Safety and tolerability of OP-724 in patients with haemophilia and liver cirrhosis due to HIV/HCV coinfection: an investigator-initiated, open-label, non-randomised, single-centre, phase I study.

OBJECTIVE: Patients with haemophilia and HIV who acquire hepatitis C virus (HCV) after receiving contaminated blood products can experience accelerated progression of liver fibrosis and a poor prognosis, making liver disease a prominent cause of mortality among these patients. In the current study, we aimed to evaluate the safety and tolerability of the potential antifibrotic agent OP-724-a CREB-binding protein/ß-catenin inhibitor-in this patient subset. DESIGN: In this single-centre, open-label, non-randomised, phase I trial, we sequentially enrolled patients with cirrhosis following HIV/HCV coinfection classified as Child-Pugh (CP) class A or B. Five patients received an intravenous infusion of OP-724 at doses of 140 or 280 mg/m2 for 4 hours two times weekly over 12 weeks. The primary endpoint was the incidence of serious adverse events (SAEs). Secondary endpoints included the incidence of AEs and improved liver stiffness measure (LSM), as determined by vibration-controlled transient elastography. This study was registered at ClinicalTrials.gov (NCT04688034). RESULTS: Between 9 February 2021 and 5 July 2022, five patients (median age: 51 years) were enrolled. All five patients completed 12 cycles of treatment. SAEs were not observed. The most common AEs were fever (60%) and gastrointestinal symptoms (diarrhoea: 20%, enterocolitis: 20%). Improvements in LSM and serum albumin levels were also observed. CONCLUSION: In this preliminary assessment, intravenous administration of 140 or 280 mg/m2/4 hours OP-724 over 12 weeks was well tolerated by patients with haemophilia combined with cirrhosis due to HIV/HCV coinfection. Hence, the antifibrotic effects of OP-724 warrant further assessment in patients with cirrhosis. TRIAL REGISTRATION NUMBER: NCT04688034.

Effectiveness of Chemotherapy on Long-Term Survival in a Case of Advanced Juvenile Hepatocellular Carcinoma Without Viral Hepatitis Infection.

Hepatocellular carcinoma (HCC) usually occurs in settings of cirrhosis and chronic hepatitis B or C virus (HBV and HCV, respectively) infection; it is extremely rare in patients <40 years of age since viral- or alcohol-induced chronic hepatitis develops over a prolonged period. Juvenile HCC is mostly associated with persistent HBV infection; cases unrelated to HBV or HCV infection (non-B, non-C juvenile HCC) are sporadic and treated in the same way as classical HCC. A woman in her late 30s was diagnosed with HCC in a healthy liver; her imaging findings were typical of HCC with bone metastasis. She was administered a combination of tyrosine kinase inhibitors, immune checkpoint inhibitors, and vascular endothelial growth factor inhibitors. Throughout chemotherapy, the liver reserve was Grade A on the Child-Pugh classification and tumor markers remained under control without marked elevation. Our patient is the first reported long-term survivor of unresectable non-B, non-C juvenile HCC following chemotherapeutic treatment.

Alpha-fetoprotein-producing gastric cancer with ruptured liver metastasis mimicking hepatocellular carcinoma.

Alpha-fetoprotein (AFP)-producing gastric cancer is a rare subtype of gastric cancer known for its aggressive nature. We present an uncommon case of a 60s male with multiple liver tumors, initially suggested as hepatocellular carcinoma (HCC) by imaging. However, a subsequent gastric biopsy revealed a poorly differentiated adenocarcinoma with hepatoid features, and liver biopsy mirrored these findings. The disease progressed swiftly, with the patient representing owing to the spontaneous rupture of a metastatic liver tumor, an extremely rare occurrence, especially in metastatic liver cancers. Such ruptures in AFP-producing gastric cancer may be attributed to the tumor's rich blood flow. Distinctly differentiating this subtype from HCC is pivotal for apt management, as was evident in our case. The diagnosis was particularly challenging due to the similarities in imaging presentations between AFP-producing gastric cancer liver metastasis and HCC. This case underscores the need for vigilant diagnosis, emphasizing the importance of liver biopsy, especially in the absence of chronic liver disease. It also highlights the potential complications, like spontaneous rupture, associated with this rare form of gastric cancer.

