Takahiro Okuno

Anti-PD-L1 treatment enhances antitumor effect of everolimus in a mouse model of renal cell carcinoma.

Immunotherapy based on blockade of the programmed death‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) axis has shown promising clinical activity for renal cell carcinoma (RCC) patients; however, the most effective use of these agents in combination with conventional targeted therapy remains to be resolved. Here we evaluated the therapeutic efficacy of the combination of the mTOR inhibitor everolimus (EVE) and anti‐PD‐L1 using an immunocompetent mouse model of RCC. We first assessed the in vitro effect of EVE on PD‐L1 expression in the human 786‐O and mouse RENCA RCC cell lines and found that EVE upregulated PD‐L1 expression in these RCC cell lines. We then treated RENCA tumor‐bearing mice with EVE and found that PD‐L1 expression was also increased in tumor cells after EVE treatment. To determine the antitumor effects of EVE alone, anti‐PD‐L1 alone, and EVE in combination with anti‐PD‐L1, we evaluated their antitumor effects on RENCA tumor‐bearing mice. A significant decrease in the tumor burden was observed in the EVE alone but not in the anti‐PD‐L1 alone treatment group compared with the control group. Importantly, the combination of EVE with anti‐PD‐L1 significantly reduced tumor burden compared with the EVE alone treatment, increasing tumor infiltrating lymphocytes (TILs) and the ratio of cytotoxic CD8+ T cells to TILs. The results of the present study demonstrated that anti‐PD‐L1 treatment enhanced the antitumor effect of EVE in a mouse model, supporting a direct translation of this combination strategy to the clinic for the treatment of RCC.

B cells in tertiary lymphoid structures are associated with favorable prognosis in gastric cancer

BACKGROUND The role of tumor-infiltrating B cells in the tumor microenvironment is still unclear. Recent studies have reported that B cells and tertiary lymphoid structures (TLSs) that contain B cell follicles correlate with the favorable prognosis of cancer patients. The aim of this study was to investigate the association between tumor-infiltrating B cells and clinicopathological features in gastric cancer. METHODS Tumor blocks were obtained from 226 patients with stage Ib to stage IV gastric cancer. The density of CD20+ B cells within the tumor and in the invasive margin area was assessed using immunohistochemistry. We also evaluated CD3+ T cells, CD21+ follicular dendritic cells, Bcl6+ germinal center B cells, and PNAd+ high endothelial venules to show the presence of TLSs. RESULTS Tumor-infiltrating B cells were mostly organized as clusters that were surrounded by CD3+ T cells. The B cell area contained follicular dendritic cells and some clusters contained Bcl6+ B cells. High endothelial venules were present around follicles. We identified these follicles as TLSs. A high number of CD20+ B cells were associated with significantly better overall survival, and multivariate analysis also showed that CD20 high was one of the independent predictors of prognosis. In addition, there was a significant correlation between CD20+ B cell and CD8+ T cell infiltration. CONCLUSIONS B cells mostly infiltrated tumors as TLSs and were associated with better prognosis in patients with gastric cancer.

Proteome Characteristics of Non-Alcoholic Steatohepatitis Liver Tissue and Associated Hepatocellular Carcinomas

To uncover mechanisms of nonalcoholic steatohepatitis (NASH) associated hepatocarcinogenesis, we compared the proteomes of human NASH-associated liver biopsies, resected hepatocellular carcinomas (HCCs) and HCCs of HCV+ patients with normal liver tissue of patients with gastrointestinal tumor metastasis, in formalin-fixed paraffin-embedded samples obtained after surgery in our hospital during the period from 2006 to 2011. In addition, proteome analysis of liver tumors in male STAM NASH-model mice was performed. Similar changes in the proteome spectrum such as overexpression of enzymes involved in lipid, cholesterol and bile acid biosynthesis and examples associated with suppression of fatty acid oxidation and catabolism, alcohol metabolism, mitochondrial function as well as low expression levels of cytokeratins 8 and 18 were observed in both human NASH biopsies and NASH HCCs, but not HCV+ HCCs. Alterations in downstream protein expression pointed to significant activation of transforming growth factor β, SMAD family member 3, β-catenin, Nrf2, SREBP-LXRα and nuclear receptor-interacting protein 1 (NRIP1), and inhibition of PPARs and p53 in human NASH biopsies and/or HCCs, suggesting their involvement in accumulation of lipids, development of fibrosis, oxidative stress, cell proliferation and suppression of apoptosis in NASH hepatocarcinogenesis. In STAM mice, PPARs inhibition was not obvious, while expression of cytokeratins 8 and 18 was elevated, indicative of essential differences between human and mouse NASH pathogenesis.

