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Hepatocellular carcinoma (HCC) activates platelets through the action of adjacent sinusoidal cells. Activated platelets bind to tumor-associated endothelial cells and release growth factors that promote tumor progression. We hypothesized that platelets encapsulated with tumor inhibitors would function as drug carriers for tumor therapy. We propose a therapeutic strategy for HCC using autologous platelets encapsulating multiple tyrosine kinase inhibitors in a rat chemically induced HCC model. Sorafenib or lenvatinib was encapsulated in platelets isolated from tumor-bearing rats in vitro. The rats were divided into groups that received repeated intravenous injections (twice a week for 10 weeks) of the following materials: placebo, sorafenib (SOR), lenvatinib (LEN), autologous platelets, autologous platelets encapsulating sorafenib (SOR-PLT) and autologous platelets encapsulating lenvatinib (LEN-PLT). The therapeutic effect was then analyzed by ultrasonography (US) and histopathological analysis. Histopathological and US analysis demonstrated extensive tumor necrosis in the tumor tissue of SOR-PLT or LEN-PLT, but not in other experimental groups. By liquid chromatography-mass spectrometry, more abundant sorafenib was detected in tumor tissues after SOR-PLT administration than in surrounding normal tissues, but no such difference in sorafenib level was observed with SOR administration. Therefore, the use of autologous platelets encapsulating drugs might be a novel therapeutic strategy for HCC.

A 63-year-old man with hepatitis C was treated with atezolizumab plus bevacizumab for unresectable diffuse hepatocellular carcinoma (HCC). After four cycles of atezolizumab plus bevacizumab, the diffuse HCC markedly shrank; however, he complained of general fatigue, loss of appetite, and slight loss of muscle strength in the lower legs. He was diagnosed with isolated adrenocorticotropic hormone deficiency (IAD), hypothyroidism, and myopathy, suggesting multisystem immune-related adverse events (irAEs). After administration of hydrocortisone, the clinical symptoms rapidly disappeared. Patients with multisystem irAEs can have favorable outcomes; thus, to continue immune-checkpoint inhibitors therapy, a correct diagnosis and management of multisystem irAEs are important.

Background and aim Because of their survival benefits, immune checkpoint inhibitors (ICIs) are widely administered to patients with various advanced-stage malignancies. During ICI treatment, drug-induced liver injury (DILI) occasionally occurs. In particular, hepatic immune-related adverse events (irAEs) are rare but serious and fatal. In patients with hepatic irAEs, immediate steroid treatment is generally recommended; however, the risk factors for ICI-associated DILI remain unknown. In the present study, we identified a risk factor for ICI-associated DILI. Methods We retrospectively analyzed 135 patients treated with anti-programmed cell death-1 (PD-1) antibodies, such as nivolumab and pembrolizumab, at Asahikawa Medical University Hospital. We investigated grade ≥ 2 hepatotoxic AEs during anti-PD-1 therapy, and PD-1 inhibitor-associated DILI was then diagnosed according to the Digestive Disease Week Japan (DDW-J) 2004 scale. The risk factors for PD-1 inhibitor-associated DILI were identified by Cox hazard analysis. Results Thirty-six patients developed grade ≥ 2 hepatic AEs during anti-PD-1 therapy. Among them, eight patients were diagnosed with PD-1 inhibitor-associated DILI based on the DDW-J 2004 scale. Cox hazard analysis revealed that non-alcoholic fatty liver disease (NAFLD) was a risk factor for PD-1 inhibitor-associated DILI. In addition, we revealed that the outcomes of patients with the DDW-J 2004 score = 3 were improved without steroid treatment. Conclusions NAFLD is a potential risk factor for PD-1 inhibitor-associated DILI based on the DDW-J 2004 scale. The DDW-J 2004 scale might be useful for determining whether steroid treatment is required in patients with PD-1 inhibitor-associated DILI.

The patient was a 76-year-old man who was treated with nivolumab due to recurrent gastric cancer. A blood examination revealed grade 3 alkaline phosphatase (ALP) elevation. A histopathological examination revealed marked portal infiltration, including eosinophils and CD4+ and CD8+ T lymphocytes, suggesting nivolumab-related cholangitis accompanied by the features of both an immune-related adverse event (irAE) and drug-induced liver injury (DILI) with allergic reaction. The patient's ALP level immediately decreased after the administration of prednisolone. Although nivolumab-related cholangitis, a rare irAE, has been reported to be refractory to steroid therapy, patients with features of irAE and allergic DILI might immediately respond to prednisolone.