Stem Cell Growth Factor-ß as a Predictive Biomarker of Response to Chemotherapy and Prognosis in Patients with Advanced-Stage Hepatocellular Carcinoma: A Retrospective Study.

In this retrospective study, we investigated the potential application of serum stem cell growth factor beta (SCGF-ß) as a biomarker for predicting the therapeutic response and prognosis in patients with hepatocellular carcinoma (HCC) undergoing atezolizumab and bevacizumab (Atz/Bev) combination therapy. Pre- and post-treatment serum SCGF-ß levels were measured and analyzed in relation to treatment outcomes and overall survival (OS). Pretreatment SCGF-ß levels were associated with treatment response. Patients with SCGF-ß levels exceeding the 163,295 pg/mL cutoff experienced significantly reduced OS, with a median OS of 12.03 months, compared to 28.87 months in those with SCGF-ß levels at or below this threshold. These findings suggest that SCGF-ß can serve as a predictive marker for clinical outcomes in HCC treatment, highlighting the need for prospective studies to further validate these results and clarify the mechanisms underlying SCGF-ß-related therapeutic resistance.

Molecular insights of a CBP/ß-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

Rapidly Progressing Sarcomatoid Hepatocellular Carcinoma after Needle Biopsy and Radiofrequency Ablation.

BACKGROUND Sarcomatoid hepatocellular carcinoma is a rare, primary malignant liver cancer. Its pathogenesis is unknown, but it often occurs in patients who have undergone repeated antitumor therapies for hepatocellular carcinoma. Sarcomatoid hepatocellular carcinoma is more likely to recur and has a worse prognosis than that of hepatocellular carcinoma. As no specific features have been identified in the symptoms, serological findings, or imaging findings, it is difficult to accurately diagnose the disease before surgical resection or autopsy. CASE REPORT An 83-year-old woman was diagnosed with hepatocellular carcinoma 20 years ago. Radiofrequency ablation was initially performed. Thereafter, invasive, non-surgical treatments were repeated. The most recent treatment was 4 years ago, during which computed tomography suggested recurrent hepatocellular carcinoma. However, upon needle biopsy, histological examination revealed spindle-shaped tumor cells and actively mitotic cells. Immunohistochemical analysis showed negative results for Arginase-1, HepPar1, and Glypican3 and positive results for AE1/AE3, CK7, and vimentin. Therefore, sarcomatoid hepatocellular carcinoma was diagnosed, which was treated with radiofrequency ablation but progressed rapidly thereafter. Considering the rapid disease progression, the patient was treated conservatively. However, the patient's general condition gradually deteriorated, resulting in death. CONCLUSIONS Compared with hepatocellular carcinoma, sarcomatoid hepatocellular carcinoma is more prone to recurrence and has a poorer prognosis. Therefore, aggressive surgical resection seems to be the most appropriate treatment for sarcomatoid hepatocellular carcinoma at present. Additional hepatic resection or follow-up imaging in a short period should be considered at the time of diagnosis of sarcomatoid hepatocellular carcinoma by biopsy, considering the risk of seeding or recurrence.

Prevalence of hepatitis C virus infection among men who have sex with men with human immunodeficiency virus-1 infection between 2010 and 2020 in Japan: A single-center retrospective cohort study.

