Integrins in biliary injury and fibrosis.

PURPOSE OF REVIEW: Current treatment options for cholangiopathies are severely limited and there is thus a critical need to identify and develop therapies. This review discusses the role of integrins in biliary injury and fibrosis and their potential as therapeutic targets. RECENT FINDINGS: There are a diverse set of roles that integrins play in biliary injury and fibrosis. Some integrins activate TGF-ß signaling or are involved in sensing of the extracellular matrix, making them attractive targets for biliary fibrosis. In recent work, autoantibodies to α v ß 6 were identified in patients with PSC, supporting the relevance of this integrin in the disease. In addition, a role for α 2 ß 1 in cyst formation was identified in a mouse model of polycystic liver disease. Leukocyte integrins (e.g. α E ß 7 and α 4 ß 7 ) contribute to lymphocyte trafficking, making them potential targets for biliary inflammation; however, this has not yet translated to the clinic. SUMMARY: While all members of the same family of proteins, integrins have diverse roles in the pathogenesis of biliary disease. Targeting one or multiple of these integrins may slow or halt the progression of biliary injury and fibrosis by simultaneously impacting different pathologic cells and processes.

Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

Stratification of Tamoxifen Synergistic Combinations for the Treatment of ER+ Breast Cancer.

Breast cancer alone accounts for the majority of cancer deaths among women, with the most commonly diagnosed subtype being estrogen receptor positive (ER+). Survival has greatly improved for patients with ER+ breast cancer, due in part to the development of antiestrogen compounds, such as tamoxifen. While treatment of the primary disease is often successful, as many as 30% of patients will experience recurrence and metastasis, mainly due to developed endocrine therapy resistance. In this study, we discovered two tamoxifen combination therapies, with simeprevir and VX-680, that reduce the tumor burden in animal models of ER+ breast cancer more than either compound or tamoxifen alone. Additionally, these tamoxifen combinations reduced the expression of HER2, a hallmark of tamoxifen treatment, which can facilitate acquisition of a treatment-resistant phenotype. These combinations could provide clinical benefit by potentiating tamoxifen treatment in ER+ breast cancer.

24-hydroxycholesterol replacement rate measured in blood is a non-invasive biomarker of brain demyelination and remyelination in cuprizone-treated mice.

In mice, dietary cuprizone causes brain demyelination with subsequent spontaneous remyelination upon return to normal chow. Heavy water (2H2O) labeling with mass spectrometric analysis can be used to measure brain de novo synthesis of several myelin components including cholesterol, phospholipids, galactocereboside (GalC) and myelin-associated proteins. 24-hydroxycholesterol (24-OHC), the major metabolite of brain cholesterol, is detected in blood and is believed to be specifically derived from CNS cholesterol metabolism. We assessed changes in syntheses of myelin components in brain and of blood sterols during cuprizone-induced experimental demyelination and remyelination, with and without thyroid hormone (T3) treatment. Mice were fed cuprizone for 4 weeks, then returned to control diet and treated with either placebo or T3 (0.005 mg/day). 2H2O was administered for the last 14 days of cuprizone diet, and for either 6, 12 or 19 days of treatment during recovery from cuprizone, after which blood and corpus callosum (CC) samples were collected (n = 5/time point/treatment). 2H incorporation into cholesterol and 24-OHC in blood and CC, and incorporation into phospholipid (PL)-palmitate, GalC, myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) in CC were measured. Cuprizone significantly (p < 0.05) decreased syntheses of cholesterol, 24-OHC, GalC, MBP, CNPase and PL-palmitate in the CC and these effects were all reversed during recovery. T3 treatment significantly (p < 0.05) increased syntheses of cholesterol, 24-OHC and palmitate compared to placebo. 24-OHC and cholesterol turnover rates in brain and blood were nearly identical and 24-OHC rates in blood paralleled rates in CC, indicating that blood 24-OHC derives primarily from the brain and reflects oligodendrocyte function. In summary, changes in synthesis of several lipid and protein components in brain during cuprizone-induced demyelination and remyelination are measurable through stable isotope labeling. Blood 24-OHC turnover rates closely reflect flux rates of brain cholesterol in response to cuprizone and T3, which alter oligodendrocyte function. Labeling of blood 24-OHC has potential as a non-invasive marker of brain de novo cholesterol synthesis and breakdown rates in demyelinating conditions.

