Treatment of Retinoblastoma 1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6 Inhibitor Combination Therapy.

BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.

Non-occlusive mesenteric ischemia during bevacizumab treatment for glioblastoma: a case report.

Glioblastoma is one of the most aggressive brain tumors in adults. The standard treatment is radiotherapy and chemotherapy based on the Stupp regimen after maximal safe resection. One effective chemotherapeutic drug is bevacizumab, which can prolong progression-free survival in glioblastoma patients but not overall survival. Adverse events of bevacizumab include hypertension, proteinuria, delayed wound healing, bleeding of the nose and gums, and thromboembolism resulting in gastrointestinal perforation. Herein, we describe an autopsy case of a patient with glioblastoma who died from non-occlusive mesenteric ischemia that was presumably caused by bevacizumab.

Diversity in cell differentiation, histology, phenotype and vasculature of mass-forming intrahepatic cholangiocarcinomas.

AIMS: Mass-forming intrahepatic cholangiocarcinomas (MF-iCCAs), involving small bile ducts, bile ductules or canals of Hering, remain treated as a single entity. We aimed to examine the diversity in histology, phenotype and tumour vasculature of MF-iCCAs. METHODS AND RESULTS: Based on morphology and immunophenotype, we classified MF-iCCAs into small bile duct (SBD), cholangiolocarcinoma (CLC), ductal plate malformation (DPM) and hepatocellular carcinoma (HCC)-like subtypes. Genetic correlations among the histological subtypes were examined by multi-region tumour sequencing. Vasculatures and other clinicopathological features were compared among tumour groups with various proportions of the histological subtypes in 62 MF-iCCAs. Cases of pure SBD, CLC, DPM and HCC-like subtypes numbered 18 (29%), seven (11.3%), none (0%) and two (3%), respectively; the remaining 35 (56.4%) cases comprised several components. Genetic alterations, isocitrate dehydrogenase (IDH)1/2, KRAS, TP53, polybromo-1 (PBRM1) and BRCA1-associated protein 1 (BAP1), were shared among SBD, CLC, DPM and hepatoid components within a tumour. We uncovered distinct vascularisation mechanisms among SBD, CLC and DPM subtypes with a prominent vessel co-option in CLC tumours. iCCA with a DPM pattern had the highest vascular densities (mean microvascular density,140/mm2 ; arterial vessel density, 18.3/mm2 ). Increased CLC component was correlated with longer overall survival time (r = 0.44, P = 0.006). Pure SBD tumours had a lower 5-year overall survival rate compared with MF-iCCA with CLC pattern (30.5 versus 72.4%, P = 0.011). CONCLUSIONS: MF-iCCAs comprise four histological subtypes. Given their sharing some driver gene alterations, indicating they can have a common cell origin, SBD, CLC and DPM subtypes, however, differ in cell differentiation, histology, phenotype or tumour vasculature.

Notch-Hes1 signaling activation in Caroli disease and polycystic liver disease.

The Notch signaling pathway plays a key role in the morphogenesis of the biliary tree, but its involvement in cystic biliary diseases, such as Caroli disease (CD) and polycystic liver disease (PLD), has yet to be determined. Immunostaining was performed using liver sections of CD and PLD, and the results were compared with those of congenital hepatic fibrosis (CHF) and von Meyenburg complex (VMC). The expression of Notch receptor 1 (Notch1) was increased in the nuclei of biliary epithelial cells in all cases of CD and PLD, whereas it remained at a low level in CHF and VMC. In addition, Notch2 and Notch3 were preferably expressed in the nuclei of biliary epithelial cells of PLD. Accordingly, the Notch effector Hes1 was highly expressed in biliary epithelial cells of CD and PLD, and the cell proliferative activity was significantly higher in CD and PLD. The expression of the Notch ligand Delta-like 1 was significantly increased in biliary epithelial cells of CD and PLD, which may be causally associated with the nuclear overexpression of Notch1 and Hes1. These results indicate that aberrant activation of the Notch-Hes1 signaling pathway may be responsible for the progression of biliary cystogenesis in CD and PLD.

Blockade of Hedgehog Signaling Attenuates Biliary Cystogenesis in the Polycystic Kidney (PCK) Rat.

Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease.

The PD-1/PD-L1 axis may be aberrantly activated in occupational cholangiocarcinoma.

An outbreak of cholangiocarcinoma in a printing company was reported in Japan, and these cases were regarded as an occupational disease (occupational cholangiocarcinoma). This study examined the expression status of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) in occupational cholangiocarcinoma. Immunostaining of PD-1, PD-L1, CD3, CD8, and CD163 was performed using tissue sections of occupational cholangiocarcinoma (n = 10), and the results were compared with those of control cases consisting of intrahepatic (n = 23) and extrahepatic (n = 45) cholangiocarcinoma. Carcinoma cells expressed PD-L1 in all cases of occupational cholangiocarcinoma, whereas the detection of PD-L1 expression in cholangiocarcinoma cells was limited to a low number of cases (less than 10%) in the control subjects. In cases of occupational cholangiocarcinoma, occasional PD-L1 expression was also noted in precancerous/preinvasive lesions such as biliary intraepithelial neoplasia and intraductal papillary neoplasm of the bile duct. Additionally, tumor-associated macrophages and tumor-infiltrating T cells expressed PD-L1 and PD-1, respectively. The number of PD-L1-positive mononuclear cells, PD-1-positive lymphocytes, and CD8-positive lymphocytes infiltrating within the tumor was significantly higher in occupational cholangiocarcinoma compared with that in control cases. These results indicate that immune escape via the PD-1/PD-L1 axis may be occurring in occupational cholangiocarcinoma.