INTRODUCTION: The high prevalence of hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) is an important health issue. The purpose of this study is to investigate the actual prevalence of HCV infection among HIV-positive MSM in Japan. METHODS: This study is a single-center retrospective cohort study. We collected data of HIV-infected MSM who visited our hospital from January 2010 to December 2020, and evaluated HCV prevalence, course of HCV infection, and direct-acting antiviral (DAA) treatment efficacy in HIV-infected MSM. RESULTS: Overall, 1135 HIV-infected MSM had HCV antibody (Ab) tests during the observation period. The first anti-HCV Ab positive rate in HIV-infected MSM was 4% (45/1135), and the seroconversion rate of HCV antibody was 3.6% (39/1090). Treponema pallidum hemagglutination antigen positivity (odds ratio [OR], 5.28; 95% confidence interval [CI], 2.9 to 10.5) and intravenous drug injection (OR, 19; 95% CI, 3.4 to 149) were identified as factors associated with HCV Ab positivity. Spontaneous elimination of HCV infection was observed in 17.9% (7/39) of patients. DAA treatment was performed in 43 cases, and the overall sustained virologic response 12 (SVR12) rate for DAA treatment was 93% (40/43). CONCLUSION: A high HCV infection rate among HIV-infected MSM was observed in Japan. The DAA treatment response rate in patients with HIV/HCV co-infection was the high response rate.

Phosphotungstic Acid-treated Picrosirius Red Staining Improves Whole-slide Quantitative Analysis of Collagen in Histological Specimens.

We tried to prevent nonspecific nuclear staining (NS-NS) of picrosirius red (PSR) staining by treating the specimens with one of the heteropoly acids phosphotungstic acid (PTA). We analyzed a total of 35 cases of non-cancerous liver tissue for fibrosis and NS-NS under PSR-alone, phosphomolybdic acid (PMA)-pretreated PSR (PMA + PSR), or PTA-pretreated PSR (PTA + PSR) condition. In addition, we analyzed the photosensitivity of PMA or PTA single stain specimens. PTA + PSR significantly suppressed NS-NS compared with PSR. The color of the specimens did not change into blue by 30 times the exposure to whole slide scanner (WSS) light. The PTA + PSR condition showed the highest correlation with the Ishak score (pathological evaluation of liver fibrosis) compared with other conditions. Furthermore, Sirius Red-positive percentage (SRP%) in PSR was increased in the NS-NS observed cases. SRP% in PMA + PSR was significantly affected by WSS light exposure time. Moreover, the deposition of non-polarized PSR-stained substances (NP-PSR+S) clinging to the collagen fibers potentially explains why SRP% seemed bigger under PSR than PTA + PSR. Our protocol enabled us to analyze the whole slide image of PSR staining by high magnification, which would contribute to the accurate analysis of collagen amount in the tissue sections.

Feasibility, safety and tolerability of the CREB-binding protein/ß-catenin inhibitor OP-724 in patients with advanced primary biliary cholangitis: an investigator-initiated, open-label, non-randomised, two-centre, phase 1 study.

OBJECTIVE: This study aimed to evaluate the safety and tolerability of OP-724, a CREB-binding protein/ß-catenin inhibitor, in patients with advanced primary biliary cholangitis (PBC). DESIGN: An open-label, non-randomised, phase 1 trial was conducted at two hospitals in Japan. Patients with advanced PBC classified as stage III or higher according to the Scheuer classification by liver biopsy between 4 September 2019 and 21 September 2021 were enrolled. Seven patients received intravenous OP-724 infusions at escalating dosages of 280 and 380 mg/m2/4 hours two times weekly for 12 weeks. The primary endpoint was the incidence of serious adverse events (SAEs). The secondary endpoints were the incidence of AEs and the improvement in the modified Histological Activity Index (mHAI) score. RESULTS: Seven patients (median age, 68 years) were enrolled. Of these seven patients, five completed twelve cycles of treatment, one discontinued prematurely for personal reasons in the 280 mg/m2/4 hours cohort, and one in the 380 mg/m2/4 hours cohort was withdrawn from the study due to drug-induced liver injury (grade 2). Consequently, the recommended dosage was determined to be 280 mg/m2/4 hours. SAEs did not occur. The most common AEs were abdominal discomfort (29%) and abnormal hepatic function (43%). OP-724 treatment was associated with histological improvements in the fibrosis stage (2/5 (40%)) and mHAI score (3/5 (60%)) on histological analysis. CONCLUSION: Administration of intravenous OP-724 infusion at a dosage of 280 mg/m2/4 hours two times weekly for 12 weeks was well tolerated by patients with advanced PBC. However, further evaluation of antifibrotic effects in patients with PBC is warranted. TRIAL REGISTRATION NUMBER: NCT04047160.