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions.

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

Intrahepatic quantification of HBV antigens in chronic hepatitis B reveals heterogeneity and treatment-mediated reductions in HBV core-positive cells.

Background & Aims: Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Methods: Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n = 39) and HBeAg-negative (n = 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Results: Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Conclusions: Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). Impact and Implications: HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.

Discovering Synergistic Compounds with BYL-719 in PI3K Overactivated Basal-like PDXs.

Basal-like triple-negative breast cancer (TNBC) tumor cells are difficult to eliminate due to resistance mechanisms that promote survival. While this breast cancer subtype has low PIK3CA mutation rates when compared to estrogen receptor-positive (ER+) breast cancers, most basal-like TNBCs have an overactive PI3K pathway due to gene amplification or high gene expression. BYL-719 is a PIK3CA inhibitor that has been found to have low drug-drug interactions, which increases the likelihood that it could be useful for combinatorial therapy. Alpelisib (BYL-719) with fulvestrant was recently approved for treating ER+ breast cancer patients whose cancer had developed resistance to ER-targeting therapy. In these studies, a set of basal-like patient-derived xenograft (PDX) models was transcriptionally defined with bulk and single-cell RNA-sequencing and clinically actionable mutation profiles defined with Oncomine mutational profiling. This information was overlaid onto therapeutic drug screening results. BYL-719-based, synergistic two-drug combinations were identified with 20 different compounds, including everolimus, afatinib, and dronedarone, which were also found to be effective at minimizing tumor growth. These data support the use of these drug combinations towards cancers with activating PIK3CA mutations/gene amplifications or PTEN deficient/PI3K overactive pathways.

Assessments of Somatic Variant Classification Using the Association for Molecular Pathology/American Society of Clinical Oncology/College of American Pathologists Guidelines: A Report from the Association for Molecular Pathology.

To assess the clinical implementation of the 2017 Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists, identify content that may result in classification inconsistencies, and evaluate implementation barriers, an Association for Molecular Pathology Working Group conducted variant interpretation challenges and a guideline implementation survey. A total of 134 participants participated in the variant interpretation challenges, consisting of 11 variants in four cancer cases. Results demonstrate 86% (range, 54% to 94%) of the respondents correctly classified clinically significant variants, variants of uncertain significance, and benign/likely benign variants; however, only 59% (range, 39% to 84%) of responses agreed with the working group's consensus intended responses regarding both tiers and categories of clinical significance. In the implementation survey, 71% (157/220) of respondents have implemented the 2017 guidelines for variant classification and reporting either with or without modifications. Collectively, this study demonstrates that, although they may not yet be optimized, the 2017 guideline recommendations are being adopted for standardized somatic variant classification. The working group identified significant areas for future guideline improvement, including the need for a more granular and comprehensive classification system and education resources to meet the growing needs of both laboratory professionals and medical oncologists.

The basics of commonly used molecular techniques for diagnosis, and application of molecular testing in cytology.

Molecular diagnostics has expanded to become the standard of care for a variety of solid tumor types. With limited diagnostic material, it is often desirable to use cytological preparations to provide rapid and accurate molecular results. This review covers important pre-analytic considerations and limitations, and a description of common techniques that the modern cytopathologist should understand when ordering and interpreting molecular tests in practice.

Acute megakaryoblastic leukemia with trisomy 3 and CBFA2T3::GLIS2: A case report.

