immune and cellular disturbances triggered by the acute and subacute periods.

Neoadjuvant durvalumab plus radiation versus durvalumab alone in stages I-III non-small cell lung cancer: survival outcomes and molecular correlates of a randomized phase II trial.

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway.

Introduction: In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen. Methods: We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes. Results: The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-ß (TGF-ß) to activate collagen synthesis in the HLFs. Unlike TGF-ß, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-ß have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis. Discussion: It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Persistent alveolar type 2 dysfunction and lung structural derangement in post-acute COVID-19.

SARS-CoV-2 infection can manifest as a wide range of respiratory and systemic symptoms well after the acute phase of infection in over 50% of patients. Key questions remain on the long-term effects of infection on tissue pathology in recovered COVID-19 patients. To address these questions we performed multiplexed imaging of post-mortem lung tissue from 12 individuals who died post-acute COVID-19 (PC) and compare them to lung tissue from patients who died during the acute phase of COVID-19, or patients who died with idiopathic pulmonary fibrosis (IPF), and otherwise healthy lung tissue. We find evidence of viral presence in the lung up to 359 days after the acute phase of disease, including in patients with negative nasopharyngeal swab tests. The lung of PC patients are characterized by the accumulation of senescent alveolar type 2 cells, fibrosis with hypervascularization of peribronchial areas and alveolar septa, as the most pronounced pathophysiological features. At the cellular level, lung disease of PC patients, while distinct, shares pathological features with the chronic pulmonary disease of IPF. which may help rationalize interventions for PC patients. Altogether, this study provides an important foundation for the understanding of the long-term effects of SARS-CoV-2 pulmonary infection at the microanatomical, cellular, and molecular level.

The pathogenesis of coronavirus-19 disease.

Severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) is the causal agent of coronavirus disease-2019 (COVID-19), a systemic illness characterized by variably severe pulmonary symptoms, cardiac conduction abnormalities, diarrhea, and gastrointestinal bleeding, as well as neurologic deficits, renal insufficiency, myalgias, endocrine abnormalities, and other perturbations that reflect widespread microvascular injury and a pro-inflammatory state. The mechanisms underlying the various manifestations of viral infection are incompletely understood but most data suggest that severe COVID-19 results from virus-driven perturbations in the immune system and resultant tissue injury. Aberrant interferon-related responses lead to alterations in cytokine elaboration that deplete resident immune cells while simultaneously recruiting hyperactive macrophages and functionally altered neutrophils, thereby tipping the balance from adaptive immunity to innate immunity. Disproportionate activation of these macrophages and neutrophils further depletes normal activity of B-cells, T-cells, and natural killer (NK) cells. In addition, this pro-inflammatory state stimulates uncontrolled complement activation and development of neutrophil extracellular traps (NETS), both of which promote the coagulation cascade and induce a state of "thrombo-inflammation". These perturbations have similar manifestations in multiple organ systems, which frequently show pathologic findings related to microvascular injury and thrombosis of large and small vessels. However, the pulmonary findings in patients with severe COVID-19 are generally more pronounced than those of other organs. Not only do they feature inflammatory thromboses and endothelial injury, but much of the parenchymal damage stems from failed maturation of alveolar pneumocytes, interactions between type 2 pneumocytes and non-resident macrophages, and a greater degree of NET formation. The purpose of this review is to discuss the pathogenesis underlying organ damage that can occur in patients with SARS-CoV-2 infection. Understanding these mechanisms of injury is important to development of future therapies for patients with COVID-19, many of which will likely target specific components of the immune system, particularly NET induction, pro-inflammatory cytokines, and subpopulations of immune cells.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster after either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely affected the olfactory bulb (OB) and olfactory epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month after viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Angiopoietin 2 Is Associated with Vascular Necroptosis Induction in Coronavirus Disease 2019 Acute Respiratory Distress Syndrome.

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)-induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

Global evolution of the tumor microenvironment associated with progression from preinvasive invasive to invasive human lung adenocarcinoma.

To investigate changes in the tumor microenvironment (TME) during lung cancer progression, we interrogate tumors from two chest computed tomography (CT)-defined groups. Pure non-solid (pNS) CT density nodules contain preinvasive/minimally invasive cancers, and solid density nodules contain invasive cancers. Profiling data reveal a dynamic interaction between the tumor and its TME throughout progression. Alterations in genes regulating the extracellular matrix and genes regulating fibroblasts are central at the preinvasive state. T cell-mediated immune suppression is initiated in preinvasive nodules and sustained with rising intensity through progression to invasive tumors. Reduced T cell infiltration of the cancer cell nests is more frequently associated with preinvasive cancers, possibly until tumor evolution leads to a durable, viable invasive phenotype accompanied by more varied and robust immune suppression. Upregulation of immune checkpoints occurs only in the invasive nodules. Throughout progression, an effector immune response is present but is effectively thwarted by the immune-suppressive elements.

System-wide transcriptome damage and tissue identity loss in COVID-19 patients.

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.

Integrative network analysis of early-stage lung adenocarcinoma identifies aurora kinase inhibition as interceptor of invasion and progression.

Here we focus on the molecular characterization of clinically significant histological subtypes of early-stage lung adenocarcinoma (esLUAD), which is the most common histological subtype of lung cancer. Within lung adenocarcinoma, histology is heterogeneous and associated with tumor invasion and diverse clinical outcomes. We present a gene signature distinguishing invasive and non-invasive tumors among esLUAD. Using the gene signatures, we estimate an Invasiveness Score that is strongly associated with survival of esLUAD patients in multiple independent cohorts and with the invasiveness phenotype in lung cancer cell lines. Regulatory network analysis identifies aurora kinase as one of master regulators of the gene signature and the perturbation of aurora kinases in vitro and in a murine model of invasive lung adenocarcinoma reduces tumor invasion. Our study reveals aurora kinases as a therapeutic target for treatment of early-stage invasive lung adenocarcinoma.

Expression of the mono-ADP-ribosyltransferase ART1 by tumor cells mediates immune resistance in non-small cell lung cancer.

Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.