Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Protein kinase C delta enhances the diagnostic performance of hepatocellular carcinoma.

BACKGROUND: The conventional markers for hepatocellular carcinoma (HCC), α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), have several limitations; both have low sensitivity in patients with early-stage HCC; low sensitivity for AFP with HCC after eliminating hepatitis C virus (HCV); low specificity for DCP in patients with non-viral HCC, which is increasing worldwide; low specificity for AFP in patients with liver injury; and low specificity for DCP in patients treated with warfarin. To overcome these issues, the identification of novel biomarkers is an unmet need. OBJECTIVE: This study aimed to assess the usefulness of serum protein kinase C delta (PKCδ) for detecting these HCCs. METHODS: PKCδ levels were measured using a sandwich enzyme-linked immunosorbent assay in 363 chronic liver disease (CLD) patients with and without HCC. RESULTS: In both viral and non-viral CLD, PKCδ can detect HCCs with high sensitivity and specificity, particularly in the very early stages. Notably, the value and sensitivity of PKCδ were not modified by HCV elimination status. Liver injury and warfarin administration, which are known to cause false-positive results for conventional markers, did not modify PKCδ levels. CONCLUSIONS: PKCδ is an enhanced biomarker for the diagnosis of HCC that compensates for the drawbacks of conventional markers.

Kinetics of the hepatitis B core-related antigen and treatment responses in chronic hepatitis B patients treated with tenofovir alafenamide.

AIM: An association between hepatitis B core-related antigen (HBcrAg) kinetics and hepatocarcinogenesis during nucleoside (t)id analog (NA) treatment has recently been reported. HBcrAg kinetics and factors associated with HBcrAg response during tenofovir alafenamide (TAF) administration remain unclear. In this multicenter retrospective study, we aimed to clarify the efficacy and safety of TAF in treatment-naïve patients with chronic hepatitis B, focusing on the reduction in HBcrAg levels. METHODS: Patients were treated with TAF monotherapy for 96 weeks, and the kinetics of HBcrAg during treatment and the factors associated with HBcrAg response (defined as a change in HBcrAg of -1 log IU/mL from baseline) were evaluated. RESULTS: The study population comprised 241 patients, 36.9% of whom were HBeAg-positive. The median baseline HBcrAg level was 4.7 log IU/mL. The median change in HBcrAg from baseline was -1.1 log IU/mL at 96 weeks after treatment. The HBcrAg response rate at 96 weeks was 56.6% (43/76). Multivariate analysis revealed high alanine transaminase level as an independent baseline factor associated with HBcrAg response at 96 weeks of treatment (p = 4.53 × 10-6). No correlation was found between the HBcrAg and hepatitis B surface antigen kinetics in patients treated with TAF monotherapy. CONCLUSIONS: In TAF monotherapy for patients with chronic hepatitis B, HBcrAg levels were significantly decreased and baseline alanine transaminase level is an important factor associated with HBcrAg reduction. As no correlation was found between HBcrAg and reduced hepatitis B surface antigen levels in this study, HBcrAg kinetics in addition to hepatitis B surface antigen may need to be monitored during TAF treatment.

Osteosarcopenia predicts poor survival in patients with cirrhosis: a retrospective study.

BACKGROUND: Osteosarcopenia, defined as the coexistence of sarcopenia and osteoporosis, is associated with adverse clinical outcomes. The present study investigated the prognostic significance of osteosarcopenia in patients with cirrhosis. METHODS: This retrospective study evaluated 126 patients with cirrhosis. Participants were classified into three groups based on the presence or absence of (1) sarcopenia and/or osteoporosis; and (2) Child-Pugh (CP) class B/C cirrhosis and/or osteosarcopenia, and the cumulative survival rates were compared between the groups. Cox proportional hazards model was used to identify independent factors associated with mortality. Sarcopenia and osteoporosis were diagnosed according to the Japan Society of Hepatology and the World Health Organization criteria, respectively. RESULTS: Among the 126 patients, 24 (19.0%) had osteosarcopenia. Multivariate analysis identified osteosarcopenia as a significant and independent prognostic factor. The cumulative survival rates were significantly lower in patients with osteosarcopenia than in those without (1/3/5-year survival rates = 95.8%/73.7%/68.0% vs. 100%/93.6%/86.5%, respectively; p = 0.020). Patients with osteosarcopenia, but not sarcopenia or osteoporosis alone, had significantly lower cumulative survival rates than those without both conditions (p = 0.019). Furthermore, patients with both CP class B/C and osteosarcopenia had significantly lower cumulative survival rates than those without both (p < 0.001) and with either condition (p < 0.001). CONCLUSIONS: Osteosarcopenia was significantly associated with mortality in patients with cirrhosis. The cumulative survival rates were lower in patients with osteosarcopenia than in those without both conditions. Additionally, comorbid osteosarcopenia worsened the prognosis of patients with CP class B/C. Therefore, simultaneous evaluation of both sarcopenia and osteoporosis is crucial to better predict the prognosis.