Inhibition of CBP/ß-catenin signaling ameliorated fibrosis in cholestatic liver disease.

Chronic cholestatic liver diseases are characterized by injury of the bile ducts and hepatocytes caused by accumulated bile acids (BAs) and inflammation. Wnt/ß-catenin signaling is implicated in organ fibrosis; however, its role in cholestatic liver fibrosis remains unclear. Therefore, we explored the effect of a selective cAMP response element-binding protein-binding protein (CBP)/ß-catenin inhibitor, PRI-724, on murine cholestatic liver fibrosis. PRI-724 suppressed liver fibrosis induced by multidrug resistance protein 2 knockout (KO), bile duct ligation, or a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet; it also suppressed BA synthesis and macrophage infiltration. The expression of early growth response-1 (Egr-1), which plays a key role in BA synthesis, was increased in the hepatocytes of patients with cholestatic liver disease. PRI-724 inhibited Egr-1 expression induced by cholestasis, and adenoviral shEgr-1-mediated Egr-1 knockdown suppressed BA synthesis and fibrosis in DDC diet-fed mice, suggesting that PRI-724 exerts its effects, at least in part, by suppressing Egr-1 expression in hepatocytes. Hepatocyte-specific CBP KO in mice suppressed BA synthesis, liver injury, and fibrosis, whereas hepatocyte-specific KO of P300, a CBP homolog, exacerbated DDC-induced fibrosis. Intrahepatic Egr-1 expression was also decreased in hepatocyte-specific CBP-KO mice and increased in P300-KO mice, indicating that Egr-1 is located downstream of CBP/ß-catenin signaling. Conclusion: PRI-724 inhibits cholestatic liver injury and fibrosis by inhibiting BA synthesis in hepatocytes. These results highlight the therapeutic effect of CBP/ß-catenin inhibition in cholestatic liver diseases.

Safety, tolerability, and anti-fibrotic efficacy of the CBP/ß-catenin inhibitor PRI-724 in patients with hepatitis C and B virus-induced liver cirrhosis: An investigator-initiated, open-label, non-randomised, multicentre, phase 1/2a study.

BACKGROUND: We conducted an exploratory study to assess the safety tolerability, and anti-fibrotic effects of PRI-724, a CBP/ß-catenin inhibitor, in patients with hepatitis C virus (HCV)- and hepatitis B virus (HBV)-induced cirrhosis. METHODS: This multicentre, open-label, non-randomised, non-placebo-controlled phase 1/2a trial was conducted at three hospitals in Japan. Between July 27, 2018, and July 13, 2021, we enrolled patients with HCV- and HBV-induced cirrhosis classified as Child-Pugh (CP) class A or B. In phase 1, 15 patients received intravenous infusions of PRI-724 at escalating doses of 140, 280, and 380 mg/m2/4 h twice weekly for 12 weeks. In phase 2a, 12 patients received the recommended PRI-724 dose. The primary endpoints of phases 1 and 2a were the frequency and severity of adverse events and efficacy in treating cirrhosis based on liver biopsy. This study was registered at ClinicalTrials.gov (no. NCT03620474). FINDINGS: Three patients from phase 1 who received the recommended PRI-724 dose were evaluated to obtain efficacy and safety data in phase 2a. Serious adverse events occurred in three patients, one of which was possibly related to PRI-724. The most common adverse events were diarrhoea and nausea. PRI-724 did not decrease hepatic fibrosis with any statistical significance, either by ordinal scoring or measurement of collagen proportionate area at 12 weeks; however, we observed statistically significant improvements in liver stiffness, Model for End-stage Liver Disease score, and serum albumin level. INTERPRETATION: Intravenous administration of 280 mg/m2/4 h PRI-724 over 12 weeks was preliminarily assessed to be well tolerated; however, further evaluation of anti-fibrotic effects in patients with cirrhosis is warranted. FUNDING: AMED, Ohara Pharmaceutical.