Non-Down-syndrome-related acute megakaryoblastic leukemia (non-DS-AMKL) is a rare form of leukemia that can present with a variety of initial symptoms, including fever, rash, bruising, bleeding, or other more clinically challenging symptoms. Herein, we describe a 19-month-old female patient who presented with left lower extremity pain and language regression who was diagnosed with AMKL, not otherwise specified (NOS), on the basis of peripheral blood and bone marrow analysis, as well as cytogenetic and molecular diagnostic phenotyping. Of note, in addition to this patient's karyotype showing trisomy 3, a fusion between CBFA2T3 (core-binding factor, alpha subunit 2, translocated to, 3) on chromosome 16 and GLIS2 (GLIS family zinc finger protein 2), also on chromosome 16, was observed. Patients with AMKL who have trisomy 3 with CBFA2T3::GLIS2 fusions are rare, and it is not known if the co-occurrence of these abnormalities is coincidental or biologically related. This highlights the continued need for further expansion of genetic testing in individuals with rare disease to establish the groundwork for identifying additional commonalities that could potentially be used to identify therapeutic targets or improve prognostication.

Severe Graft-Versus-Host Disease Following Solid-Organ Transplant Confirmed by Chimerism Studies and Cytogenetic Analyses.

Graft-versus-host disease is an uncommon complication of solid-organ transplant and is associated with a high rate of mortality. Here, we describe a female patient with primary biliary cholangitis who developed graft-versus-host disease following an orthotopic liver and renal allotransplant from a deceased male donor. Systemic donor lymphoid chimerism is one of several important findings to confirm a diagnosis of graft-versus-host disease after solid-organ transplant, along with clinical and histologic findings. In this case, cytogenetic analyses and chimerism studies performed on blood, blood components, and bone marrow specimens obtained at several timepoints supported the diagnosis of graft-versus-host disease and demonstrated sustained near-complete donor engraftment of the lymphoid compartment. This case report illustrates the utility of chimerism testing to rapidly diagnose this serious condition in patients who have received a solid-organ transplant.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.

Background & Aims: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms. Methods: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline. Results: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations. Conclusions: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC. Lay summary: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection.

BACKGROUND & AIMS: Chronic HBV is clinically categorized into 4 phases by a combination of serum HBV DNA levels, HBeAg status and alanine aminotransferase (ALT): immunotolerant (IT), immune-active (IA), inactive carrier (IC) and HBeAg-negative hepatitis (ENEG). Immune and virological measurements in the blood have proven useful but are insufficient to explain the interrelation between the immune system and the virus since immune dynamics differ in the blood and liver. Furthermore, the inflammatory response in the liver and parenchymal cells cannot be fully captured in blood. METHODS: Immunological composition and transcriptional profiles of core needle liver-biopsies in chronic HBV phases were compared to those of healthy controls by multiplex immunofluorescence and RNA-sequencing (n = 37 and 78, respectively) analyses. RESULTS: Irrespective of the phase-specific serological profiles, increased immune-gene expression and frequency was observed in chronic HBV compared to healthy livers. Greater transcriptomic deregulation was seen in IA and ENEG (172 vs. 243 DEGs) than in IT and IC (13 vs. 35 DEGs) livers. Interferon-stimulated genes, immune-activation and exhaustion genes (ICOS, CTLA4, PDCD1) together with chemokine genes (CXCL10, CXCL9) were significantly induced in IA and ENEG livers. Moreover, distinct immune profiles associated with ALT elevation and a more accentuated immune-exhaustion profile (CTLA4, TOX, SLAMF6, FOXP3) were observed in ENEG, which set it apart from the IA phase (LGALS9, PDCD1). Interestingly, all HBV phases showed downregulation of metabolic pathways vs. healthy livers (fatty and bile acid metabolism). Finally, increased leukocyte infiltrate correlated with serum ALT, but not with HBV DNA or viral proteins. CONCLUSION: Our comprehensive multi-parametric analysis of human livers revealed distinct inflammatory profiles and pronounced differences in intrahepatic gene profiles across all chronic HBV phases in comparison to healthy liver. LAY SUMMARY: Immunological studies on chronic HBV remain largely restricted to assessment of peripheral responses due to the limited access to the site of infection, the liver. In this study, we comprehensively analyzed livers from a well-defined cohort of patients with chronic HBV and uninfected controls with state-of-the-art techniques, and evaluated the differences in gene expression profiles and inflammation characteristics across distinct disease phases in patients with chronic HBV.

Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection.

BACKGROUND & AIMS: We aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel. METHOD: Matching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment. RESULTS: Using unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments. CONCLUSION: These data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure. LAY SUMMARY: Liver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure.

Randomized, open-label, phase 2 study of andecaliximab plus nivolumab versus nivolumab alone in advanced gastric cancer identifies biomarkers associated with survival.

BACKGROUND: Matrix metalloproteinase-9 (MMP9) selectively cleaves extracellular matrix proteins contributing to tumor growth and an immunosuppressive microenvironment. This study evaluated andecaliximab (ADX), an inhibitor of MMP9, in combination with nivolumab (NIVO), for the treatment of advanced gastric cancer. METHODS: Phase 2, open-label, randomized multicenter study evaluating the efficacy, safety, and pharmacodynamics of ADX+NIVO versus NIVO in patients with pretreated metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and adverse events (AEs). We explored the correlation of efficacy outcomes with biomarkers. RESULTS: 144 patients were randomized; 141 were treated: 81% white, 69% male, median age was 61 years in the ADX+NIVO group and 62 years in the NIVO-alone group. The ORR was 10% (95% CI 4 to 19) in the ADX+NIVO group and 7% (95% CI 2 to 16) in the NIVO-alone group (OR: 1.5 (95% CI 0.4 to 6.1; p=0.8)). There was no response or survival benefit associated with adding ADX. AE rates were comparable in both treatment groups; the most common AEs were fatigue, decreased appetite, nausea, and vomiting. Programmed cell death ligand 1, interferon-γ (IFN), and intratumoral CD8+ cell density were not associated with treatment response or survival. The gene signature most correlated with shorter survival was the epithelial-to-mesenchymal gene signature; high transforming growth factor (TGF)-ß fibrosis score was negatively associated with OS (p=0.036). Gene expression analysis of baseline tumors comparing long-(1+ years) and short-term (<1 year) survivors showed that GRB7 was associated with survival beyond 1 year. Human epidermal growth factor receptor 2 (HER2)-positive disease was associated with significantly longer survival (p=0.0077). Median tumor mutation burden (TMB) was 2.01; patients with TMB ≥median had longer survival (p=0.0025) and improved PFS (p=0.016). Based on a model accounting for TMB, TGF-ß fibrosis, and HER2, TMB was the main driver of survival in this patient population. CONCLUSION: Combination of ADX+NIVO had a favorable safety profile but did not improve efficacy compared with NIVO alone in patients with pretreated metastatic gastric or GEJ adenocarcinoma. HER2 positivity, higher TMB or GRB7, and lower TGF-ß were associated with improved outcomes. TRIAL REGISTRATION NUMBER: NCT02864381 or GS-US-296--2013.

Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

Exceptionally high temperature spin crossover in amide-functionalised 2,6-bis(pyrazol-1-yl)pyridine iron(ii) complex revealed by variable temperature Raman spectroscopy and single crystal X-ray diffraction.

