Citations

Abstract Background Chromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis. Methods We hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes. Results After translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage. Conclusion We demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.

ImportanceAdrenalectomy is the definitive treatment for multiple adrenal abnormalities. Advances in technology and genomics and an improved understanding of adrenal pathophysiology have altered operative techniques and indications.ObjectiveTo develop evidence-based recommendations to enhance the appropriate, safe, and effective approaches to adrenalectomy.Evidence ReviewA multidisciplinary panel identified and investigated 7 categories of relevant clinical concern to practicing surgeons. Questions were structured in the framework Population, Intervention/Exposure, Comparison, and Outcome, and a guided review of medical literature from PubMed and/or Embase from 1980 to 2021 was performed. Recommendations were developed using Grading of Recommendations, Assessment, Development and Evaluation methodology and were discussed until consensus, and patient advocacy representation was included.FindingsPatients with an adrenal incidentaloma 1 cm or larger should undergo biochemical testing and further imaging characterization. Adrenal protocol computed tomography (CT) should be used to stratify malignancy risk and concern for pheochromocytoma. Routine scheduled follow-up of a nonfunctional adrenal nodule with benign imaging characteristics and unenhanced CT with Hounsfield units less than 10 is not suggested. When unilateral disease is present, laparoscopic adrenalectomy is recommended for patients with primary aldosteronism or autonomous cortisol secretion. Patients with clinical and radiographic findings consistent with adrenocortical carcinoma should be treated at high-volume multidisciplinary centers to optimize outcomes, including, when possible, a complete R0 resection without tumor disruption, which may require en bloc radical resection. Selective or nonselective α blockade can be used to safely prepare patients for surgical resection of paraganglioma/pheochromocytoma. Empirical perioperative glucocorticoid replacement therapy is indicated for patients with overt Cushing syndrome, but for patients with mild autonomous cortisol secretion, postoperative day 1 morning cortisol or cosyntropin stimulation testing can be used to determine the need for glucocorticoid replacement therapy. When patient and tumor variables are appropriate, we recommend minimally invasive adrenalectomy over open adrenalectomy because of improved perioperative morbidity. Minimally invasive adrenalectomy can be achieved either via a retroperitoneal or transperitoneal approach depending on surgeon expertise, as well as tumor and patient characteristics.Conclusions and RelevanceTwenty-six clinically relevant and evidence-based recommendations are provided to assist surgeons with perioperative adrenal care.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease globally in tandem with the growing obesity epidemic. However, there is a lack of data on the relationship between historical weight changes 10 years ago and at present on NAFLD prevalence at the population level. Therefore, we sought to evaluate the relationship between weight classes and the prevalence of NAFLD. Methods: Data were used from the United States National Health and Nutrition Examination Survey (NHANES) from 1999 to 2018. Univariate and multivariate general linear model analyses were used to obtain risk ratio (RR) estimations of NAFLD events. Results: In total, 34,486 individuals were analysed, with those who were lean at both time points as the control group. Overweight (RR: 14.73, 95%CI: 11.94 to 18.18, p < 0.01) or obese (RR: 31.51, 95%CI: 25.30 to 39.25, p < 0.01) individuals at both timepoints were more likely to develop NAFLD. Residual risk exists where previously obese individuals became overweight (RR: 14.72, 95%CI: 12.36 to 17.52, p < 0.01) or lean (RR: 2.46, 95%CI: 1.40 to 4.31, p = 0.02), and previously overweight individuals who became lean (RR 2.24, 95%CI 1.42 to 3.54, p = 0.01) had persistent elevated risk of developing NAFLD despite weight regression. Sensitivity analysis identified that a higher proportion of individuals with regression in weight class were diabetics and Mexican Americans, while fewer African Americans saw weight-class regression. Conclusions: Residual risk exists in patients who lost weight despite the smaller magnitude of effect, and targeted weight reductions should still be used to mitigate the risk of NAFLD at the population level.