Anti-tumor Activity of the Small Molecule Inhibitor PRI-724 Against ß-Catenin-activated Hepatocellular Carcinoma.

BACKGROUND/AIM: CBP is a transcriptional coactivator in the Wnt/ß-catenin pathway that is related to cell kinetics and differentiation. This study aimed to characterize ß-catenin-activated hepatocellular carcinoma (HCC) and evaluate the direct effects of PRI-724 (a selective inhibitor of Wnt/ß-catenin/CBP signaling) on HCC. MATERIALS AND METHODS: Immunohistochemistry for ß-catenin was performed in 199 HCC resected samples. Moreover, using cultured HCC cell lines, cell kinetics and its related proteins were analyzed after treatment of cells with C-82 (active form of PRI-724). RESULTS: Nuclear ß-catenin expression was found in 18% of HCC cases and the tumor sizes in these positive samples were larger. In HCC cell lines with a constitutively activated ß-catenin, C-82 inhibited cell proliferation. C-82 led to an increase in the percentage of cells in the G0/G1 phase of the cell cycle. The percentage of cells in the sub-G1 phase also increased. Moreover, C-82 treatment significantly decreased the expression of cell proliferating markers and increased the expression of apoptosis-related proteins. CONCLUSION: PRI-724(C-82) may be a novel drug for ß-catenin-activated HCC therapy.

Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light.

Picrosirius red (PSR) staining is generally used to evaluate liver fibrosis; however, PSR sometimes causes nonspecific nuclear staining. In this study, we evaluated the ability of phosphomolybdic acid (PMA) pretreatment to prevent nonspecific nuclear staining by PSR. In a manual evaluation of 27 non-tumor samples from patients with hepatocellular carcinoma, nonspecific nuclear staining was observed in 3.7% of PMA-treated specimens, compared with 85.2% of untreated specimens. Conversely, computer-assisted image analysis (CAIA) identified nonspecific nuclear staining in 0% of PMA-treated samples, vs 44.4% of untreated samples. Surprisingly, after mounting, PMA-treated specimens exhibited a blue tinge because of molybdenum blue (MB) production following sunlight exposure or virtual slide scanning. Using UV cut film, MB production induced by sunlight exposure was prevented; however, the film did not prevent MB production during virtual slide scanning. Moreover, only blue light-emitting diode exposure resulted in a blue tinge in PMA solution. Our data indicated that PMA pretreatment is effective for evaluating liver fibrosis using CAIA. Meanwhile, improvements in virtual slide scanning protocols would directly improve the quality of PMA-pretreated specimens subjected to CAIA.

A prospective trial of vaccine to prevent hepatitis B virus reactivation after hematopoietic stem cell transplantation.

Hepatitis B virus (HBV) reactivation reportedly occurs frequently after hematopoietic stem cell transplantation (HSCT) in resolved HBV-infected patients. Here, 50 patients with resolved HBV infections and scheduled to undergo HSCT were enrolled; all subjects were vaccinated with three doses of hepatitis B vaccine 12 months after HSCT and the incidence of HBV reactivation was monitored. The patients' characteristics were: median age, 61 (34-72) years; male/female, 27/19; allogeneic/autologous, 40/6; bone marrow/peripheral blood stem cells/cord blood, 26/16/4. Of the 46 patients who underwent HSCT, 19 were excluded and did not make it to vaccination due to relapse of underlying disease, HBV reactivation within 12 months of HSCT, or transfer of patients. The remaining 27 were vaccinated 12 months after HSCT and monitored for 2 years. Six showed HBV reactivation, with a 2-year cumulative reactivation incidence of 22.2%; the same incidence was 27.3% only in allogeneic HSCT patients. Factors associated with HBV reactivation included the discontinuation of immunosuppressants (P = 0.0379) and baseline titers of antibody against hepatitis B surface antigen (P = 0.004). HBV reactivation with vaccination following HSCT could occur despite maintenance of serum anti-HBs at more than protective levels.

