Distention of the Immature Left Ventricle Triggers Development of Endocardial Fibroelastosis: An Animal Model of Endocardial Fibroelastosis Introducing Morphopathological Features of Evolving Fetal Hypoplastic Left Heart Syndrome

Background. Endocardial fibroelastosis (EFE), characterized by a diffuse endocardial thickening through collagen and elastin fibers, develops in the human fetal heart restricting growth of the left ventricle (LV). Recent advances in fetal imaging indicate that EFE development is directly associated with a distended, poorly contractile LV in evolving hypoplastic left heart syndrome (HLHS). In this study, we developed an animal model of EFE by introducing this human fetal LV morphopathology to an immature rat heart. Methods and Results. A neonatal donor heart, in which aortic regurgitation (AR) was created, was heterotopically transplanted into a recipient adult rat. AR successfully induced the LV morphology of evolving HLHS in the transplanted donor hearts, which resulted in the development of significant EFE covering the entire LV cavity within two weeks postoperatively. In contrast, posttransplants with a competent aortic valve displayed unloaded LVs with a trace of EFE. Conclusions. We could show that distention of the immature LV in combination with stagnant flow triggers EFE development in this animal model. This model would serve as a robust tool to develop therapeutic strategies to treat EFE while providing insight into its pathogenesis

The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5

Equal Efficacy and Safety Profile in Elderly Patients with Hepatocellular Carcinoma Receiving Palliative Treatment

Palliative treatment of elderly patients with hepatocellular carcinoma (HCC) is often challenging due to comorbidities or frailty, and data about the outcome and overall survival (OS) in these patients are limited. This was a retrospective single centre study. Patients were grouped according to their age as young (&lt;60 years; YP), intermediate (60&ndash;70 years; IP) or elderly (&gt;70 years; EP). Administration of chemotherapy or transarterial chemoembolization (TACE) was defined as palliative treatment. Therapy-related adverse events (AE) were assessed via CTCAE 5.0. Out of 656 patients analyzed, n = 359 received palliative treatment: YP: n = 90; IP: n = 127 and EP: n = 142. The median OS (months) in patients receiving TACE (n = 254) was 17 vs. 18 vs. 20 months for YP, IP, and EP, respectively (p = 0.44) and 15 vs. 16 vs. 17 months (p = 0.56), respectively, in patients receiving chemotherapy (n = 105). AEs differed non-significantly between the subgroups. Multivariate analysis revealed impaired liver function and advanced tumor stage as significant factors for impaired OS. In this study, the mOS and rate of AEs were equal between elderly and younger HCC patients receiving palliative treatment. Therefore, we propose regular palliative treatment stratification in spite of the high age of patients

EpCAM-positive circulating tumor cells and serum AFP levels predict outcome after curative resection of hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) has high recurrence rates exceeding 50% despite curative resection. The serum biomarker alpha-fetoprotein (AFP) is a well-known prognostic marker for HCC. EpCAM-positive circulating tumor cells (CTC) have a high predictive value for early HCC recurrence after curatively intended resection, most likely indicating micro-metastases at the time of resection. However, sensitivity remains low. The objective of this study was to evaluate a composite test comprising both CTC and AFP to identify patients at high risk for early HCC recurrence. We prospectively enrolled 58 patients undergoing curative intended resection for HCC at a tertiary referral center. Blood specimens were obtained prior to resection and analyzed for EpCAM-positive CTC and serum AFP levels. A positive result was defined as either detection of CTC or AFP levels ≥ 400 ng/ml. Eight patients tested positive for CTC, seven for AFP, and two for both markers. A positive composite test was significantly associated with shorter early recurrence-free survival (5 vs. 16 months, p = 0.005), time to recurrence (5 vs. 16 months, p = 0.011), and overall survival (37 vs. not reached, p = 0.034). Combining CTC and AFP identified patients with poor outcome after surgical resection, for whom adjuvant or neoadjuvant therapies may be particularly desirable

Biallelic FRA10AC1 variants cause a neurodevelopmental disorder with growth retardation.

