The Tissue Systems Pathology Test Objectively Risk-Stratifies Patients With Barrett's Esophagus: Results From a Multicenter US Clinical Experience Study.

BACKGROUND: Barrett's esophagus (BE) is a diagnosis of esophageal intestinal metaplasia, which can progress to esophageal adenocarcinoma (EAC), and guidelines recommend endoscopic surveillance for early detection and treatment of EAC. However, current practices have limited effectiveness in risk-stratifying patients with BE. AIM: This study aimed to evaluate use of the TSP-9 test in risk-stratifying clinically relevant subsets of patients with BE in clinical practice. METHODS: TSP-9 results for tests ordered by 891 physicians for 8080 patients with BE with clinicopathologic data were evaluated. Orders were from nonacademic (94.3%) and academic (5.7%) settings for nondysplastic BE (NDBE; n=7586; 93.9%), indefinite for dysplasia (IND, n=312, 3.9%), and low-grade dysplasia (LGD, n=182, 2.3%). RESULTS: The TSP-9 test scored 83.2% of patients with low risk, 10.6% intermediate risk, and 6.2% high risk, respectively, for progression to HGD/EAC within 5 years. TSP-9 provided significant risk-stratification independently of clinicopathologic features, within NDBE, IND, and LGD subsets, male and female, and short- and long-segment subsets of patients. TSP-9 identified 15.3% of patients with NDBE as intermediate/high-risk for progression, which was 6.4 times more than patients with a pathology diagnosis of LGD. Patients with NDBE who scored intermediate or high risk had a predicted 5-year progression risk of 8.1% and 15.3%, respectively, which are similar to and higher than published progression rates in patients with BE with confirmed LGD. CONCLUSIONS: The TSP-9 test identified a high-risk subset of patients with NDBE who were predicted to progress at a higher rate than confirmed LGD, enabling early detection of patients requiring management escalation to reduce the incidence of EAC. TSP-9 scored the majority of patients with NDBE as low risk, providing support to adhere to 3- to 5-year surveillance per guidelines.

The Tissue Systems Pathology Test Outperforms Pathology Review in Risk Stratifying Patients With Low-Grade Dysplasia.

BACKGROUND & AIMS: Low-grade dysplasia (LGD) is associated with an increased risk of progression in Barrett's esophagus (BE); however, the diagnosis of LGD is limited by substantial interobserver variability. Multiple studies have shown that an objective tissue systems pathology test (TissueCypher Barrett's Esophagus Test, TSP-9), can effectively predict neoplastic progression in patients with BE. This study aimed to compare the risk stratification performance of the TSP-9 test vs benchmarks of generalist and expert pathology. METHODS: A blinded cohort study was conducted in the screening cohort of a randomized controlled trial of patients with BE with community-based LGD. Biopsies from the first endoscopy with LGD were assessed by the TSP-9 test and independently reviewed by 30 pathologists from 5 countries per standard practice. The accuracy of the test and the diagnoses in predicting high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) were compared. RESULTS: A total of 154 patients with BE (122 men), mean age 60.9 ± 9.8 years were studied. Twenty-four patients progressed to HGD/EAC within 5 years (median time of 1.7 years) and 130 did not progress to HGD/EAC within 5 years (median 7.8 years follow-up). The TSP-9 test demonstrated higher sensitivity (71% vs mean 63%, range 33%-88% across 30 pathologists), than the pathology review in detecting patients who progressed (P = .01186). CONCLUSIONS: The TSP-9 test outperformed the pathologists in risk stratifying patients with BE with LGD. Care guided by the test can provide an effective solution to variable pathology review of LGD, improving health outcomes by upstaging care to therapeutic intervention for patients at high risk for progression, while reducing unnecessary interventions in low-risk patients.

A Tissue Systems Pathology Test Outperforms the Standard-of-Care Variables in Predicting Progression in Patients With Barrett's Esophagus.

INTRODUCTION: Objective risk stratification is needed for patients with Barrett's esophagus (BE) to enable risk-aligned management to improve health outcomes. This study evaluated the predictive performance of a tissue systems pathology [TSP-9] test (TissueCypher) vs current clinicopathologic variables in a multicenter cohort of patients with BE. METHODS: Data from 699 patients with BE from 5 published studies on the TSP-9 test were evaluated. Five hundred nine patients did not progress during surveillance, 40 were diagnosed with high-grade dysplasia/esophageal adenocarcinoma (HGD/EAC) within 12 months, and 150 progressed to HGD/EAC after 12 months. Age, sex, segment length, hiatal hernia, original and expert pathology review diagnoses, and TSP-9 risk classes were collected. The predictive performance of clinicopathologic variables and the TSP-9 test was compared, and the TSP-9 test was evaluated in clinically relevant patient subsets. RESULTS: The sensitivity of the TSP-9 test in detecting progressors was 62.3% compared with 28.3% for expert-confirmed low-grade dysplasia (LGD), while the original diagnosis abstracted from medical records did not provide any significant risk stratification. The TSP-9 test identified 57% of progressors with nondysplastic Barrett's esophagus (NDBE) ( P < 0.0001). Patients with NDBE who scored TSP-9 high risk progressed at a similar rate (3.2%/yr) to patients with expert-confirmed LGD (3.7%/yr). The TSP-9 test provided significant risk stratification in clinically low-risk patients (NDBE, female, short-segment BE) and clinically high-risk patients (IND/LGD, male, long-segment BE) ( P < 0.0001 for comparison of high-risk classes vs low-risk classes). DISCUSSION: The TSP-9 test predicts risk of progression to HGD/EAC independently of current clinicopathologic variables in patients with BE. The test provides objective risk stratification results that may guide management decisions to improve health outcomes for patients with BE.