Programmed cell death ligand 1 (PD-L1) blockade attenuates metastatic colon cancer growth in cAMP-response element-binding protein (CREB)-binding protein (CBP)/ß-catenin inhibitor-treated livers.

Immune checkpoint blockade with specific antibodies can accelerate anti-tumor immunity, resulting in clinical responses in patients with various types of cancer. However, these antibodies achieve only partial tumor regression. Thus, a wide variety of treatment combinations based on programmed death-ligand 1 (PD-L1) pathway inhibition are under development to enhance such therapeutic effects. In this study, the effects of combination treatment using PRI-724, a selective inhibitor of CBP/ß-catenin, and an anti-PD-L1 antibody were examined in a mouse model of colon cancer liver metastasis. Mice were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. The combination treatment resulted in regression of tumor growth, whereas monotherapy with each treatment individually failed to exhibit any anti-tumor activity. In addition, co-administration of the inhibitor and antibody induced CD8+CD44lowCD62Llow cells and interferon (IFN)-γ production in CD8+ T-cells in the liver compared with that in control mice. Administration of an anti-CD8 antibody mitigated the anti-tumor effects of the combined treatment of PRI-724 and anti-PD-L1 antibody. In conclusion, targeting CBP/ß-catenin, combined with PD-1/PD-L1 immune checkpoint blockade, shows potential as a new therapeutic strategy for treating liver metastasis during colon cancer.

Bone morphogenetic protein 4 provides cancer-supportive phenotypes to liver fibroblasts in patients with hepatocellular carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are essential constituents of cancer-supportive microenvironments. The high incidence of hepatocellular carcinoma (HCC) in advanced fibrosis patients implies that fibroblasts have a promoting effect on HCC development. We aimed to explore the regulators of phenotypes and function of CAFs in the liver. METHODS: We established primary cancer-associated fibroblasts (CAFs) and non-cancerous liver fibroblasts (NFs) from 15 patients who underwent HCC resection. We compared phenotypes, capacity of cytokine/chemokine production and gene expression profiles between pairs of CAFs and NFs from the same donors. We examined resected tissue from additional 50 patients with HCC for immunohistochemical analyses. RESULTS: The CAFs expressed more ACTA2 and COL1A1 than the NFs, suggesting that CAFs are more activated phenotype. The CAFs produced larger amounts of IL-6, IL-8 and CCL2 than the NFs, which led to invasiveness of HuH7 in vitro. We found that Bone Morphogenetic Protein-4 (BMP4) is up-regulated in CAFs compared to NFs. The CAF phenotype and function were gained by BMP4 over-expression or recombinant BMP4 given to fibroblasts, all of which decreased with BMP4 knockdown. In tissues obtained from the patients, BMP4-positive cells are mainly observed in encapsulated fibrous lesions and HCC. Positive expression of BMP4 in HCC in resected tissues, not in fibroblasts, was associated with poorer postoperative overall survival in patients with HCC. CONCLUSION: Endogenous and exogenous BMP4 activate liver fibroblasts to gain capacity of secreting cytokines and enhancing invasiveness of cancer cells in the liver. BMP4 is one of the regulatory factors of CAFs functioning in the microenvironment of HCC.

Image analysis of the nuclear characteristics of emerin protein and the correlation with nuclear grooves and intranuclear cytoplasmic inclusions in lung adenocarcinoma.