Poor tolerability of lenvatinib in elderly patients ≥80 years old with hepatocellular carcinoma: A multicenter observational study.

INTRODUCTION: Management of elderly patients with cancer has become a global issue. We investigated the safety and tolerability of lenvatinib in hepatocellular carcinoma (HCC) patients ≥80 years old. METHODS: We retrospectively evaluated 61 HCC patients and divided them into 2 groups: an elderly group (n = 13, ≥80 years old) and a younger group (n = 48, <80 years old). We compared the adverse events (AEs), administration period, dose intensity, objective response, and progression-free survival (PFS) between the two groups. RESULTS: The discontinuation of lenvatinib due to AEs was more frequent in the elderly group (8/13, 61.5%) than in the younger group (10/48, 20.8%) (P = 0.0043). Fatigue and appetite loss accounted for half of the cases discontinued due to AEs in the elderly group. The elderly group had a significantly lower 8-week-delivered dose intensity/body surface area ratio (147.2) and 8-week-relative dose intensity (50.0%) than those in the younger group (267.4, 67%) (P = 0.003, 0.029). The objective response rate was significantly lower in the elderly group (15.4%) than in the younger group (61.5%) (P = 0.021). The PFS in the elderly group tended to be shorter than that in the younger group (P = 0.058, hazard ratio [HR] 1.98). The modified albumin-bilirubin (mALBI) grade (hepatic function) (HR, 2.60; P = 0.01) and objective response (HR, 0.41; P = 0.011) were independently associated with the PFS in the multivariate analysis. CONCLUSION: The management of AEs is crucial for adherence and maintaining the dose intensity of lenvatinib in elderly HCC patients.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Extracellular PKCδ signals to epidermal growth factor receptor for tumor proliferation in liver cancer cells.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR-expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C-terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ-EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ-targeting therapy for liver cancer.

Misunderstanding of hepatitis C virus (HCV) infection status by non-specialized medical doctors in patients who achieved sustained virologic response to anti-HCV therapy.

INTRODUCTION: With the increase in the number of patients with sustained virologic response (SVR) in whom hepatitis C virus (HCV) was eradicated by the anti-HCV therapy, there are now many individuals in whom serum HCV RNA is absent despite positive serum HCV antibodies. However, in general clinical practice, HCV infection remains usually screened by measurement of serum HCV antibodies and patients with SVR can be misunderstood regarding HCV infection status. METHODS: In the multicenter study, we conducted interviews with administered questionnaires to SVR individuals who had regular hospital visits after SVR. The prevalence of experiencing an incorrect diagnosis of HCV infection after SVR was assessed. Individuals who experienced this misunderstanding were further asked where they experienced it and how it made them feel. RESULTS: In a survey of 2,246 SVR individuals, 197 individuals (8.8%) were misunderstood as having persistent HCV infection by medical doctors due to positive HCV antibody, despite the absence of HCV viremia. These misunderstandings occurred most prevalently at a private clinic (55.3%). More than half (53.3%) of these individuals felt anxious about their HCV infection with becoming unsure about their HCV eradication status. CONCLUSIONS: Misunderstanding HCV status is commonly occurred in SVR individuals. Specialists in hepatology and infectious diseases should broadly emphasize the fact that most patients with HCV antibodies are now HCV-free because of the use of anti-HCV therapy.

IgM response is a prognostic biomarker of primary biliary cholangitis treated with ursodeoxycholic acid and bezafibrate.