Background and aim Several reports have demonstrated that skeletal muscle mass influences mortality in patients with hepatocellular carcinoma (HCC) receiving sorafenib treatment; however, there is still controversy with regard to whether skeletal muscle and adipose tissue are associated with the prognosis in HCC patients. We examined the relationship between body composition and prognosis in HCC patients. Methods We retrospectively analyzed 82 patients with unresectable HCC receiving sorafenib treatment. The skeletal muscle area and adipose tissue area were measured by computed tomography. Patients with low skeletal muscle index (male ≤36.2 cm2/m2, female ≤29.6 cm2/m2) and high visceral to subcutaneous adipose tissue area ratio (VSR) (male ≥ 1.33, female ≥ 0.93) were diagnosed as low skeletal muscle mass (LSMM) and high VSR, respectively. Results A total of 16 and 34 patients were classified as LSMM and high VSR, respectively. LSMM patients frequently experienced serious adverse events (SAEs) and thus had a shorter duration of sorafenib treatment than non-LSMM patients. High VSR was a significant factor for progression-free survival. LSMM patients less frequently received additional/subsequent therapies combined with sorafenib than non-LSMM patients. Multivariate Cox hazard analysis demonstrated that LSMM was a significant factor for the duration of sorafenib treatment. The treatment duration and receiving of additional/subsequent therapies were significantly associated with overall survival (OS) but not with LSMM or high VSR. Conclusion LSMM was associated with the frequency of SAEs, treatment tolerability, and treatment duration. LSMM patients were less likely to receive additional/subsequent therapies than non-LSMM patients. Thus, LSMM could identify a subgroup of patients with poor OS.

Background IBD are chronic immune disorders of unclear etiology. TNF inhibitors are effective for IBD treatment but are much more expensive than traditional treatments. A large number of clinical trials of infliximab biosimilar (CT-P13) have suggested that the administration of biosimilars provides high efficacy and safety similar to that of the originators, with a lower cost, so switching from the originator to the biosimilar is recommended. While several abnormalities of blood examination have been observed in patients with CT-P13 administration, no cases of drug-induced liver injury (DILI) caused by CT-P13 has been reported. Case presentations: A 32-year-old woman had been diagnosed with Crohn’s disease and was treated with original infliximab (O-IFX) for 9 years. She developed severe jaundice one month after switching from O-IFX to CT-P13. Serologic tests of auto-immune and hepatitis viruses were negative, and ultrasonography, computed tomography and magnetic resonance cholangiopancreatography revealed no abnormalities. A liver biopsy showed prominent pericentral canalicular cholestasis, without features of steatosis or sclerosing cholangitis, which was consistent with drug-induced cholestasis. The cholestasis improved 10 weeks after the discontinuation of CT-P13, and no DILI redeveloped even after re-switching from CT-P13 to O-IFX. This is the first report of DILI due to switching from O-IFX to CT-P13. Conclusions This is the first report of DILI due to switching from O-IFX to CT-P13, and our case suggests the different immunogenicity between O-IFX and CT-P13. While the efficacy and safety of CT-P13 are considered equal to those of O-IFX, clinicians need to be alert for certain severe DILIs when switching from O-IFX to CT-P13 with careful monitoring and appropriate treatment.

