Allele-specific expression reveals genetic drivers of tissue regeneration in mice.

In adult mammals, skin wounds typically heal by scarring rather than through regeneration. In contrast, "super-healer" Murphy Roths Large (MRL) mice have the unusual ability to regenerate ear punch wounds; however, the molecular basis for this regeneration remains elusive. Here, in hybrid crosses between MRL and non-regenerating mice, we used allele-specific gene expression to identify cis-regulatory variation associated with ear regeneration. Analyzing three major cell populations (immune, fibroblast, and endothelial), we found that genes with cis-regulatory differences specifically in fibroblasts were associated with wound-healing pathways and also co-localized with quantitative trait loci for ear wound-healing. Ectopic treatment with one of these proteins, complement factor H (CFH), accelerated wound repair and induced regeneration in typically fibrotic wounds. Through single-cell RNA sequencing (RNA-seq), we observed that CFH treatment dramatically reduced immune cell recruitment to wounds, suggesting a potential mechanism for CFH's effect. Overall, our results provide insights into the molecular drivers of regeneration with potential clinical implications.

Color Data v2: a user-friendly, open-access database with hereditary cancer and hereditary cardiovascular conditions datasets.

Publicly available genetic databases promote data sharing and fuel scientific discoveries for the prevention, treatment and management of disease. In 2018, we built Color Data, a user-friendly, open access database containing genotypic and self-reported phenotypic information from 50 000 individuals who were sequenced for 30 genes associated with hereditary cancer. In a continued effort to promote access to these types of data, we launched Color Data v2, an updated version of the Color Data database. This new release includes additional clinical genetic testing results from more than 18 000 individuals who were sequenced for 30 genes associated with hereditary cardiovascular conditions as well as polygenic risk scores for breast cancer, coronary artery disease and atrial fibrillation. In addition, we used self-reported phenotypic information to implement the following four clinical risk models: Gail Model for 5-year risk of breast cancer, Claus Model for lifetime risk of breast cancer, simple office-based Framingham Coronary Heart Disease Risk Score for 10-year risk of coronary heart disease and CHARGE-AF simple score for 5-year risk of atrial fibrillation. These new features and capabilities are highlighted through two sample queries in the database. We hope that the broad dissemination of these data will help researchers continue to explore genotype-phenotype correlations and identify novel variants for functional analysis, enabling scientific discoveries in the field of population genomics. Database URL: https://data.color.com/.

Targeted lymphodepletion with a CD45-directed antibody radioconjugate as a novel conditioning regimen prior to adoptive cell therapy.

Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion in vivo following infusion, and represents potential point of intervention to decrease serious toxicities following CAR-T treatment. In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.

Magnetic resonance elastography vs. point shear wave ultrasound elastography for the assessment of renal allograft dysfunction.

PURPOSE: To investigate the utility of magnetic resonance elastography (MRE) vs. ultrasound (US) point shear wave elastography (pSWE) for the assessment of chronic renal allograft dysfunction, prediction of outcome and determine the correlation with Banff pathology scores. METHODS: In this IRB approved prospective study, 27 enrolled patients with functional (n = 15) and chronic dysfunctional (n = 12) renal allografts underwent same day 2D MRE and pSWE. Histogram parameters [including mean, median, standard deviation, kurtosis and skewness] of the magnitude of the complex shear modulus (MRE) and median Young's modulus (pSWE) were measured in the cortex (MRE and pSWE) and combined corticomedullary regions (MRE). Histopathology was available for 16 patients (4 functional, 12 dysfunctional). RESULTS: MRE and pSWE stiffness were not significantly different between functional and dysfunctional groups (p range 0.139-0.347). The skewness of MRE corticomedullary stiffness was significantly lower (p = 0.04) in patients with chronic dysfunction and correlated significantly with Banff histopathologic scores (range r=-0.518-0.567, p = 0.035-0.040). MRE cortical and corticomedullary mean stiffness showed strong performance in predicting graft loss/relist (AUC 0.958, p = 0.011 for both). Reliable pSWE measurements were obtained in 13 patients (48 %). pSWE stiffness did not correlate with Banff scores and did not predict outcome. CONCLUSIONS: The skewness of MRE corticomedullary stiffness is sensitive to changes in chronic allograft dysfunction, while mean/median MRE renal stiffness and median US stiffness did not differentiate patients with stable function vs those with chronic renal allograft dysfunction. MRE corticomedullary mean stiffness appears to be a predictor of graft loss/relist. pSWE was not found to be a useful method for assessing renal allografts.