Nuclear size and shape are important components in the diagnosis of pathological specimens. However, a qualitative evaluation is typically applied rather than a quantitative evaluation technique. In the present study, we sought to evaluate the nuclear morphological characteristics of lung adenocarcinoma using whole-slide imaging (WSI) and computer-assisted image analysis (IA). We evaluated the nuclear characteristics of 106 cases of surgically resected lung adenocarcinoma according to Feulgen staining and immunohistochemistry (IHC) for the inner nuclear membrane protein emerin. According to the Feulgen reaction, although the nuclear area (size) of the carcinoma cells was correlated with the nuclear perimeter (NP) (R=0.8973), the nuclear staining intensity of carcinoma cells was not correlated with the nuclear area. Using emerin IHC, we used IA software that was able to detect both the NP and the emerin-stained nuclear membrane length (ENML) in the nucleus, and found that the more nuclei exhibited a longer ENML relative to the NP, the more nuclear grooves and intranuclear cytoplasmic inclusions were present. In addition, the nuclear area was correlated with the percentage of nuclei that had a longer ENML compared to the NP against the total nuclei (R=0.7759). Furthermore, the emerin low expression group showed an enlarged nuclear area (P=0.0264), elongated NP (P=0.0091), and lower shape factor (P=0.0486) compared with the normal emerin expression group. Our data indicated the usefulness of WSI and IA for pathological specimen analysis. In addition, this study is the first to report that the low expression of emerin in cancer cell results in an oval shape of nuclei and nuclear enlargement in clinical samples.

Tumor necrosis factor-α-mediated hepatocyte apoptosis stimulates fibrosis in the steatotic liver in mice.

Hepatocyte apoptosis has been implicated in the progression of nonalcoholic steatohepatitis. However, it is unclear whether the induction of tumor necrosis factor (TNF)-α-mediated hepatocyte apoptosis in the simple fatty liver triggers liver fibrosis. To address this question, high-fat diet-fed mice were repeatedly administered D-galactosamine, which increases the sensitivity of hepatocytes to TNF-α-mediated apoptosis. In mice treated with a high-fat diet plus D-galactosamine, hepatocyte apoptosis and liver fibrosis were induced, whereas both apoptosis and fibrosis were inhibited in these mice following gut sterilization with antimicrobials or knockout of TNF-α. Furthermore, liver fibrosis was diminished when hepatocyte apoptosis was inhibited by expressing a constitutively active inhibitor of nuclear factor κB kinase subunit ß. Thus, hepatocyte apoptosis induced by intestinal dysbiosis or TNF-α up-regulation in the steatotic liver caused fibrosis. Organ fibrosis, including liver fibrosis, involves the interaction of cyclic adenosine monophosphate-response element-binding protein-binding protein (CBP) and ß-catenin. Here, hepatocyte-specific CBP-knockout mice showed reduced liver fibrosis accompanied by hepatocyte apoptosis diminution; notably, liver fibrosis was also decreased in mice in which CBP was specifically knocked out in collagen-producing cells because the activation of these cells was now suppressed. Conclusion: TNF-α-mediated hepatocyte apoptosis induced fibrosis in the steatotic liver, and inhibition of CBP/ß-catenin signaling attenuated the liver fibrosis due to the reduction of hepatocyte apoptosis and suppression of the activation of collagen-producing cells. Thus, targeting CBP/ß-catenin may represent a new therapeutic strategy for treating fibrosis in nonalcoholic steatohepatitis. (Hepatology Communications 2018;2:407-420).

Reduced therapeutic effect of antiviral drugs in patients with hepatitis B virus reactivation after hematopoietic stem cell transplantation.