The major spliceosome mediates pre-mRNA splicing by recognizing the highly conserved sequences at the 5' and 3' splice sites and the branch point. More than 150 proteins participate in the splicing process and are organized in the spliceosomal A, B, and C complexes. FRA10AC1 is a peripheral protein of the spliceosomal C complex and its ortholog in the green alga facilitates recognition or interaction with splice sites. We identified biallelic pathogenic variants in FRA10AC1 in five individuals from three consanguineous families. The two unrelated Patients 1 and 2 with loss-of-function variants showed developmental delay, intellectual disability, and no speech, while three siblings with the c.494_496delAAG (p.Glu165del) variant had borderline to mild intellectual disability. All patients had microcephaly, hypoplasia or agenesis of the corpus callosum, growth retardation, and craniofacial dysmorphism. FRA10AC1 transcripts and proteins were drastically reduced or absent in fibroblasts of Patients 1 and 2. In a heterologous expression system, the p.Glu165del variant impacts intrinsic stability of FRA10AC1 but does not affect its nuclear localization. By co-immunoprecipitation, we found ectopically expressed HA-FRA10AC1 in complex with endogenous DGCR14, another component of the spliceosomal C complex, while the splice factors CHERP, NKAP, RED, and SF3B2 could not be co-immunoprecipitated. Using an in vitro splicing reporter assay, we did not obtain evidence for FRA10AC1 deficiency to suppress missplicing events caused by mutations in the highly conserved dinucleotides of 5' and 3' splice sites in an in vitro splicing assay in patient-derived fibroblasts. Our data highlight the importance of specific peripheral spliceosomal C complex proteins for neurodevelopment. It remains possible that FRA10AC1 may have other and/or additional cellular functions, such as coupling of transcription and splicing reactions

Hepatocellular carcinoma: Intratumoral EpCAM-positive cancer stem cell heterogeneity identifies high-risk tumor subtype

Background!#!The translational interest in the intratumoral heterogeneity of hepatocellular carcinoma (HCC) has been increasing. The dismal prognosis of this pathology is linked to the features of the HCC harbouring cancer stem cells (CSC), represented by EpCAM-expression. However, the extent of the impact of intratumoral distribution of CSC-features, both on the recurrence after curative resection and on clinical outcome, remains unknown. To address this, we investigated the spatial heterogeneity of CSC-features with the aim of identifying the unique HCC patient subgroups amenable to adjuvant treatment.!##!Methods!#!We designed a tissue microarray (TMA) from patients who had received liver resection between 2011 and 2017. Tumor specimens were sampled at multiple locations (n = 3-8). EpCAM-positivity was assessed for intensity and proportion by applying a score dividing three groups: (i) negative (E-/-); (ii) heterogeneous (E-/+); and (iii) homogeneous (E+/+). The groups were further analysed with regard to time-to-recurrence (TTR) and recurrence-free-survival (RFS).!##!Results!#!We included 314 tumor spots from 69 patients (76.8% male, median age 66, liver cirrhosis/fibrosis 75.8%). The risk factors were alcohol abuse (26.2%), NASH (13.1%), HBV (15.5%), HCV (17.9%) and others (27.4%), representative of a typical Western cohort. E+/+ patients experienced significantly shorter TTR and RFS compared to E+/- and E-/- patients (TTR 5 vs. 19 months, p = 0.022; RFS 5 vs. 14 vs. 21 months, p = 0.016). Only homogeneous EpCAM-positivity correlated with higher AFP levels (&amp;gt; 400 ng/ml, p = 0.031).!##!Conclusions!#!Spatial heterogeneity of EpCAM-expression was markedly present in the cohort. Of note, only homogeneous EpCAM-expression correlated significantly with early recurrence, whereas heterogeneous EpCAM-expression was associated with clinical endpoints comparable to EpCAM-negativity. We identified a unique HCC subtype associated with a high risk of tumor recurrence