Background and Aim Some single nucleotide polymorphisms (SNPs) are associated with the development of non-alcoholic fatty liver disease (NAFLD). As one of the genetic factors, PNPLA3 rs738409 (I148M) is important to associate with pathogenesis of NAFLD. Since other SNPs remain unclear in Japan, we performed high-throughput sequencing, which targeted more than 1,000 genes to identify a novel genetic variant in Japanese patients with NAFLD. Methods The present study in 36 NAFLD patients and 27 healthy volunteers (HVs) was performed. A high-throughput sequencer was used to detect the gene variations. Candidate genes were validated by TaqMan SNP genotyping assay in 53 NAFLD patients and 41 HVs. To investigate the function of candidate gene, biochemical analyses were performed in cultured hepatocytes and liver tissues. Results EXO1 rs1047840, PTPRD rs35929428, IFNAR2 rs2229207, CPOX rs1131857, IL23R rs1884444, IL10RA rs2228055, and FAM3B rs111988437 were identified as candidate genetic variants and PTPRD rs35929428 was only extracted as a SNP predicting to cause protein dysfunction. In validation analysis, PTPRD rs35929428 associated with the development of NAFLD (p=0.015, OR=5.00, 95%CI: 1.33-18.70). In addition, PTPRD rs35929428 was associated with Fib-4 index and with hepatic fat droplets. Biochemical analyses indicated that PTPRD rs35929428 promoted dephosphorylation of tyrosine 705 Signal Transducer and Activator of Transcription 3 (STAT3) (Tyr 705) in hepatocytes. Conclusion PTPRD rs35929428 was a novel SNP in patients with NAFLD. Through exacerbation of the dephosphorylation of STAT3 (Tyr 705) in hepatocytes, PTPRD rs35929428 might play a role in hepatic lipid accumulation and fibrosis, followed by the development of NAFLD.

Background Intraductal papillary neoplasm of the bile duct (IPNB) is a type of tumor that presents in the intra- or extrahepatic bile ducts. Cystic-type intrahepatic IPNB often mimics simple liver cysts, making the diagnosis difficult. Because the growth of IPNB is slow, careful follow-up and timely therapeutic intervention is recommended. There are few reports with a follow-up period longer than a decade; thus, we report the case of a patient with an IPNB that grew for over 13 years. Case summary A 65-year-old man was diagnosed, 13 years prior with a cystic hepatic tumor with abnormal imaging findings. The targeted tumor biopsy results showed no malignancy. Biannual follow-up examinations were performed because of the potential for malignancy. The cystic lesions showed gradual enlargement over 11 years and a 4 mm papillary proliferation appeared on the cyst wall, which is compatible with IPNB. The tumor was observed for another 2 years because of the patient's wishes. The imaging findings showed enlargement to 8 mm and a new 9 mm papillary proliferation of the cystic tumor. Contrast-enhanced ultrasonography showed hyperenhancement during the arterial phase in both cyst walls, indicating intraductal tumor progression in both tumors. Thus, liver segment 8 subsegmentectomy was performed. The pathological findings indicated that the tumors contained mucin, and high-grade atypia was observed in the papillary lesions, showing IPNB. Conclusion The development of IPNB should be monitored in patients with cystic lesions and ultrasonography are useful tool for the evaluation.

Chemopreventative properties of traditional medicines and underlying mechanisms of action are incompletely investigated. This study demonstrates that dietary daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper effectively suppresses intestinal tumor development and progression in the azoxymethane (AOM) and APCmin/+ mouse models. For the AOM model, TU-100 was provided after the first of six biweekly AOM injections. Mice were sacrificed at 30 weeks. APCmin/+ mice were fed diet without or with TU-100 starting at 6 weeks, and sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In APC min/+ mice, the number of small intestinal tumors was significantly decreased. In the AOM model, both TU-100 and Japanese ginseng decreased colon tumor numbers. Decreased Ki-67 and β-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression were observed. EGF receptor expression and stimulation/phosphorylation in vitro were investigated in C2BBe1 cells. TU-100, ginger, and 6-gingerol suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol inhibited EGF ERK1/2 activation. TU-100 and some of its components and metabolites of these components inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation. Copyright © 2016 John Wiley & Sons, Ltd.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/） 2016 Jun 30.Epub, BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is frequently accompanied by iron overload. However, because of the complex hepcidin-regulating molecules, the molecular mechanism underlying iron overload remains unknown. To identify the key molecule involved in NAFLD-associated iron dysregulation, we performed whole-RNA sequencing on the livers of obese mice. METHODS: Male C57BL/6 mice were fed a regular or high-fat diet for 16 or 48 weeks. Internal iron was evaluated by plasma iron, ferritin or hepatic iron content. Whole-RNA sequencing was performed by transcriptome analysis using semiconductor high-throughput sequencer. Mouse liver tissues or isolated hepatocytes and sinusoidal endothelial cells were used to assess the expression of iron-regulating molecules. RESULTS: Mice fed a high-fat diet for 16 weeks showed excess iron accumulation. Longer exposure to a high-fat diet increased hepatic fibrosis and intrahepatic iron accumulation. A pathway analysis of the sequencing data showed that several inflammatory pathways, including bone morphogenetic protein (BMP)-SMAD signaling, were significantly affected. Sequencing analysis showed 2314 altered genes, including decreased mRNA expression of the hepcidin-coding gene Hamp. Hepcidin protein expression and SMAD phosphorylation, which induces Hamp, were found to be reduced. The expression of BMP-binding endothelial regulator (BMPER), which inhibits BMP-SMAD signaling by binding BMP extracellularly, was up-regulated in fatty livers. In addition, immunohistochemical and cell isolation analyses showed that BMPER was primarily expressed in the liver sinusoidal endothelial cells (LSECs) rather than hepatocytes. CONCLUSIONS: BMPER secretion by LSECs inhibits BMP-SMAD signaling in hepatocytes and further reduces hepcidin protein expression. These intrahepatic molecular interactions suggest a novel molecular basis of iron overload in NAFLD.