Enhanced Susceptibility of Ogg1 Mutant Mice to Multiorgan Carcinogenesis

The role of deficiency of oxoguanine glycosylase 1 (Ogg1) Mmh homolog, a repair enzyme of the 8-hydroxy-2’-deoxyguanosine (8-OHdG) residue in DNA, was investigated using the multiorgan carcinogenesis bioassay in mice. A total of 80 male and female six-week-old mice of C57BL/6J background carrying a mutant Mmh allele of the Mmh/Ogg1 gene (Ogg1−/−) and wild type (Ogg1+/+) mice were administered N-diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), N-bis (2-hydroxypropyl) nitrosamine (DHPN) and 1,2-dimethylhydrazine dihydrochloride (DMH) (DMBDD) to induce carcinogenesis in multiple organs, and observed up to 34 weeks. Significant increase of lung adenocarcinomas incidence was observed in DMBDD-treated Ogg1−/− male mice, but not in DMBDD-administered Ogg1+/+ animals. Furthermore, incidences of lung adenomas were significantly elevated in both Ogg1−/− males and females as compared with respective Ogg1−/− control and DMBDD-treated Ogg1+/+ groups. Incidence of total liver tumors (hepatocellular adenomas, hemangiomas and hemangiosarcomas) was significantly higher in the DMBDD-administered Ogg1−/− males and females. In addition, in DMBDD-treated male Ogg1−/− mice, incidences of colon adenomas and total colon tumors showed a trend and a significant increase, respectively, along with significant rise in incidence of simple hyperplasia of the urinary bladder, and a trend to increase for renal tubules hyperplasia in the kidney. Furthermore, incidence of squamous cell hyperplasia in the forestomach of DMBDD-treated Ogg1−/− male mice was significantly higher than that of Ogg1+/+ males. Incidence of small intestine adenomas in DMBDD Ogg1−/− groups showed a trend for increase, as compared to the wild type mice. The current results demonstrated increased susceptibility of Ogg1 mutant mice to the multiorgan carcinogenesis induced by DMBDD. The present bioassay could become a useful tool to examine the influence of various targets on mouse carcinogenesis.

Progression of Hepatic Adenoma to Carcinoma in Ogg1 Mutant Mice Induced by Phenobarbital

The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1) responsible for the repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Mmh homozygous mutant (Ogg1−/−) and wild-type (Ogg1+/+) male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs) were found in PB-treated Ogg1−/− mice, while Ogg1+/+ animals developed only hepatocellular adenomas (HCAs) at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1−/− mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1−/− mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1−/− and Ogg1+/+ animals. Treatment of Ogg1−/− mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1−/− mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

Clinicopathological features of neuroblastic tumors with opsoclonus-myoclonus-ataxia syndrome: Follicular structure predicts a better neurological outcome

Neuroblastic tumors (NT) with opsoclonus‐myoclonus syndrome (OMS) display characteristic histological features, such as lymphocytic infiltration with lymphoid follicles, indicating an underlying immune response. We retrospectively assessed NT patients from 2001 to 2016. Five cases of NT with OMS and 76 cases of NT without OMS were histopathologically reviewed in this study. The grade of lymphocytic infiltration was evaluated. The number of follicles was counted and the presence or absence of lymphoid follicles was recorded for each case. We also confirmed the presence or absence of follicular dendritic cells (FDCs). We investigated the relationship between the histopathological and clinical findings of NT with OMS. Lymphocytic infiltration was observed in all cases; however, the precise follicular structure was occasionally unclear. Patients with clear follicular structures displayed germinal centers including tingible body macrophages and FDCs. All patients without neurological sequelae demonstrated a clear follicular structure with a FDC meshwork pattern. The interval between OMS onset and the detection and initial treatment of NT was typically longer in patients with neurological sequelae compared to those without neurological sequelae. Early detection and treatment of NT with OMS at the phase of a clear follicular formation with multiple FDC may provide favorable neurological outcomes.

A carcinogenicity study of diphenylarsinic acid in F344 rats in drinking water for 104 weeks

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical used as a chemical warfare agent, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. We previously demonstrated that DPAA promotes diethylnitrosamine-induced liver carcinogenesis in a medium-term rat liver bioassay. The purpose of the present study was to evaluate the potential carcinogenicity of DPAA, including investigation of whether the bile duct hyperplasia in the liver that was observed in a previous 52 week rat chronic study develops into a tumor, when administered to rats in their drinking water for 104 weeks. DPAA was administered to groups 1-4 at concentrations of 0, 5, 10, and 20 ppm in their drinking water for 104 weeks. A significant decrease in survival rate was found for females in the 20 ppm DPAA group. Body weights of males in the 20 ppm and females in the 10 and 20 ppm DPAA groups were significantly decreased compared to the controls. Overall histopathological evaluation of neoplasms in all tissues showed no significant increase of tumor incidence in any organ or tissue of the 5, 10, or 20 ppm DPAA-treated male or female F344 rats. In conclusion, the present study demonstrated that DPAA is not a complete carcinogen in male or female F344 rats.