BACKGROUND AND AIM: Primary biliary cholangitis (PBC) patients who are refractory to ursodeoxycholic acid (UDCA) are at risk for progression to cirrhosis and liver failure. Bezafibrate could be an alternative second-line therapeutic option in these patients. This study aimed to evaluate the long-term outcome(s) of combined UDCA and bezafibrate therapy in UDCA-refractory PBC patients and identify prognostic factors. METHODS: Among 445 patients treated with UDCA, 150 patients inadequately responded to UDCA monotherapy and received long-term UDCA plus bezafibrate (median, 15 years). Data from these patients were used for this retrospective analysis. RESULTS: Combination therapy resulted in significant improvements in serum biochemistry and liver transplantation risk estimated using the UK-PBC-risk and the GLOBE scores. The cumulative normalization rates of alkaline phosphatase, gamma-glutamyltransferase, and immunoglobulin M (IgM) were significantly higher in patients without cirrhosis-related symptoms or liver-related events than in those with them. Overall, IgM constantly emerged as a significant factor associated with cirrhosis-related symptoms and liver-related events at all time points. Cumulative survival rates were significantly lower in patients with IgM ≥ 240 mg/dL than in patients with IgM < 240 mg/dL. Thus, normalization of IgM levels was a good surrogate predictor of long-term prognosis. None of the patients discontinued combination therapy due to any adverse events during the follow-up period. CONCLUSIONS: Our findings point to the beneficial effects of long-term UDCA plus bezafibrate combination therapy for UDCA-refractory PBC patients, and IgM response can be a useful predictive biomarker of long-term clinical outcomes.

Evaluation of 8-week glecaprevir/pibrentasvir treatment in direct-acting antiviral-naïve noncirrhotic HCV genotype 1 and 2infected patients in a real-world setting in Japan.

Based on high efficacy and safety demonstrated in clinical trials, treatment with glecaprevir/pibrentasvir (G/P) for 8 weeks is recommended for hepatitis C virus (HCV)-infected patients who are direct-acting antiviral (DAA) naïve, genotype 1 or 2, and noncirrhotic. The aim of this study was to validate real-world experience with 8-week G/P treatment in Japan. We conducted a prospective observational cohort study in 554 patients who underwent 8-week treatment from among 1,022 patients who initiated G/P therapy. The majority (54.5%) were male, with a median age of 66 years, and HCV genotype distribution was genotype 1, 43.8%; genotype 2, 55.3%; and mixed subtype, 0.9%. Overall, the sustained virologic response rate at 12 weeks (SVR12) was 92.8% (530/571) in the intention-to-treat population and 99.3% (526/530) in the per-protocol population. The SVR12 rates by subgroups were as follows: subtype 1a, 100% (6/6); 1b, 100% (189/189); 2a, 99.3% (150/151); 2b, 99.0% (103/104); and mixed subtype, 50% (2/4). Among four patients with virologic failure following 8-week treatment with G/P, none had baseline polymorphisms or treatment-emergent amino acid substitutions in NS3. However, 2 of 4 patients with virologic failure had treatment-emergent amino acid substitutions in NS5A. Adverse events (AEs) were reported in 21.5% of patients and 1.2% of patients discontinued due to drug-related AEs. In conclusion, G/P treatment for 8 weeks was safe and effective for DAA-naïve noncirrhotic genotype 1 or 2 patients in a real-world clinical setting in Japan.

CD4+ T cells from patients with primary biliary cholangitis show T cell activation and differentially expressed T-cell receptor repertoires.

AIM: Primary biliary cholangitis (PBC) is an autoimmune liver disease with unknown pathogenesis. In PBC, activation of T-cell receptor (TCR) signaling is associated with inflammatory cytokine production through N-Ras upregulation. Although the CD4+ T cell TCR repertoire could be associated with PBC pathogenesis, it has not been evaluated. Thus, we analyzed the PBC-CD4+ T cell TCR repertoire using next generation sequencing (NGS). METHODS: Four PBC patients (one treatment-naïve and three receiving ursodeoxycholic acid) and three healthy individuals were enrolled. NRAS expression in CD4+ T cells was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). N-Ras dynamics in CD4+ T cells were assessed by qRT-PCR and GTP-N-Ras activation assay. The TCR α- (TRA) and ß-chain (TRB) repertoires on CD4+ T cells were analyzed by NGS and profiled using hierarchical analysis. Motif analysis was undertaken to elucidate the structure of PBC-specific TCRs. RESULTS: NRAS was upregulated in PBC relative to control CD4+ T cells (P < 0.05), and N-Ras enhanced T cell activation in CD4+ T cells. Among 2668 TRAs and 841 TRBs, 20 and 11, respectively, were differentially expressed in PBC compared to that in controls (P < 0.05, fold-change >2). Among them, TRAV29/J22, TRBV6-5/J2-6, and TRBV10-1/J2-1 were expressed in PBC but the expression was negligible in the controls, with more mature and longer forms observed in PBC-CD4+ T cells. CONCLUSIONS: N-Ras was upregulated in PBC-CD4+ T cells, and it enhanced TCR activation, indicating that PBC-CD4+ T cells were activated by N-Ras upregulation with differentially expressed TCR repertoires on their surfaces.