An Automated Tissue Systems Pathology Test Can Standardize the Management and Improve Health Outcomes for Patients With Barrett's Esophagus.

INTRODUCTION: Low-grade dysplasia (LGD) in Barrett's esophagus (BE) is associated with an increased risk of progression to high-grade dysplasia or esophageal adenocarcinoma. However, because of substantial interobserver variability in the diagnosis of LGD, a patient's management plan and health outcome depend largely on which pathologist reviews their case. This study evaluated the ability of a tissue systems pathology test that objectively risk stratifies patients with BE (TissueCypher, TSP-9) to standardize management in a manner consistent with improved health outcomes for patients with BE. METHODS: A total of 154 patients with BE with community-based LGD from the prospectively followed screening cohort of the SURF trial were studied. Management decisions were simulated 500 times with varying generalist (n = 16) and expert (n = 14) pathology reviewers to determine the most likely care plan with or without use of the TSP-9 test for guidance. The percentage of patients receiving appropriate management based on the known progression/nonprogression outcomes was calculated. RESULTS: The percentage of patients with 100% of simulations resulting in appropriate management significantly increased from 9.1% for pathology alone, to 58.4% when TSP-9 results were used with pathology, and further increased to 77.3% of patients receiving appropriate management when only TSP-9 results were used. Use of the test results also significantly increased the consistency of management decisions for patients when their slides were reviewed by different pathologists ( P < 0.0001). DISCUSSION: Management guided by the TSP-9 test can standardize care plans by increasing the early detection of progressors who can receive therapeutic interventions, while also increasing the percentage of nonprogressors who can avoid unnecessary therapy and be managed by surveillance alone.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis.

Retinal diseases are frequently characterized by the accumulation of excessive scar tissue found throughout the neural retina. However, the pathophysiology of retinal fibrosis remains poorly understood, and the cell types that contribute to the fibrotic response are incompletely defined. Here, we show that myofibroblast differentiation of mural cells contributes directly to retinal fibrosis. Using lineage tracing technology, we demonstrate that after chemical ocular injury, Myh11+ mural cells detach from the retinal microvasculature and differentiate into myofibroblasts to form an epiretinal membrane. Inhibition of TGFßR attenuates Myh11+ retinal mural cell myofibroblast differentiation, and diminishes the subsequent formation of scar tissue on the surface of the retina. We demonstrate retinal fibrosis within a murine model of oxygen-induced retinopathy resulting from the intravitreal injection of adipose Myh11-derived mesenchymal stem cells, with ensuing myofibroblast differentiation. In this model, inhibiting TGFßR signaling does not significantly alter myofibroblast differentiation and collagen secretion within the retina. This work shows the complexity of retinal fibrosis, where scar formation is regulated both by TGFßR and non-TGFßR dependent processes involving mural cells and derived mesenchymal stem cells. It also offers a cautionary note on the potential deleterious, pro-fibrotic effects of exogenous MSCs once intravitreally injected into clinical patients.

An ultrasensitive fiveplex activity assay for cellular kinases.

Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt-IKK-JNK-MEK-MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes.

Cellular responses to stimuli involve dynamic and localized changes in protein kinases and phosphatases. Here, we report a generalized functional assay for high-throughput profiling of multiple protein phosphatases with subcellular resolution and apply it to analyze coxsackievirus B3 (CVB3) infection counteracted by interferon signaling. Using on-plate cell fractionation optimized for adherent cells, we isolate protein extracts containing active endogenous phosphatases from cell membranes, the cytoplasm, and the nucleus. The extracts contain all major classes of protein phosphatases and catalyze dephosphorylation of plate-bound phosphosubstrates in a microtiter format, with cellular activity quantified at the end point by phosphospecific ELISA. The platform is optimized for six phosphosubstrates (ERK2, JNK1, p38α, MK2, CREB, and STAT1) and measures specific activities from extracts of fewer than 50,000 cells. The assay was exploited to examine viral and antiviral signaling in AC16 cardiomyocytes, which we show can be engineered to serve as susceptible and permissive hosts for CVB3. Phosphatase responses were profiled in these cells by completing a full-factorial experiment for CVB3 infection and type I/II interferon signaling. Over 850 functional measurements revealed several independent, subcellular changes in specific phosphatase activities. During CVB3 infection, we found that type I interferon signaling increases subcellular JNK1 phosphatase activity, inhibiting nuclear JNK1 activity that otherwise promotes viral protein synthesis in the infected host cell. Our assay provides a high-throughput way to capture perturbations in important negative regulators of intracellular signal-transduction networks.

Computational Models of Reactive Oxygen Species as Metabolic Byproducts and Signal-Transduction Modulators.

Reactive oxygen species (ROS) are widely involved in intracellular signaling and human pathologies, but their precise roles have been difficult to enumerate and integrate holistically. The context- and dose-dependent intracellular effects of ROS can lead to contradictory experimental results and confounded interpretations. For example, lower levels of ROS promote cell signaling and proliferation, whereas abundant ROS cause overwhelming damage to biomolecules and cellular apoptosis or senescence. These complexities raise the question of whether the many facets of ROS biology can be joined under a common mechanistic framework using computational modeling. Here, we take inventory of some current models for ROS production or ROS regulation of signaling pathways. Several models captured non-intuitive observations or made predictions that were later verified by experiment. There remains a need for systems-level analyses that jointly incorporate ROS production, handling, and modulation of multiple signal-transduction cascades.