Repeat Next-Generation Sequencing Testing on Progression in Men With Metastatic Prostate Cancer Can Identify New Actionable Alterations.

PURPOSE: There are limited data available on the real-world patterns of molecular testing in men with advanced prostate cancer. We thus sought to evaluate next-generation sequencing (NGS) testing in the United States, focused on single versus serial NGS testing, the different disease states of testing (hormone-sensitive v castration-resistant, metastatic vs nonmetastatic), tissue versus plasma circulating tumor DNA (ctDNA) assays, and how often actionable data were found on each NGS test. METHODS: The Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort clinical-genomic database was used for this retrospective analysis, including 1,597 patients across 15 institutions. Actionable NGS data were defined as including somatic alterations in homologous recombination repair genes, mismatch repair deficiency, microsatellite instability (MSI-high), or a high tumor mutational burden ≥10 mut/MB. RESULTS: Serial NGS testing (two or more NGS tests with specimens collected more than 60 days apart) was performed in 9% (n = 144) of patients with a median of 182 days in between test results. For the second NGS test and beyond, 82.1% (225 of 274) of tests were from ctDNA assays and 76.1% (217 of 285) were collected in the metastatic castration-resistant setting. New actionable data were found on 11.1% (16 of 144) of second NGS tests, with 3.5% (5 of 144) of tests detecting a new BRCA2 alteration or MSI-high. A targeted therapy (poly (ADP-ribose) polymerase inhibitor or immunotherapy) was given after an actionable result on the second NGS test in 31.3% (5 of 16) of patients. CONCLUSION: Repeat somatic NGS testing in men with prostate cancer is infrequently performed in practice and can identify new actionable alterations not present with initial testing, suggesting the utility of repeat molecular profiling with tissue or blood of men with metastatic castration-resistant prostate cancer to guide therapy choices.

Pathogenic Germline Mutational Landscape in Patients With Renal Cell Carcinoma and Associated Clinicopathologic Features.

PURPOSE: A subset of renal cell carcinoma (RCC) cases occur because of a hereditary predisposition. However, the prevalence and profiling of germline alterations in RCC have not been fully characterized. Additionally, clinicopathologic factors associated with pathogenic or likely pathogenic (P/LP) germline variants in patients with RCC remain poorly understood. METHODS: A retrospective analysis of patients with RCC who underwent genetic evaluation was performed. The frequency of P/LP germline variants and genes was evaluated in this cohort. The association between genetic testing outcomes and clinicopathologic features was also assessed. RESULTS: A total of 321 patients with RCC who had germline testing were identified. Within this cohort, 42 patients (13.1%) had P/LP variants. Genes with the most frequent germline mutations were FLCN (n = 10, 3.1%), SDHB (n = 4, 1.2%), VHL (n = 4, 1.2%), MLH1 (n = 3, 0.9%), and CHEK2 (n = 4, 1.2%). Among patients with P/LP variants, 19 (45.2%) had a potentially targetable mutation. The presence of bilateral or multifocal tumors was associated with P/LP variants (P = .0012 and P = .0098, respectively). Patients who had targeted gene testing had higher rates of P/LP variants compared with multigene panel testing (P = .015). Age and family history of cancers (RCC and non-RCC) did not have any statistically significant association with germline testing outcomes. CONCLUSION: Among patients with RCC, unselected for a known familial predisposition, 13.4% had P/LP variants. Almost half of patients with P/LP variants had a potentially targetable mutation. Targeted gene panel testing is a feasible option for patients, particularly if syndromic features are present. Age and family history were not associated with P/LP variants. Future studies are needed to optimize current genetic evaluation criteria to expand the detection of patients with RCC who may have germline mutations.

PROBABILISTIC LEARNING OF TREATMENT TREES IN CANCER.

Accurate identification of synergistic treatment combinations and their underlying biological mechanisms is critical across many disease domains, especially cancer. In translational oncology research, preclinical systems such as patient-derived xenografts (PDX) have emerged as a unique study design evaluating multiple treatments administered to samples from the same human tumor implanted into genetically identical mice. In this paper, we propose a novel Bayesian probabilistic tree-based framework for PDX data to investigate the hierarchical relationships between treatments by inferring treatment cluster trees, referred to as treatment trees (Rx-tree). The framework motivates a new metric of mechanistic similarity between two or more treatments accounting for inherent uncertainty in tree estimation; treatments with a high estimated similarity have potentially high mechanistic synergy. Building upon Dirichlet Diffusion Trees, we derive a closed-form marginal likelihood encoding the tree structure, which facilitates computationally efficient posterior inference via a new two-stage algorithm. Simulation studies demonstrate superior performance of the proposed method in recovering the tree structure and treatment similarities. Our analyses of a recently collated PDX dataset produce treatment similarity estimates that show a high degree of concordance with known biological mechanisms across treatments in five different cancers. More importantly, we uncover new and potentially effective combination therapies that confer synergistic regulation of specific downstream biological pathways for future clinical investigations. Our accompanying code, data, and shiny application for visualization of results are available at: https://github.com/bayesrx/RxTree.