Comparative assessment of sarcopenia using the JSH, AWGS, and EWGSOP2 criteria and the relationship between sarcopenia, osteoporosis, and osteosarcopenia in patients with liver cirrhosis.

BACKGROUND: Sarcopenia and osteoporosis reduce life quality and worsen prognosis in patients with liver cirrhosis (LC). When these two complications coexist, a diagnosis of osteosarcopenia is made. We aimed to investigate the actual situations of sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture, and to clarify the relationship among these events in patients with LC. METHODS: We describe a cross-sectional study of 142 patients with LC. Sarcopenia was defined according to the Japan Society of Hepatology (JSH) criteria, Asian Working Group for Sarcopenia (AWGS) criteria, and European Working Group on Sarcopenia in Older People (EWGSOP2) criteria. The skeletal muscle mass index (SMI) and handgrip strength were assessed using bioelectrical impedance analysis and a digital grip strength dynamometer, respectively. Bone mineral density (BMD) was measured using dual energy X-ray absorptiometry, and vertebral fracture was evaluated using spinal lateral X-rays. The severity of LC was assessed using the Child-Pugh classification. RESULTS: Among the 142 patients, the prevalence of sarcopenia was 33.8% (48/142) according to the JSH and AWGS criteria and 28.2% (40/142) according to the EWGSOP2 criteria. The number of patients with osteoporosis, osteosarcopenia, and vertebral fracture was 49 (34.5%), 31 (21.8%), and 41 (28.9%), respectively. Multivariate analysis revealed a close association between sarcopenia and osteoporosis. Osteoporosis was independently associated with sarcopenia [odds ratio (OR) = 3.923, P = 0.010]. Conversely, sarcopenia was independently associated with osteoporosis (OR = 5.722, P < 0.001). Vertebral fracture occurred most frequently in patients with osteosarcopenia (19/31; 61.3%) and least frequently in those without both sarcopenia and osteoporosis (12/76; 15.8%). The SMI and handgrip strength values were significantly correlated with the BMD of the lumbar spine (r = 0.55 and 0.51, respectively; P < 0.001 for both), femoral neck, (r = 0.67 and 0.62, respectively; P < 0.001 for both), and total hip (r = 0.67 and 0.61, respectively; P < 0.001 for both). CONCLUSIONS: Sarcopenia, osteoporosis, osteosarcopenia, and vertebral fracture were highly prevalent and closely associated with one another in patients with LC. Specifically, patients with osteosarcopenia had the highest risk of vertebral fractures. Early diagnosis of these complications is essential for treatment intervention.

Serum Wisteria floribunda agglutinin-positive Mac-2 binding protein more reliably distinguishes liver fibrosis stages in non-alcoholic fatty liver disease than serum Mac-2 binding protein.

AIM: Serum Mac-2 binding protein (M2BP) and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) are used to estimate the liver fibrosis stage in chronic liver diseases. However, few head-to-head studies have been carried out to compare the two biomarkers in non-alcoholic fatty liver disease (NAFLD). METHODS: Serum M2BP and WFA+ -M2BP levels were compared against clinical characteristics and liver histological manifestations in the same samples collected from 213 biopsy-proven NAFLD patients. RESULTS: Median levels (range) of M2BP and WFA+ -M2BP were 1.58 (0.70-7.75) pg/mL and 0.85 (0.22-11.32) cut-off index (COI), respectively. Fibrosis stages 1, 2, 3, and 4 were determined in 136, 37, 17, and 23 patients, respectively. Median levels of both biomarkers increased stepwise with fibrosis progression. The M2BP and WFA+ -M2BP levels showed a significant positive correlation (r = 0.643, P = 2.91 × 10-26 ), but a marked discrepancy between both biomarkers was noted in five stage 4 and three stage 1 patients, who had high WFA+ -M2BP but relatively low M2BP levels. Most of these outliers had findings suggestive of more advanced fibrosis. For diagnosing any fibrosis severity, WFA+ -M2BP had greater area under the receiver operating characteristic curve (AUC) and predictive accuracy than M2BP. Among eight fibrosis markers/indices, WFA+ -M2BP yielded the second highest AUC (0.832) and the highest predictive accuracy (82.2%) to diagnose cirrhosis. In addition, WFA+ -M2BP showed the second highest predictive accuracy to diagnose severe fibrosis (78.4%) and significant fibrosis (76.1%). CONCLUSION: This head-to-head comparison suggests that WFA+ -M2BP is superior to M2BP for distinguishing liver fibrosis stages in NAFLD patients. A marked discrepancy between the two biomarkers may be indicative of advanced NAFLD (UMIN000023286).