The Pre-Registry Commercial Driver Medical Examination: Screening Sensitivity and Certification Lengths for Two Safety-Related Medical Conditions.

OBJECTIVE: Use independent diagnostic data to analyze the screening effectiveness of the pre-Registry commercial driver medical examination (CDME) for obstructive sleep apnea (OSA), and its sensitivity for hypertension; analyze certification lengths where relevant. METHODS: CDME screening results for 1668 drivers were compared to polysomnogram diagnostic test results, and CDME screening results were evaluated for 1155 drivers with at least one insurance claim with a hypertension diagnostic code. Any CDME documentation of the medical condition was considered as detection by screening. RESULTS: CDME sensitivity was 20.7% for moderate OSA (AHI ≥ 15). While sensitivity was 77.5% for hypertension, 93.3% of drivers with Stage 3 hypertension were certified, contrary to Federal Motor Carrier Safety Administration standards. CONCLUSIONS: The pre-Registry CDME was ineffective in screening commercial drivers for OSA. Screening was better for hypertension; incorrect certifications were given to many hypertensive drivers.

Single-cell transcriptional diversity is a hallmark of developmental potential.

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation is challenging. Here, we demonstrate a simple, yet robust, determinant of developmental potential-the number of expressed genes per cell-and leverage this measure of transcriptional diversity to develop a computational framework (CytoTRACE) for predicting differentiation states from scRNA-seq data. When applied to diverse tissue types and organisms, CytoTRACE outperformed previous methods and nearly 19,000 annotated gene sets for resolving 52 experimentally determined developmental trajectories. Additionally, it facilitated the identification of quiescent stem cells and revealed genes that contribute to breast tumorigenesis. This study thus establishes a key RNA-based feature of developmental potential and a platform for delineation of cellular hierarchies.

Employer-mandated obstructive sleep apnea treatment and healthcare cost savings among truckers.

OBJECTIVE: To evaluate the effect of an employer-mandated obstructive sleep apnea (OSA) diagnosis and treatment program on non-OSA-program trucker medical insurance claim costs. METHODS: Retrospective cohort analysis; cohorts constructed by matching (randomly, with replacement) Screen-positive Controls (drivers with insurance screened as likely to have OSA, but not yet diagnosed) with Diagnosed drivers (n = 1,516; cases = 1,224, OSA Negatives = 292), on two factors affecting exposure to medical claims: experience level at hire and weeks of job tenure at the Diagnosed driver's polysomnogram (PSG) date (the "matching date"). All cases received auto-adjusting positive airway pressure (APAP) treatment and were grouped by objective treatment adherence data: any "Positive Adherence" (n = 932) versus "No Adherence" (n = 292). Bootstrap resampling produced a difference-in-differences estimate of aggregate non-OSA-program medical insurance claim cost savings for 100 Diagnosed drivers as compared to 100 Screen-positive Controls before and after the PSG/matching date, over an 18-month period. A two-part multivariate statistical model was used to set exposures and demographics/anthropometrics equal across sub-groups, and to generate a difference-in-differences comparison across periods that identified the effect of OSA treatment on per-member per-month (PMPM) costs of an individual driver, separately from cost differences associated with adherence choice. RESULTS: Eighteen-month non-OSA-program medical claim costs savings from diagnosing (and treating as required) 100 Screen-positive Controls: $153,042 (95% CI: -$5,352, $330,525). Model-estimated effect of treatment on those adhering to APAP: -$441 PMPM (95% CI: -$861, -$21). CONCLUSIONS: Results suggest a carrier-based mandatory OSA program generates substantial savings in non-OSA-program medical insurance claim costs.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors.

Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Multiparametric Magnetic Resonance Imaging Features Identify Aggressive Prostate Cancer at the Phenotypic and Transcriptomic Level.

