Long-term outcomes of biliary atresia patients surviving with their native livers

Publisher Copyright: © 2021 The Author(s)Portoenterostomy (PE) has remained as the generally accepted first line surgical treatment for biliary atresia (BA) for over 50 years. Currently, close to half of BA patients survive beyond 10 years with their native livers, and most of them reach adulthood without liver transplantation (LT). Despite normalization of serum bilirubin by PE, ductular reaction and portal fibrosis persist in the native liver. The chronic cholangiopathy progresses to cirrhosis, complications of portal hypertension, recurrent cholangitis or hepatobiliary tumors necessitating LT later in life. Other common related health problems include impaired bone health, neuromotor development and quality of life. Only few high-quality trials are available for evidence-based guidance of post-PE adjuvant medical therapy or management of the disease complications. Better understanding of the pathophysiological mechanisms connecting native liver injury to clinical outcomes is critical for development of accurate follow-up tools and novel therapies designed to improve native liver function and survival.Peer reviewe

Chloroquine Triggers Cell Death and Inhibits PARPs in Cell Models of Aggressive Hepatoblastoma

Background:Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB. Methods:Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level. Results:CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD(+)and aspartate in CQ treated cells. In further investigations, oxidation of NAD(+)decreased as consequence of CQ treatment and NAD(+)/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed. Conclusions:CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD(+)levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.Peer reviewe

Transcription factor GATA4 associates with mesenchymal-like gene expression in human hepatoblastoma cells

GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells

Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3′ mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17–26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7–18 years) and eight relapsed tumors (follow-up time 2.8–18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse

Analysis of non-relapsed and relapsed adult type granulosa cell tumors suggests stable transcriptomes during tumor progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3\u27 mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2

Analysis of Non-Relapsed and Relapsed Adult Type Granulosa Cell Tumors Suggests Stable Transcriptomes during Tumor Progression

Adult-type granulosa cell tumor (AGCT) is a rare ovarian malignancy characterized by slow growth and hormonal activity. The prognosis of AGCT is generally favorable, but one-third of patients with low-stage disease experience a late relapse, and over half of them die of AGCT. To identify markers that would distinguish patients at risk for relapse, we performed Lexogen QuantSeq 3′ mRNA sequencing on formalin-fixed paraffin-embedded, archival AGCT tissue samples tested positive for the pathognomonic Forkhead Box L2 (FOXL2) mutation. We compared the transcriptomic profiles of 14 non-relapsed archival primary AGCTs (follow-up time 17–26 years after diagnosis) with 13 relapsed primary AGCTs (follow-up time 1.7–18 years) and eight relapsed tumors (follow-up time 2.8–18.9 years). Non-relapsed and relapsed primary AGCTs had similar transcriptomic profiles. In relapsed tumors three genes were differentially expressed: plasmalemma vesicle associated protein (PLVAP) was upregulated (p = 0.01), whereas argininosuccinate synthase 1 (ASS1) (p = 0.01) and perilipin 4 (PLIN4) (p = 0.02) were downregulated. PLVAP upregulation was validated using tissue microarray RNA in situ hybridization. In our patient cohort with extremely long follow-up, we observed similar gene expression patterns in both primary AGCT groups, suggesting that relapse is not driven by transcriptomic changes. These results reinforce earlier findings that molecular markers do not predict AGCT behavior or risk of relapse