The synthesis of a novel amide-functionalised 2,6-bis(pyrazol-1-yl)pyridine-4-carboxamide ligand (bppCONH2) is described. The complex salts [Fe(bppCONH2)2](BF4)2 and [Fe(bppCONH2)2](ClO4)2 were synthesised and characterised by SQUID magnetometry, differential scanning calorimetry, variable temperature Raman spectroscopy and single crystal X-ray diffraction. DSC measurements of [Fe(bppCONH2)2](BF4)2 indicate a spin-crossover (SCO) transition with T↑ at 481 K and T↓ at 461 K, showing a 20 K hysteresis. DSC for the perchlorate salt shows an SCO transition with T↑ at 459 K and T↓ at 445 K with a 14 K hysteresis. For the BF4- salt analysis of low and high-spin state crystal structures at 101, 290 and 500 K, suggest stabilisation of the low spin state due to the formation of 1D hydrogen-bonded cationic chains. Variable temperature Raman studies of the BF4 salt support the presence of a high temperature SCO. It is speculated that the presence of hysteresis may be attributed to differences in the inter-molecular hydrogen bonding in the low spin and high spin states.

Factors Associated With, and Mitigation Strategies for, Health Care Disparities Faced by Patients With Headache Disorders.

OBJECTIVE: To review contemporary issues of health care disparities in headache medicine with regard to race/ethnicity, socioeconomic status (SES), and geography and propose solutions for addressing these disparities. METHODS: An Internet and PubMed search was performed and literature was reviewed for key concepts underpinning disparities in headache medicine. Content was refined to areas most salient to our goal of informing the provision of equitable care in headache treatment through discussions with a group of 16 experts from a range of headache subspecialties. RESULTS: Taken together, a multitude of factors, including racism, SES, insurance status, and geographical disparities, contribute to the inequities that exist within the health care system when treating headache disorders. Interventions such as improving public education, advocacy, optimizing telemedicine, engaging in community outreach to educate primary care providers, training providers in cultural sensitivity and competence and implicit bias, addressing health literacy, and developing recruitment strategies to increase representation of underserved groups within headache research are proposed as solutions to ameliorate disparities. CONCLUSION: Neurologists have a responsibility to provide and deliver equitable care to all. It is important that disparities in the management of headache disorders are identified and addressed.

De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle.

The biological basis of Duchenne muscular dystrophy (DMD) pathology is only partially characterized and there are still few disease-modifying therapies available, therein underlying the value of strategies to model and study DMD. Dystrophin, the causative gene of DMD, is responsible for linking the cytoskeleton of muscle fibers to the extracellular matrix beyond the sarcolemma. We posited that disease-associated phenotypes not yet captured by two-dimensional culture methods would arise by generating multinucleated muscle cells within a three-dimensional (3D) extracellular matrix environment. Herein we report methods to produce 3D human skeletal muscle microtissues (hMMTs) using clonal, immortalized myoblast lines established from healthy and DMD donors. We also established protocols to evaluate immortalized hMMT self-organization and myotube maturation, as well as calcium handling, force generation, membrane stability (i.e., creatine kinase activity and Evans blue dye permeability) and contractile apparatus organization following electrical-stimulation. In examining hMMTs generated with a cell line wherein the dystrophin gene possessed a duplication of exon 2, we observed rare dystrophin-positive myotubes, which were not seen in 2D cultures. Further, we show that treating DMD hMMTs with a ß1-integrin activating antibody, improves contractile apparatus maturation and stability. Hence, immortalized myoblast-derived DMD hMMTs offer a pre-clinical system with which to investigate the potential of duplicated exon skipping strategies and those that protect muscle cells from contraction-induced injury. STATEMENT OF SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder that is caused by mutation of the dystrophin gene. The biological basis of DMD pathology is only partially characterized and there is no cure for this fatal disease. Here we report a method to produce 3D human skeletal muscle microtissues (hMMTs) using immortalized human DMD and healthy myoblasts. Morphological and functional assessment revealed DMD-associated pathophysiology including impaired calcium handling and de novo formation of dystrophin-positive revertant muscle cells in immortalized DMD hMMTs harbouring an exon 2 duplication, a feature of many DMD patients that has not been recapitulated in culture prior to this report. We further demonstrate that this "DMD in a dish" system can be used as a pre-clinical assay to test a putative DMD therapeutic and study the mechanism of action.