Optimizing Precision Medicine for Breast Cancer Brain Metastases with Functional Drug Response Assessment.

The development of novel therapies for brain metastases is an unmet need. Brain metastases may have unique molecular features that could be explored as therapeutic targets. A better understanding of the drug sensitivity of live cells coupled to molecular analyses will lead to a rational prioritization of therapeutic candidates. We evaluated the molecular profiles of 12 breast cancer brain metastases (BCBM) and matched primary breast tumors to identify potential therapeutic targets. We established six novel patient-derived xenograft (PDX) from BCBM from patients undergoing clinically indicated surgical resection of BCBM and used the PDXs as a drug screening platform to interrogate potential molecular targets. Many of the alterations were conserved in brain metastases compared with the matched primary. We observed differential expressions in the immune-related and metabolism pathways. The PDXs from BCBM captured the potentially targetable molecular alterations in the source brain metastases tumor. The alterations in the PI3K pathway were the most predictive for drug efficacy in the PDXs. The PDXs were also treated with a panel of over 350 drugs and demonstrated high sensitivity to histone deacetylase and proteasome inhibitors. Our study revealed significant differences between the paired BCBM and primary breast tumors with the pathways involved in metabolisms and immune functions. While molecular targeted drug therapy based on genomic profiling of tumors is currently evaluated in clinical trials for patients with brain metastases, a functional precision medicine strategy may complement such an approach by expanding potential therapeutic options, even for BCBM without known targetable molecular alterations. Significance: Examining genomic alterations and differentially expressed pathways in brain metastases may inform future therapeutic strategies. This study supports genomically-guided therapy for BCBM and further investigation into incorporating real-time functional evaluation will increase confidence in efficacy estimations during drug development and predictive biomarker assessment for BCBM.

Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1ɑ expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

Ulinastatin promotes macrophage efferocytosis and ameliorates lung inflammation via the ERK5/Mer signaling pathway.

Acute lung injury (ALI) is a pneumonic response characterized by neutrophil infiltration. Macrophage efferocytosis is the process whereby macrophages remove apoptotic cells, and is required for ALI inflammation to subside. The glycoprotein ulinastatin (UTI) has an anti-inflammatory effect during the acute stages of ALI, but its effect on efferocytosis and the subinflammatory stage of ALI is unclear. Extracellular signal-regulated kinase 5 (ERK5) is a key protein in efferocytosis, and we thus hypothesized that it may be activated by UTI to regulate efferocytosis and the resolution of pneumonia. To test this hypothesis, here we monitored phagocytosis of macrophages through in vivo and in vitro experiments. Pulmonary edema, neutrophil infiltration, protein exudation, and inflammatory factor regression were observed on days 1, 3, 5, and 7 in vivo. RAW264.7 cells were pretreated with different concentrations of UTI and ERK5 inhibitors, and the expression of tyrosine-protein kinase Mer (Mer) protein on macrophage membrane was detected. UTI increased the phagocytosis of apoptotic neutrophils by macrophages in vitro and in vivo, and promoted the resolution of pneumonia. The protein expression of ERK5 and Mer increased with UTI concentration, while the expression of Mer was down-regulated by ERK5 inhibitors. Therefore, our results suggest that UTI enhances efferocytosis and reduces lung inflammation and injury through the ERK5/Mer signaling pathway, which may be one of the targets of UTI in the treatment of lung injury.

Identification of Hub Genes in Type 2 Diabetes Mellitus Using Bioinformatics Analysis.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases in the world with complicated pathogenesis. This study aimed to identify differentially expressed genes (DEGs) and molecular pathways in T2DM using bioinformatics analysis. MATERIALS AND METHODS: To explore potential therapeutic targets for T2DM, we analyzed three microarray datasets (GSE50397, GSE38642, and GSE44035) acquired from the Gene Expression Omnibus (GEO) database. DEGs between T2DM islet and normal islet were picked out by the GEO2R tool and Venn diagram software. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to identify the pathways and functional annotation of DEGs. Then, protein-protein interaction (PPI) of these DEGs was visualized by Cytoscape with Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). RESULTS: In total, we identified 36 DEGs in the three datasets, including 32 up-regulated genes and four down-regulated genes. The improved functions and pathways of the DEGs enriched in cytokine-cytokine receptor interaction, pathways in cancer, PI3K-Akt signaling pathway, and Rheumatoid arthritis. Among them, ten hub genes with a high degree of connectivity were selected. Furthermore, via the re-analysis of DAVID, four genes (IL6, MMP3, MMP1, and IL11) were significantly enriched in the Rheumatoid arthritis pathway. CONCLUSION: Our study, based on the GEO database, identified four significant up-regulated DEGs and provided novel targets for diagnosis and treatment of T2DM.