PURPOSE: Multiparametric magnetic resonance imaging is a diagnostic tool for prostate cancer with limited data on prognostic use. We sought to determine whether multiparametric magnetic resonance could predict aggressive prostate cancer features. MATERIALS AND METHODS: We retrospectively analyzed the records of 206 patients who underwent radical prostatectomy between 2013 and 2017. All patients had available RNA expression data on the final pathology specimen obtained from a location corresponding to a lesion location on multiparametric magnetic resonance imaging. The association between the PIRADS™ (Prostate Imaging Reporting and Data System) score and adverse pathology features were analyzed. We also performed differential transcriptomic analysis between the PIRADS groups. Factors associated with adverse pathology were analyzed using a multivariable logistic regression model. RESULTS: Lesion size (p = 0.03), PIRADS score (p = 0.02) and extraprostatic extension (p = 0.01) associated significantly with the Decipher® score. Multivariable analysis showed that the PIRADS score (referent PIRADS 3, OR 8.1, 95% CI 1.2-57.5, p = 0.04), the Gleason Grade Group (referent 3, OR 5.6, 95% CI 1.5-21.1, p = 0.01) and prostate specific antigen (OR 1.103, 95% CI 1.011-1.203) were risk factors for adverse pathology findings. The difference between PIRADS 4 and 5 did not reach significance (OR 1.9, 95% CI 0.8-4.5, p = 0.12). However, the PI3K-AKT-mTOR, WNT-ß and E2F signaling pathways were more active in PIRADS 5 than in PIRADS 4 cases. CONCLUSIONS: The PIRADS score is associated with adverse pathology results, increased metastatic risk and differential genomic pathway activation.

Independent erosion of conserved transcription factor binding sites points to shared hindlimb, vision and external testes loss in different mammals.

Genetic variation in cis-regulatory elements is thought to be a major driving force in morphological and physiological changes. However, identifying transcription factor binding events that code for complex traits remains a challenge, motivating novel means of detecting putatively important binding events. Using a curated set of 1154 high-quality transcription factor motifs, we demonstrate that independently eroded binding sites are enriched for independently lost traits in three distinct pairs of placental mammals. We show that these independently eroded events pinpoint the loss of hindlimbs in dolphin and manatee, degradation of vision in naked mole-rat and star-nosed mole, and the loss of external testes in white rhinoceros and Weddell seal. We additionally show that our method may also be utilized with more than two species. Our study exhibits a novel methodology to detect cis-regulatory mutations which help explain a portion of the molecular mechanism underlying complex trait formation and loss.

Cutting Edge: Selective Oral ROCK2 Inhibitor Reduces Clinical Scores in Patients with Psoriasis Vulgaris and Normalizes Skin Pathology via Concurrent Regulation of IL-17 and IL-10.

Targeted inhibition of Rho-associated kinase (ROCK)2 downregulates the proinflammatory T cell response while increasing the regulatory arm of the immune response in animals models of autoimmunity and Th17-skewing human cell culture in vitro. In this study, we report that oral administration of a selective ROCK2 inhibitor, KD025, reduces psoriasis area and severity index scores by 50% from baseline in 46% of patients with psoriasis vulgaris, and it decreases epidermal thickness as well as T cell infiltration in the skin. We observed significant reductions of IL-17 and IL-23, but not IL-6 and TNF-α, whereas IL-10 levels were increased in peripheral blood of clinical responders after 12 wk of treatment with KD025. Collectively, these data demonstrate that an orally available selective ROCK2 inhibitor downregulates the Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via a defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.

M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity.

Variant pathogenicity classifiers such as SIFT, PolyPhen-2, CADD, and MetaLR assist in interpretation of the hundreds of rare, missense variants in the typical patient genome by deprioritizing some variants as likely benign. These widely used methods misclassify 26 to 38% of known pathogenic mutations, which could lead to missed diagnoses if the classifiers are trusted as definitive in a clinical setting. We developed M-CAP, a clinical pathogenicity classifier that outperforms existing methods at all thresholds and correctly dismisses 60% of rare, missense variants of uncertain significance in a typical genome at 95% sensitivity.

Nonadherence with Employer-Mandated Sleep Apnea Treatment and Increased Risk of Serious Truck Crashes.

