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OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

As of October 2020, there are over 40 million confirmed cases, and more than 1 million confirmed deaths of Covid-19 worldwide. The main cause of death in hospitalized patients is a respiratory failure due to acute respiratory distress syndrome. It has been suggested that the very intense immune response induces diffuse alveolar damage that far exceeds the harm that would have been caused by virus replication per se, resulting in lethal tissue destruction. We present a detailed report of the histopathological findings on cryo transbronchial biopsy in the patient with persistent (3 months) interstitial pneumonitis and severe CD8 positive cell infiltration in the lungs due to SARS-CoV-2 infection. CD8 positive T-lymphocytes have a great potential to damage tissue either through direct cytotoxicity or through cytokines release.

Purpose c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

Summary This study identified molecular differences between cisplatin-resistant and parental H460 lung cancer cells by using microarray expressionanalysis.Cisplatinresistant cells illustrated more rapidinvivotumorgrowthand greater survival following treatment with cisplatin or radiation than parental H460 cells. Cisplatin-resistant cells also demonstrated decreased expression of insulin-like growth factor binding protein3 and increased IGF-1R signaling. Cisplatin resistance was reversed by treating cisplatin-resistant cells with human recombinant IGF binding protein-3. siRNA Purpose: The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in nonesmall-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. Methods and Materials: H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. Results: Cisplatin-R cells illustrated greater expression of the markers CD133 and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposideinduced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. Conclusions: The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment. 2012 Elsevier Inc.

Introduction: Survivin, an inhibitor of apoptosis protein and key regulator of mitosis, is up-regulated in a variety of cancers and is often associated with a worse prognosis. Terameprocol down-regulates the Sp1-mediated transcription of survivin and Cdk1, which is important for cell cycle progression and many other proteins. Survivin inhibition has previously been shown to result in the induction of apoptosis and radiosensitization. Methods: This study examined the effects of terameprocol administration on survivin transcription and expression in HCC2429 and H460 lung cancer cells. We also examined the combined effects of radiation and terameprocol on apoptosis and radiosensitivity. Results: Using immunoblot analysis and luciferase assays, we confirmed that terameprocol decreases survivin transcription and protein expression. Ultimately, however, decreases in survivin expression failed to correlate with an increase in apoptosis. Nonetheless, clonogenic assay revealed that terameprocol induces increased radiosensitization in HCC2429 (dose enhancement ratio=1.26, p = 0.019) and H460 (dose enhancement ratio=1.18, p = 0.001) cells. Additionally, the data show no effect of terameprocol on cell cycle in either HCC2429 or H460 cells. Conclusions: Terameprocol significantly enhances the sensitivity of non-small cell lung carcinoma cell lines to radiation therapy, although the mechanism of action remains unclear. Further study is warranted to assess the potential of terameprocol as an agent that may enhance the therapeutic ratio of radiotherapy in lung cancer.

Glucocorticoid receptors (GRs) affect both gene induction and gene repression. The disparities of receptor binding to DNA and increased vs. decreased gene expression have suggested significant mechanistic differences between GR-mediated induction and repression. Numerous transcription factors are known to modulate three parameters of gene induction: the total activity (Vmax) and position of the dose-response curve with glucocorticoids (EC50) and the percent partial agonist activity with antiglucocorticoids. We have examined the effects on GR-mediated repression of five modulators (coactivators TIF2 [GRIP1, SRC-2] and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol [DAC]) of very different structure, and an inhibitor of histone deacetylation (trichostatin A [TSA]). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Vmax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps.

Varying the concentration of selected factors alters the induction properties of steroid receptors by changing the position of the dose–response curve (or the value for half-maximal induction = EC50) and the amount of partial agonist activity of antisteroids. We now describe a rudimentary mathematical model that predicts a simple Michaelis–Menten curve for the multi-step process of steroid-regulated gene induction. This model suggests that steps far downstream from receptor binding to steroid can influence the EC50 of agonist-complexes and partial agonist activity of antagonist-complexes. We therefore asked whether inhibitors of three possible downstream steps can reverse the effects of increased concentrations of two factors: glucocorticoid receptors (GRs) and Ubc9. The downstream steps (with inhibitors in parentheses) are protein deacetylation (TSA and VPA), DNA unwinding (CPT), and CTD phosphorylation of RNA polymerase II (DRB and H8). None of the inhibitors mimic or prevent the effects of added GRs. However, inhibitors of DNA unwinding and CTD phosphorylation do reverse the effects of Ubc9 with high GR concentrations. These results support our earlier conclusion that different rate-limiting steps operate at low and high GR concentrations versus high GR with Ubc9. The present data also suggest that downstream steps can modulate the EC50 of GR-mediated induction, thus both supporting the utility of our mathematical model and widening the field of biochemical processes that can modify the EC50.

Background Non-small-cell lung cancer frequently exhibits mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionising radiation and chemotherapy. There is an urgent need for the development of novel therapies to treat KRAS-mutant lung cancer. Activation through Mek-Erk-dependent signaling occurs through cyclin D1 induction and subsequent assembly of the cyclin D1-CDK4/CDK6 complex, which results in Rb phosphorylation and Rb-E2F dissociation and G1-S cell cycle. MEK inhibitors do not engage the cell-cycle checkpoint, resulting in tumour stasis but not regression, despite robust apoptosis. The aim of the current study was to test the hypothesis that MAP kinase inhibitors and CDK4 inhibitors will offer clear therapeutic benefit when integrated into radiotherapy for treatment of KRAS-mutant lung cancer. Methods Clonogenic survival assays were done with the H358, A549, and H460 cell lines using the MEK inhibitor trametinib and the CDK4 inhibitor PD033291. Cell viability was assessed by tetrazolium-based CellTiter 96 aqueous non-radioactive cell proliferation assay (MTS). DNA repair was analysed using immunocytochemistry. Western blots were used to investigate the effects on downstream signaling. Flow cytometry was used to analyse cell cycle and apoptosis. Growth delay was used to determine the effects of trametinib and PD0332991 on in vivo tumour radiosensitivity. Findings Preradiation treatment with trametinib and PD0332991 significantly decreased clonogenic survival compared with either agent alone. Dose modifying factors at a surviving fraction for trametinib and PD0332991 were 2.38, 1.62, and 1.77 for H358, A549, and H460, respectively. Cell proliferation was decreased by treatment with trametinib and PD0332991 as compared with either agent alone. Flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest and enhanced apoptosis in these cell lines. The expression of phospho-Rb and phospho-Erk1/2 were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumour growth delay in A549 xenografts. Interpretation These data suggest that trametinib can be used with CDK inhibitors to augment the radiation response in KRAS-mutant non-small-cell lung cancer.

Purpose/Objective(s): Combination of platinum-based chemotherapy and radiation is currently the standard treatment for locally advanced non-small cell lung cancer (NSCLC) patients. However, this approach often results in therapeutic resistance and disease relapse, partly because of the presence of cancer stem cells (CSCs) within the tumor. To investigate the CSCs hypothesis of chemoradiation resistance in NSCLC, we used high-throughput microarray assay to profile CSCs-like cells versus parental cancer cells to identify the candidate genes for cancer therapy.