Case report: pericardial effusion with constrictive physiology in a patient with wet beriberi.

Wet beriberi-induced pericardial effusion has rarely been previously described. Little is known about the effect of beriberi-induced pericardial effusion on hemodynamics. Here we present a case of wet beriberi with pericardial effusion that exhibited constrictive physiology, which was dramatically improved after treatment. A 61-year-old male patient was admitted to our hospital for progressive leg edema, dyspnea on exertion, and lower-extremity muscle weakness. Echocardiography showed a hyperkinetic left ventricle and a moderate amount of pericardial effusion. Hemodynamic measurements, including simultaneous measurement of left and right ventricular pressures, revealed high output heart failure and constrictive physiology. Blood test showed lactic acidosis, and low level of serum thiamine levels; consistent with a diagnosis of wet beriberi. After thiamine replacement therapy, the patient's hemodynamic state rapidly improved. Additionally, pericardial effusion decreased and constrictive physiology was successfully resolved. No other possible causes of pericardial effusion could be identified, with the exception of thiamine deficiency. This case illustrates the importance of considering wet beriberi as a possible cause of pericardial effusion with constrictive physiology.

Hilar cholangiocarcinoma with extensive immunoglobulin G4 reaction.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a systemic fibroinflammatory disorder that can involve multiple organs. It is often challenging to distinguish IgG4-related sclerosing cholangitis (IgG4-SC) from cholangiocarcinoma because of overlap in their clinical findings. A 75-year-old man presented to a hospital for a detailed examination of the elevation of some biliary enzymes. Radiographic examination revealed segmental bile duct with wall thickening of the common hepatic bile duct, and dilation of the peripheral branches. Transampullary biopsy showed a non-specific inflammatory reaction with several IgG4-positive cell infiltrations. There were no signs of malignancy. The liver biopsy showed bile duct injury accompanied by IgG4-positive cell infiltration. We then performed bile duct biopsy and finally diagnosed the patient with cholangiocarcinoma. We should remember that the IgG4 reaction is neither completely sensitive nor specific for IgG4-RD and avoid resting solely on the IgG4 reaction to precisely distinguish IgG4-SC from cholangiocarcinoma.

The cyclin­dependent kinase pathway involving CDK1 is a potential therapeutic target for cholangiocarcinoma.

Cholangiocarcinoma is an aggressive malignancy with high mortality, and effective therapeutic agents for this cancer are limited. Cyclin­dependent kinase (CDK) pathways are therapeutic targets for various types of cancers; however, their involvement in cholangiocarcinoma remains unclear. The present study examined the biological significance of CDK pathways in cholangiocarcinoma. An immunohistochemical analysis of cholangiocarcinoma tissue sections revealed the upregulated expression of phosphorylated cyclin­dependent kinase 1 (p­CDK1), p­CDK2, cyclin B1, and cyclin E1 in carcinoma cells. The nuclear expression of p­CDK1 and cyclin B1 was positively correlated with the presence of lymph node metastasis and the clinical stage, and p­CDK1 expression was also associated with poor patient survival. The treatment of human cholangiocarcinoma cell lines (CCKS­1, TFK­1 and HUCCT­1) with the multi­CDK inhibitor roscovitine decreased p­CDK1 expression, inhibited cell proliferation, arrested the cell cycle at the G1 or G2/M phase, and significantly inhibited carcinoma cell invasion. In vivo studies using a murine xenograft model revealed that an intraperitoneal injection of roscovitine significantly inhibited cholangiocarcinoma cell growth. Roscovitine induced apoptosis in cholangiocarcinoma cells in vitro and in vivo. These results demonstrated the potential of the CDK pathway involving CDK1 as a therapeutic target for cholangiocarcinoma. Furthermore, the immunohistochemical expression of p­CDK1 may be a useful prognostic marker of cholangiocarcinoma.

Immunosuppressive tumor microenvironment in occupational cholangiocarcinoma: Supportive evidence for the efficacy of immune checkpoint inhibitor therapy.

BACKGROUND: Occupational cholangiocarcinoma (CCA) was first described in patients who were working at a printing company in Osaka, Japan. Recently, the therapeutic efficacy and safety of a PD1 inhibitor nivolumab are being evaluated in patients with occupational CCA in an investigator-initiated clinical trial. The therapeutic effects of immune checkpoint inhibitors are closely associated with immune cells. METHODS: Immunohistochemical analysis was performed to characterize immune cells in the tumor microenvironment of occupational CCA. The status of mismatch repair (MMR)/microsatellite instability (MSI) was also examined. RESULTS: The tumor stroma of occupational CCA was characterized by abundant infiltration of immune cells expressing CD3, CD4, CD8, CD163, FOXP3, and granzyme B. Additionally, lymphocytes expressing immune checkpoint receptors, such as PD1, CTLA4, LAG3, TIM3, and TIGIT, were frequently infiltrated. The loss of immunohistochemical expression of the MMR proteins (MLH1, MSH2, PMS2, and MSH6) was not observed in cases of occupational CCA, and MSI was not detected. CONCLUSIONS: The tumor microenvironment of occupational CCA had features of immunosuppression, and the occurrence of T-cell dysfunction or exhaustion was suggested. The results provide supportive evidence for the efficacy of immune checkpoint inhibitor therapy for this disease.