Prediction of 1p/19q Codeletion in Diffuse Glioma Patients Using Pre-operative Multiparametric Magnetic Resonance Imaging.

This study compared the predictive power and robustness of texture, topological, and convolutional neural network (CNN) based image features for measuring tumors in MRI. These features were used to predict 1p/19q codeletion in the MICCAI BRATS 2017 challenge dataset. Topological data analysis (TDA) based on persistent homology had predictive performance as good as or better than texture-based features and was also less susceptible to image-based perturbations. Features from a pre-trained convolutional neural network had similar predictive performances and robustness as TDA, but also performed better using an alternative classification algorithm, k-top scoring pairs. Feature robustness can be used as a filtering technique without greatly impacting model performance and can also be used to evaluate model stability.

Regulation of cellular oxidative stress and apoptosis by G protein-coupled receptor kinase-2; The role of NADPH oxidase 4.

Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial ß-adrenergic receptors (ß-ARs) is deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that GRK2, a critical regulator of myocardial ß-AR signaling, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to ß-agonist stimulation. Stimulation of H9c2 cells with a non-selective ß-agonist, isoproterenol (Iso) caused increased oxidative stress and apoptosis. There was also increased Nox4 expression, but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. Adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with Iso stimulation. These increases in oxidative stress were abolished by pre-treatment with the non-specific Nox inhibitor, apocynin, or siRNA knockdown of Nox4. Adenoviral-mediated expression of a GRK2 inhibitor prevented ROS production and apoptosis in response to Iso stimulation. ß-Arrestins are signaling proteins that function downstream of GRK2 in ß-AR uncoupling. Adenoviral-mediated overexpression of ß-arrestins increased ROS production and Nox4 expression. Chronic ß-agonist stimulation in mice increased Nox4 expression and apoptosis compared to PBS or AngII treatment. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in cardiac myocytes and provides an additional novel mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition to prevent the development of HF.

Regulation of mitochondrial oxidative stress by ß-arrestins in cultured human cardiac fibroblasts.

Oxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. ß-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that ß-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. ß-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-ß-stimulated collagen and α-SMA expression. CF ß-arrestin expression was upregulated fourfold in HF. ß-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. ß-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. ß-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of ß-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis.

Diesel exhaust particles activate the matrix-metalloproteinase-1 gene in human bronchial epithelia in a beta-arrestin-dependent manner via activation of RAS.

BACKGROUND: Diesel exhaust particles (DEPs) are globally relevant air pollutants that exert a detrimental human health impact. However, mechanisms of damage by DEP exposure to human respiratory health and human susceptibility factors are only partially known. Matrix metalloproteinase-1 (MMP-1) has been implied as an (etio)pathogenic factor in human lung and airway diseases such as emphysema, chronic obstructive pulmonary disease, chronic asthma, tuberculosis, and bronchial carcinoma and has been reported to be regulated by DEPs. OBJECTIVE: We elucidated the molecular mechanisms of DEPs' up-regulation of MMP-1. METHODS/RESULTS: Using permanent and primary human bronchial epithelial (HBE) cells at air-liquid interface, we show that DEPs activate the human MMP-1 gene via RAS and subsequent activation of RAF-MEK-ERK1/2 mitogen-activated protein kinase signaling, which can be scaffolded by beta-arrestins. Short interfering RNA mediated beta-arrestin1/2 knockout eliminated formation, subsequent nuclear trafficking of phosphorylated ERK1/2, and resulting MMP-1 transcriptional activation. Transcriptional regulation of the human MMP-1 promoter was strongly influenced by the presence of the -1607GG polymorphism, present in 60-80% of humans, which led to striking up-regulation of MMP-1 transcriptional activation. CONCLUSION: Our results confirm up-regulation of MMP-1 in response to DEPs in HBE and provide new mechanistic insight into how these epithelia, the first line of protection against environmental insults, up-regulate MMP-1 in response to DEP inhalation. These mechanisms include a role for the human -1607GG polymorphism as a susceptibility factor for an accentuated response, which critically depends on the ability of beta-arrestin1/2 to generate scaffolding and nuclear trafficking of phosphorylated ERK1/2.