STUDY OBJECTIVES: To evaluate the effect of an employer-mandated obstructive sleep apnea (OSA) program on the risk of serious preventable truck crashes. METHODS: Data are from the first large-scale, employer-mandated program to screen, diagnose, and monitor OSA treatment adherence in the US trucking industry. A retrospective analysis of cohorts was constructed: polysomnogram-diagnosed drivers (OSA positive n = 1,613, OSA negative n = 403) were matched to control drivers unlikely to have OSA (n = 2,016) on two factors affecting crash risk, experience-at-hire and length of job tenure; tenure was matched on the date of each diagnosed driver's polysomnogram. Auto-adjusting positive airway pressure (APAP) treatment was provided to all cases (i.e. OSA positive drivers); treatment adherence was objectively monitored. Cases were grouped by treatment adherence: "Full Adherence" (n = 682), "Partial Adherence" (n = 571), or "No Adherence" (n = 360). Preventable Department-of-Transportation-reportable crashes/100,000 miles were compared across study subgroups. Robustness was assessed. RESULTS: After the matching date, "No Adherence" cases had a preventable Department of Transportation-reportable crash rate that was fivefold greater (incidence rate ratio = 4.97, 95% confidence interval: 2.09, 10.63) than that of matched controls (0.070 versus 0.014 per 100,000 miles). The crash rate of "Full Adherence" cases was statistically similar to controls (incidence rate ratio = 1.02, 95% confidence interval: 0.48, 2.04; 0.014 per 100,000 miles). CONCLUSIONS: Nontreatment-adherent OSA-positive drivers had a fivefold greater risk of serious preventable crashes, but were discharged or quit rapidly, being retained only one-third as long as other subjects. Thus, the mandated program removed risky nontreatment-adherent drivers and retained adherent drivers at the study firm. Current regulations allow nonadherent OSA cases to drive at another firm by keeping their diagnosis private. COMMENTARY: A commentary on this article appears in this issue on page 961.

A phase II, multicenter, open-label study of obatoclax mesylate in patients with previously untreated myelodysplastic syndromes with anemia or thrombocytopenia.

BACKGROUND: Obatoclax mesylate is a small-molecule Bcl-2 homology domain-3 mimetic that neutralizes antiapoptotic Bcl-2-related proteins. We evaluated obatoclax in untreated MDS patients with anemia/thrombocytopenia. PATIENTS AND METHODS: Twenty-four patients with a bone marrow blast count of ≤ 10% and anemia (hemoglobin level < 10 g/dL) or thrombocytopenia (platelet count < 50 × 10(9)/L) were eligible to receive intravenous obatoclax 60 mg over 24 hours every 2 weeks. RESULTS: Response rate was 8% (2 patients; hematologic improvement). Disease stabilization/response was maintained ≥ 12 weeks in 50% (12 patients). Because the response rate was below a predetermined threshold, the study was terminated. Adverse events (any grade) included euphoric mood (63%; 15 patients), nausea (38%; 9 patients), and diarrhea (25%; 6 patients); Grade 3/4 adverse events included anemia (21%; 5 patients), thrombocytopenia (13%; 3 patients), and pneumonia (13%; 3 patients). CONCLUSIONS: Obatoclax 60 mg every 2 weeks was feasible, but had limited first-line activity in MDS.

Randomized phase II study of carboplatin and etoposide with or without obatoclax mesylate in extensive-stage small cell lung cancer.