Some single-nucleotide polymorphisms (SNPs) are associated with the development of non-alcoholic fatty liver disease (NAFLD). As one of the genetic factors, PNPLA3 rs738409 (I148M) is important to associate with pathogenesis of NAFLD. Because other SNPs remain unclear in Japan, we performed a high-throughput sequencing that targeted more than 1000 genes to identify a novel genetic variant in Japanese patients with NAFLD.The present study in 36 NAFLD patients and 27 healthy volunteers was performed. A high-throughput sequencer was used to detect the gene variations. Candidate genes were validated by TaqMan SNP genotyping assay in 53 NAFLD patients and 41 healthy volunteers. To investigate the function of candidate gene, we performed biochemical analyses in cultured hepatocytes and liver tissues.EXO1 rs1047840, PTPRD rs35929428, IFNAR2 rs2229207, CPOX rs1131857, IL23R rs1884444, IL10RA rs2228055, and FAM3B rs111988437 were identified as candidate genetic variants, and PTPRD rs35929428 was only extracted as a SNP predicting to cause protein dysfunction. In validation analysis, PTPRD rs35929428 associated with the development of NAFLD (P = 0.015, odds ratio = 5.00, 95% confidence interval: 1.33-18.70). In addition, PTPRD rs35929428 was associated with Fib-4 index and with hepatic fat droplets. Biochemical analyses indicated that PTPRD rs35929428 promoted dephosphorylation of tyrosine 705 signal transducer and activator of transcription 3 (Tyr 705) in hepatocytes.PTPRD rs35929428 was a novel SNP in patients with NAFLD. Through exacerbation of the dephosphorylation of signal transducer and activator of transcription 3 (Tyr 705) in hepatocytes, PTPRD rs35929428 might play a role in hepatic lipid accumulation and fibrosis, followed by the development of NAFLD.

Tegafur-uracil has been reported to have only minor adverse effects and is associated with liver injury in 1.79% of Japanese patients. The development of tegafur-uracil-induced hepatic fibrosis with portal hypertension is rare. Here, we report a case of a 74-year-old woman with rapidly developing tegafur-uracil-induced hepatic fibrosis. The patient had no history of liver disease and had been treated with tegafur-uracil for 8 mo after breast cancer surgery. The patient was admitted to our hospital for abdominal distension and leg edema associated with liver dysfunction. Computed tomography imaging revealed massive ascites and splenomegaly, and a non-invasive assessment of liver fibrosis indicated advanced fibrosis. The histopathological findings revealed periportal fibrosis and bridging fibrosis with septation. The massive ascites resolved after discontinuing tegafur-uracil. These findings suggest that advanced hepatic fibrosis can develop from a relatively short-term administration of tegafur-uracil and that non-invasive assessment is useful for predicting hepatic fibrosis.