GATA6 modulates the ductular reaction to bile duct ligation

Background GATA6, a transcription factor expressed in cholangiocytes, has been implicated in the response to liver injury. In biliary atresia, a disease characterized by extrahepatic bile duct obstruction, liver expression of GATA6 increases with pathological bile duct expansion and decreases after successful Kasai portoenterostomy. The aim of this study was to garner genetic evidence that GATA6 is involved in ductular formation/expansion. Methods The murine Gata6 gene was conditionally deleted using Alb-cre, a transgene expressed in hepatoblasts (the precursors of hepatocytes and cholangiocytes) and mature hepatocytes. Bile duct ligation (BDL) was used to model biliary obstruction. Results Alb-Cre;Gata6(flox/flox) mice were viable and fertile. Cre-mediated recombination of Gata6 in hepatocytes had little impact on cellular structure or function. GATA6 immunoreactivity was retained in a majority of biliary epithelial cells in adult Alb-Cre;Gata6(flox/flox) mice, implying that surviving cholangiocytes were derived from hepatoblasts that had escaped biallelic Cre-mediated recombination. Although GATA6 immunoreactivity was preserved in cholangiocytes, Alb-cre;Gata6(flox/flox) mice had a demonstrable biliary phenotype. A neutrophil-rich infiltrate surrounded newly formed bile ducts in neonatal Alb-Cre;Gata6(flox/flox) mice. Foci of fibrosis/necrosis, presumed to reflect patchy defects in bile duct formation, were observed in the livers of 37% of adult Alb-cre;Gata6(flox/flox) mice and 0% of controls (p <0.05). Most notably, Alb-cre;Gata6(flox/flox) mice had an altered response to BDL manifest as reduced survival, impaired bile ductule proliferation, increased parenchymal necrosis, reduced fibrosis, and enhanced macrophage accumulation in the portal space. Conclusions GATA6 orchestrates intrahepatic biliary remodeling and mitigates liver injury following extrahepatic bile duct obstruction. Graphic abstractPeer reviewe

Liver secretin receptor predicts portoenterostomy outcomes and liver injury in biliary atresia

Biliary atresia (BA) is a chronic neonatal cholangiopathy characterized by fibroinflammatory bile duct damage. Reliable biomarkers for predicting native liver survival (NLS) following portoenterostomy (PE) surgery are lacking. Herein we explore the utility of 22 preidentified profibrotic molecules closely connected to ductular reaction (DR) and prevailing after successful PE (SPE), in predicting PE outcomes and liver injury. We used qPCR and immunohistochemistry in a BA cohort including liver samples obtained at PE (n = 53) and during postoperative follow-up after SPE (n = 25). Of the 13 genes over-expressed in relation to cholestatic age-matched controls at PE, only secretin receptor (SCTR) expression predicted cumulative 5-year NLS and clearance of jaundice. Patients in the highest SCTR expression tertile showed 34-55% lower NLS than other groups at 1-5 years after PE (P = 0.006-0.04 for each year). SCTR expression was also significantly lower [42 (24-63) vs 75 (39-107) fold, P = 0.015] among those who normalized their serum bilirubin after PE. Liver SCTR expression localized in cholangiocytes and correlated positively with liver fibrosis, DR, and transcriptional markers of fibrosis (ACTA2) and cholangiocytes (KRT7, KRT19) both at PE and after SPE. SCTR is a promising prognostic marker for PE outcomes and associates with liver injury in BA.Peer reviewe

Prognostic and Pathophysiologic Significance of IL-8 (CXCL8) in Biliary Atresia

Interleukin (IL)-8 (CXCL8), a chemokine involved in neutrophil recruitment, has been implicated in ductular reaction and liver fibrogenesis. We studied liver and serum IL-8 expression in a large biliary atresia (BA) cohort and explored its prognostic and pathophysiological potential. IL-8 expression was assessed in liver utilizing quantitative polymerase chain reaction (qPCR), immunohistochemistry and in situ hybridization and in serum using an enzyme-linked immunosorbent assay, among 115 BA patients, 10 disease controls and 68 normal controls. Results were correlated to portoenterostomy (PE) outcomes, biochemical and histological liver injury, transcriptional markers of fibrosis and cholangiocytes, and expression of other related cytokines. IL-8 was markedly overexpressed in liver and serum of BA patients at PE (n = 88) and in serum samples obtained during postoperative follow-up (n = 40). IL-8 expression in the liver was predominantly in cholangiocytes within areas of ductular reaction. Liver IL-8 mRNA expression correlated positively with its serum concentration, bile ductular proliferation, Metavir fibrosis stage, and transcriptional markers of activated myofibroblasts (ACTA2) and cholangiocytes (KRT19). Taken together, IL-8 may mediate liver injury in BA by promoting ductular reaction and associated liver fibrogenesis. Prognostic value of serum IL-8 to predict native liver survival was limited and confined to the postoperative period after PE