OBJECTIVE: This randomized phase II study assessed the efficacy and safety of obatoclax mesylate, a small-molecule Bcl-2 inhibitor, added to carboplatin/etoposide chemotherapy as initial treatment for extensive-stage small-cell lung cancer (ES-SCLC). MATERIALS AND METHODS: Chemotherapy-naïve subjects with ES-SCLC and Eastern Cooperative Oncology Group performance status (ECOG PS) 0-2 received carboplatin/etoposide with (CbEOb) or without (CbE) obatoclax for up to six cycles. Responders to CbEOb could receive maintenance obatoclax until disease progression. The primary endpoint was objective response rate (ORR). RESULTS: 155 subjects (median age 62, 58% male, 10% ECOG PS 2) were treated with CbEOb (n=77) or CbE (n=78); 65% and 59% of subjects, respectively, completed six cycles. ORR was 62% with CbEOb versus 53% with CbE (1-sided p=0.143). Clinical benefit (ORR+ stable disease) trended better with CbEOb (81% versus 68%; p=0.054). Median progression-free survival (PFS) and overall survival (OS) were 5.8 months (95% confidence interval [CI]: 5.3-6.5) and 10.5 months (8.9-13.8) with CbEOb and 5.2 months (95% CI: 4.1-5.7) and 9.8 months (7.2-11.2) with CbE. Median OS was 10.5 months (95% CI: 8.9-13.8) and 9.8 months (7.2-11.2) with a nonsignificant hazard ratio for OS, 0.823; 1-sided p=0.121. Grade 3/4 adverse events (AEs) were primarily hematologic and similar in frequency between treatment arms. Obatoclax-related somnolence and euphoria were grade 1/2, transient, and did not require treatment discontinuation. CONCLUSION: Obatoclax was well tolerated when added to carboplatin/etoposide in first-line treatment of ES-SCLC, but failed to significantly improve ORR, PFS, or OS.

A phase I/II study of the pan Bcl-2 inhibitor obatoclax mesylate plus bortezomib for relapsed or refractory mantle cell lymphoma.

Obatoclax, a BH3 mimetic inhibitor of anti-apoptotic Bcl-2 proteins, demonstrates synergy with bortezomib in preclinical models of mantle cell lymphoma (MCL). This phase I/II study assessed obatoclax plus bortezomib in patients with relapsed/refractory MCL. Twenty-three patients received obatoclax 30 or 45 mg plus bortezomib 1.0 or 1.3 mg/m(2), administered intravenously on days 1, 4, 8 and 11 of a 21-day cycle. In phase I, the combination was feasible at all doses. Obatoclax 45 mg plus bortezomib 1.3 mg/m(2) was selected for phase II study. Common adverse events were somnolence (87%), fatigue (61%) and euphoric mood (57%), all primarily grade 1/2. Grade 3/4 events included thrombocytopenia (21%), anemia (13%) and fatigue (13%). Objective responses occurred in 4/13 (31%) evaluable patients (three complete and one partial response). Six patients (46%) had stable disease lasting ≥ 8 weeks. Obatoclax plus bortezomib was feasible, but the synergy demonstrated in preclinical models was not confirmed.

Obatoclax mesylate, a pan-bcl-2 inhibitor, in combination with docetaxel in a phase 1/2 trial in relapsed non-small-cell lung cancer.

INTRODUCTION: The proapoptotic small-molecule pan-Bcl-2 inhibitor obatoclax mesylate (GX15-070) may enhance the cytotoxicity of chemotherapy in relapsed/refractory non-small-cell lung cancer (NSCLC). METHODS: Obatoclax was administered with docetaxel in patients with relapsed or refractory NSCLC- docetaxel as a 1-hour infusion on day 1 and obatoclax as a 24-hour infusion on days 1 and 2-every 3 weeks for up to eight cycles. Four dose levels were evaluated in phase 1 (level 1: docetaxel 55 mg/m × 1 and obatoclax 30 mg × 2; levels 2-4: docetaxel 75 mg/m and obatoclax 30 mg, 45 mg, or 60 mg × 2) to identify dose-limiting toxicity and a phase 2 dose. In phase 2, response and tolerability were evaluated. RESULTS: Eighteen patients were included in phase 1. Two dose-limiting toxicities occurred during cycle 1: one febrile neutropenia each at dose levels 3 and 4. Maximum tolerated dose was not reached; 32 patients (including 3 from phase 1) were treated in phase 2 with docetaxel 75 mg/m and obatoclax 60 mg (median 2 cycles). Three patients (11%) had partial responses in phase 2; two demonstrated stable disease lasting 12 weeks or more. Median duration of response was 4.8 months. Overall, median progression-free survival was 1.4 months. Neutropenia (31%), febrile neutropenia (16%), and dyspnea (19%) were the most common grade 3/4 adverse events observed. CONCLUSIONS: Combined obatoclax mesylate plus docetaxel is tolerable in patients with NSCLC, but response was minimal and neutropenia was a common adverse event.