AIM: Patients with resolved hepatitis B virus (HBV) infection following hematopoietic stem cell transplantation (HSCT) are potentially at high risk of HBV reactivation. Although antiviral drug therapy is recommended when HBV DNA reappears in the serum, drug efficacy after HBV reactivation remains unclear. METHODS: Host immune response against HBV was investigated by immunological analyses at 12 months after entecavir (ETV) treatment in six HSCT-treated and five non-HSCT-treated patients with HBV reactivation, and 18 patients with chronic hepatitis B (CHB). Peripheral HBV-specific CD8+ T cells were analyzed for total numbers by flow cytometry and tetramer staining, as was intracellular γ-interferon (IFN-γ) production and CD107a expression in response to HBV peptides. Interleukin-10 (IL-10)-expressing CD19+ B-cell count and serum inflammatory cytokine levels were also analyzed. RESULTS: Serum HBV DNA was detectable in HSCT-treated patients with HBV reactivation at 12 months compared with other groups, indicating insufficient ETV efficacy against HBV. The HBV-specific CD8+ T-cell counts in HSCT-treated patients with HBV reactivation were significantly lower compared with those in non-HSCT patients. Additionally, IFN-γ production and CD107a expression by CD8+ T cells after incubation with HBV peptides was significantly reduced in HSCT-treated compared with CHB patients at 12 months after ETV treatment. Conversely, HSCT-treated patient serum IL-10 levels were significantly elevated compared with those in non-HSCT patients. Finally, IL-10-producing CD19+ B-cell counts were increased in HSCT-treated compared with CHB patients. CONCLUSION: After HBV reactivation, ETV efficacy was impaired in HSCT-treated patients as evidenced by low HBV-specific CD8+ T-cell counts and high B-cell IL-10 production.

Safety, Tolerability, and Preliminary Efficacy of the Anti-Fibrotic Small Molecule PRI-724, a CBP/ß-Catenin Inhibitor, in Patients with Hepatitis C Virus-related Cirrhosis: A Single-Center, Open-Label, Dose Escalation Phase 1 Trial.

BACKGROUND: There is currently no anti-fibrotic drug therapy available to treat hepatitis C virus (HCV) cirrhosis. The aim of this study was to assess the safety, tolerability, and anti-fibrotic effect of PRI-724, a small-molecule modulator of Wnt signaling, in patients with HCV cirrhosis. METHODS: In this single-center, open-label, phase 1 trial, we sequentially enrolled patients with HCV cirrhosis who were classified as Child-Pugh (CP) class A or B. PRI-724 was administered as a continuous intravenous infusion of 10, 40, or 160mg/m2/day for six cycles of 1week on and 1week off. The primary endpoints were frequency and severity of adverse events. The secondary endpoint was efficacy of PRI-724 in treating cirrhosis based on CP score and liver biopsy. This study is registered with ClinicalTrials.gov (no. NCT02195440). FINDINGS: Between Sept 3, 2014 and May 2, 2016, 14 patients were enrolled: CP class A:B, 6:8; median age, 62 (range: 43 to 74) years; male:female, 10:4. Twelve of the 14 patients completed six cycles of treatment; one was withdrawn from the study due to possible study drug-related liver injury (grade 3) in the 160mg/m2/day dose cohort and one withdrew for personal reasons. Serious adverse events occurred in three patients [21% (3/14)], one of which was possibly related to PRI-724. The most common adverse events were nausea [29% (4/14)] and fatigue [21% (3/14)]. After PRI-724 administration, the CP scores worsened by 1 point in two patients in the 10mg/m2/day cohort, improved in three patients at 1, 1, and 2 points in the 40mg/m2/day cohort, and improved in one patient by 3 points in the 160mg/m2/day cohort. The histology activity index scores of the liver tissue improved in two patients and exacerbated in two patients in the 10mg/m2/day cohort, and improved in one patient in the 40mg/m2/day cohort. INTERPRETATION: This study showed that administration of 10 or 40mg/m2/day intravenous PRI-724 over 12weeks was well-tolerated by patients with HCV cirrhosis; however, liver injury as a possible related serious adverse event was observed in the 160mg/m2/day cohort. FUNDING SOURCE: AMED.