Platelets can activate hepatocyte proliferation not only via releasing the platelet factors, but also promoting for liver sinusoidal endothelial cells (LSEC) and Kupffer cells to produce the factors such as IL-6 and TGF-α that are indispensable for hepatocyte proliferation. We previously reported that in the transgenic (TG) mice with liver specific human BRAFV600E mutation, which corresponds to the mouse BrafV637E that is highly prevalent in mouse DEN-induced hepatic tumors, exhibit increased liver weight five times larger than that of normal mice. Furthermore, hepatocytes in these mice overexpress thrombopoietin (TPO) in association with megakaryocytosis/thrombocytosis, and large numbers of platelets were deposited in the liver sinusoids. In the present study, we investigated whether platelets are deposited in liver sinusoids in hepatic tumors in human and mice. CD61 immunohistochemical staining revealed the increased numbers of platelets were deposited in the sinusoids in foci, adenomas and hepatocellular carcinomas (HCC) induced by neonatal treatment with DEN compared to surrounding normal hepatic tissues. Moreover, increased numbers of platelets were deposited in sinusoids of dysplastic nodules and well-differentiated HCC compared to normal liver tissue and cirrhotic nodules in human. In addition, we evaluated the correlation between the tumor diameters and peripheral platelets numbers in the patients who were primarily diagnosed as HCC in our institute. The result indicated that the numbers of peripheral platelets are positively correlated with the tumor diameters. These results indicate that platelets contribute to development of HCC via interacting sinusoidal cells. Citation Format: Hiroki Tanaka, Kie Horioka, Masahiro Yamamoto, Koji Sawada, Takumu Hasebe, Kosuke Yamazaki, Katsuhiro Ogawa. Deposition of platelets in sinusoids and its contribution to hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5172.

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.

Chemopreventative properties of traditional medicines, Kampo and underlying mechanisms of action have not been well explored. This study demonstrates that daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper (50/30/20 by weight), effectively suppresses development and progression of intestinal tumors in the azoxymethane (AOM) and the APCmin/+ mouse models. TU-100 was included in the diet. TU-100 was provided after first of 6, biweekly AOM injections, mice sacrificed at 30 weeks. APCmin/+ mice were fed without or with TU-100 starting at 6 weeks, sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In the APC min/+ model, the number of small intestinal tumors was also significantly decreased, whereas in the AOM model, both TU-100 and Japanese ginseng decreased tumor numbers. TU-100 and ginseng decreased Ki67 and β-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression. EGF receptor expression and stimulation/phosphorylation in vitro was investigated in Caco2BBE cells. TU-100, ginger, and 6-gingerol, but not ginseng, ginsenoside Rb1 and the bacterial metabolite compound K, nor Japanese pepper suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol, but not Japanese pepper, ginsenoside Rb1 or compound K inhibited EGF activation of ERK1/2. In conclusion, TU-100 and some of its components and metabolites inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation.

Many pharmaceutical agents not only require microbial metabolism for increased bioavailability and bioactivity, but also have direct effects on gut microbial assemblage and function. We examined the possibility that these actions are not mutually exclusive and may be mutually reinforcing in ways that enhance long‐term of these agents. Daikenchuto, TU‐100, is a traditional Japanese medicine containing ginseng. Conversion of the ginsenoside Rb1 (Rb1) to bioactive compound K (CK) requires bacterial metabolism. Diet‐incorporated TU‐100 was administered to mice over a period of several weeks. T‐RFLP and 454 pyrosequencing were performed to analyze the time‐dependent effects on fecal microbial membership. Fecal microbial capacity to metabolize Rb1 to CK was measured by adding TU‐100 or ginseng to stool samples to assess the generation of bioactive metabolites. Levels of metabolized TU‐100 components in plasma and in stool samples were measured by LC‐MS/MS. Cecal and stool short‐chain fatty acids were measured by GC‐MS. Dietary administration of TU‐100 for 28 days altered the gut microbiota, increasing several bacteria genera including members of Clostridia and Lactococcus lactis. Progressive capacity of microbiota to convert Rb1 to CK was observed over the 28 days administration of dietary TU‐100. Concomitantly with these changes, increases in all SCFA were observed in cecal contents and in acetate and butyrate content of the stool. Chronic consumption of dietary TU‐100 promotes changes in gut microbiota enhancing metabolic capacity of TU‐100 and increased bioavailability. We believe these findings have broad implications in optimizing the efficacy of natural compounds that depend on microbial bioconversion in general.