An Appraisal of Current Guidelines for Managing Malignancy in Pancreatic Intraductal Papillary Mucinous Neoplasm.

Pancreatic intraductal papillary mucinous neoplasm was originally regarded as a benign mucinous cystic tumor but certainly has a marked malignant potential. With the array of high-resolution imaging modalities that are now available, more frequent incidental asymptomatic intraductal papillary mucinous neoplasm patients can be diagnosed. Until now, our clinicians have been managing intraductal papillary mucinous neoplasm patients by referring to the international consensus guidelines which have been revised twice or American Gastroenterological Association guidelines. The aim of this review is to reassess the current guidelines for the management of malignancy in intraductal papillary mucinous neoplasm. Furthermore, we specifically discuss the problems to be solved for establishing more refined guideline for the early detection, risk stratification and better management of pancreatic cancer in intraductal papillary mucinous neoplasm patients.

Cancer Stem cells and their cellular origins in primary liver and biliary tract cancers.

UNLABELLED: Liver and biliary tract cancers are highly aggressive, are heterogeneous in their phenotypic traits, and result in clinical outcomes that are difficult to manage. Cancers have subpopulations of cells termed "cancer stem cells" (CSCs) that share common intrinsic signaling pathways for self-renewal and differentiation with normal stem cells. These CSCs likely have the potential to evolve over time and to give rise to new genetically and functionally diverse subclones by accumulating genetic mutations. Extrinsic signaling from the tumor microenvironment, including the CSC niche, has been implicated in tumor initiation/progression and heterogeneity through dynamic crosstalk. CSCs have become recognized as pivotal sources of tumor initiation/progression, relapse/metastasis, and chemoresistance. CONCLUSION: The origins of CSCs are hypothesized to derive from the transformation of normal stem/progenitors and/or from the reprogramming of adult cells that converts them to stem/progenitor traits; however, the precise mechanisms have not yet been fully elucidated. (Hepatology 2016;64:645-651).

Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder.

BACKGROUND & AIMS: Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored. METHODS: Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis. RESULTS: The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice. CONCLUSIONS: Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers.

UNLABELLED: Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4's expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2). CONCLUSION: SALL4's expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers.

Concise review: clinical programs of stem cell therapies for liver and pancreas.

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal-to-distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal-liver-derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100-150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs' effects are primarily via modulation of immune mechanisms.

Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

A novel combined C-reactive protein-albumin ratio and modified albumin-bilirubin score can predict long-term outcomes in patients with hepatocellular carcinoma after hepatic resection.

Background: Systemic inflammatory response represented by C-reactive protein and albumin ratio (CAR) and modified albumin-bilirubin (mALBI) grade both have been associated with long-term outcome in patients with hepatocellular carcinoma (HCC). In this study, we investigated the prognostic utility of combined score of CAR and mALBI score to predict the prognosis of HCC patients after hepatic resection. Methods: This study included 214 patients who had undergone primary hepatic resection for HCC between 2008 and 2018. Systemic inflammatory response and mALBI were evaluated preoperatively and patients were classified into three groups based on the combination of CAR and mALBI score: low CAR and low mALBI grade (score 0), either high CAR or high mALBI grade (score 1), and both high CAR and high mALBI grade ≥2b (score 2). Multivariate Cox proportional hazard models were conducted to assess disease-free and overall survival. Results: In multivariate analysis, sex (p < 0.01), HBsAg positivity (p < 0.01), serum AFP level ≥20 ng/mL (p < 0.01), microvascular invasion (p = 0.02), multiple tumors (p < 0.01), type of resection (p < 0.01), and CAR-mALBI score ≥2 (HR 2.19, 95% CI 1.39-3.44, p < 0.01) were independent prognostic factors of disease-free survival, while sex (p = 0.01), HBsAg positivity (p < 0.01), poor tumor differentiation (p = 0.03), multiple tumors (p < 0.01), CAR-mALBI score ≥2 (HR 2.70, 95% CI 1.51-4.83, p < 0.01) were independent prognostic factors of overall survival. Conclusions: CAR-mALBI score is associated with disease-free and overall survival in patients with HCC after hepatic resection, suggesting the importance of evaluating both hepatic functional reserve and host-inflammatory state in the risk assessment of HCC patients.