Abstract BTK plays an important role in B cell receptor and Toll-like receptor signaling pathways, which are constitutively active in primary CNS lymphomas, and hence represents an excellent therapeutic target. Ibrutinib, a first-generation BTKi, was evaluated in phase 1/2 trials for R/R PCNSL, SCNSL, and PVRL, showing limited survival benefit. GB5121 is a novel, orally available, covalent BTKi with superior specificity, CNS penetration, and CNS target occupancy in preclinical testing versus other BTKis including ibrutinib. GB5121 is well-suited for evaluation in CNS lymphoma. This is a phase 1b/2 open-label study of GB5121 in adults with R/R PCNSL, isolated SCNSL or PVRL and will be conducted in three parts: phase 1b dose-escalation, expansion, and phase 2. Eligibility criteria for phase 1b dose-escalation and expansion (N≈30 for each) include age ≥18 years, ECOG≤2, R/R PCNSL, R/R SCNSL with CNS-only relapse, or R/R PVRL. Patients with newly diagnosed PCNSL who cannot tolerate standard high-dose methotrexate-based therapies are also eligible. Patients with prior allogeneic stem cell transplant are excluded. A Bayesian optimal interval design will be employed to perform dose escalation to determine the recommended phase 2 dose (RP2D). In the absence of dose-limiting toxicity (DLT), dose levels will increase sequentially according to a modified Fibonacci approach. Safety, tolerability, PK/PD, DLT, maximum tolerated dose, and preliminary therapeutic activity will be assessed to determine the optimal biological dose informing the RP2D. Phase 1b expansion will further explore therapeutic activity and characterize safety and tolerability of GB5121 at the RP2D. Phase 2 will initiate following RP2D determination. This is a single-arm, open-label study to investigate GB5121 safety and efficacy in patients with R/R PCNSL. Adverse events will be graded per CTCAE v5.0. Clinical response will be determined using International Primary CNS Lymphoma Collaborative Group criteria. Progression-free and overall survival will be evaluated. Enrollment begins May 2022 (NCT05242146).

AbstractNitric oxide (NO) is thought to increase cardiac contractility by increasing cytosolic Ca2+ concentration ([Ca2+]cyt) during excitation. Alternatively, NO could increase the sensitivity of the contractile response to [Ca2+]cyt (Ca2+ sensitivity). Arginase regulates NO production by competing with NO synthase (NOS), and thus, arginase inhibition should increase cardiac contractility by increasing NO production. We hypothesized that arginase inhibition increases cardiac contractility by increasing both [Ca2+]cyt and Ca2+ sensitivity. [Ca2+]cyt and contractile (sarcomere length [SL] shortening) responses to electrical stimulation were measured simultaneously in isolated rat cardiomyocytes using an IonOptix system. In the same cardiomyocytes, measurements were obtained at baseline, following 3‐min exposure to an arginase inhibitor (S‐[2‐boronoethyl]‐l‐cysteine; BEC) and following 3‐min exposure to BEC plus a NOS inhibitor (NG‐nitro‐l‐arginine‐methyl ester; l‐NAME). These responses were compared to time‐matched control cardiomyocytes that were untreated. Compared to baseline, BEC increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, whereas addition of l‐NAME mitigated these effects. The [Ca2+]cyt at 50% contraction and relaxation were not different across treatment groups indicating no effect of BEC on Ca2+ sensitivity. The [Ca2+]cyt and SL shortening responses in time‐matched controls did not vary with time. Arginase inhibition by BEC significantly increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects of BEC were mitigated by l‐NAME. Together, these results indicate an effect of NO on [Ca2+]cyt responses that then increase the contractile response of cardiomyocytes.

Abstract SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene ‘islands’ that drive differentiation to other lineages. The ‘unearthing’ of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1.

Background and objectives C3 glomerulopathy and idiopathic Ig-associated membranoproliferative GN are kidney diseases characterized by abnormal glomerular complement C3 deposition. These conditions are heterogeneous in outcome, but approximately 50% of patients develop kidney failure within 10 years. Design, setting, participants, & measurements To improve identification of patients with poor prognosis, we performed a detailed analysis of percutaneous kidney biopsies in a large cohort of patients. Using a validated histologic scoring system, we analyzed 156 native diagnostic kidney biopsies from a retrospective cohort of 123 patients with C3 glomerulopathy and 33 patients with Ig-associated membranoproliferative GN. We used linear regression, survival analysis, and Cox proportional hazards models to assess the relationship between histologic and clinical parameters with outcome. Results Frequent biopsy features were mesangial expansion and hypercellularity, glomerular basement membrane double contours, and endocapillary hypercellularity. Multivariable analysis showed negative associations between eGFR and crescents, interstitial inflammation, and interstitial fibrosis/tubular atrophy. Proteinuria positively associated with endocapillary hypercellularity and glomerular basement membrane double contours. Analysis of second native biopsies did not demonstrate associations between immunosuppression treatment and improvement in histology. Using a composite outcome, risk of progression to kidney failure associated with eGFR and proteinuria at the time of biopsy, cellular/fibrocellular crescents, segmental sclerosis, and interstitial fibrosis/tubular atrophy scores. Conclusions Our detailed assessment of kidney biopsy data indicated that cellular/fibrocellular crescents and interstitial fibrosis/tubular atrophy scores were significant determinants of deterioration in kidney function.