Papers - Kurebayashi, Yutaka

Comparative Immunohistochemical Analysis of Clinicopathological Subgroups in Hepatocellular Carcinomas from Japan and Indonesia

Effendi K., Rahadiani N., Stephanie M., Kurebayashi Y., Tsujikawa H., Jasirwan C.O.M., Syaiful R.A., Sakamoto M.

Journal of Clinical and Experimental Hepatology (Journal of Clinical and Experimental Hepatology)  14 ( 6 ) 101451 2024.11

ISSN  09736883

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Background: Standardized pathological evaluation based on immunohistochemical (IHC) analysis could improve hepatocellular carcinoma (HCC) diagnoses worldwide. We evaluated differences in clinicopathological subgroups in HCCs from two academic institutions in Tokyo-Japan, and Jakarta-Indonesia. Methods: Clinicopathological parameters and molecular expression patterns were evaluated in 35 HCCs from Indonesia and 41 HCCs from Japan. IHC analysis of biliary/stem cell (B/S) markers (cytokeratin 19, sal-like protein 4, epithelial cell adhesion molecule) and Wnt/β-catenin (W/B) signaling-related molecules (β-catenin, glutamine synthetase) could determine the IHC-based subgroups. For immuno-subtypes categorization, CD3/CD79α double immunohistochemistry was done to evaluate the infiltration of T and B cells. CD34 staining allowed identification of vessels that encapsulated tumor clusters (VETC). Results: Indonesian HCC patients were mostly <60 years old (66%) with a hepatitis B virus (HBV) background (82%), in contrast to Japanese HCC patients (8% and 19%, respectively, both P < 0.001). In comparison with Japanese, Indonesian cases more frequently had >5 cm tumor size (74% vs 23%, P = 0.001), poor differentiation (40% vs 24%), portal vein invasion (80% vs 61%), and α-fetoprotein levels >500 ng/ml (45% vs 13%, P = 0.005). No significant differences were found in the proportions of B/S, W/B, and −/− subgroups from both countries. No immune-high tumors were observed among Indonesian cases, and immune-low tumors (66%) were more common than in Japanese cases (54%). VETC-positive tumors in Indonesia were significantly more common (29%), and most were in the HBV (90%) and −/− subgroups (90%), whereas Japanese VETC cases (10%, P = 0.030) were nonviral (100%) and W/B subgroups (75%). Conclusion: IHC-based analysis more precisely reflected the clinicopathological differences of HCCs in Japan and Indonesia. These findings provide new insights into standardization attempts and HCC heterogeneity among countries.

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Spatial Dynamics of T and B Cell Response Predicts Clinical Outcome of Resectable and Unresectable Hepatocellular Carcinoma.

Kurebayashi Y, Sugimoto K, Tsujikawa H, Matsuda K, Nomura R, Ueno A, Masugi Y, Yamazaki K, Effendi K, Takeuchi H, Itoi T, Hasegawa Y, Abe Y, Kitago M, Ojima H, Sakamoto M

Clinical cancer research : an official journal of the American Association for Cancer Research  2024.10

ISSN  1078-0432

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Glomus Tumor of Intrathoracic Chest Wall: a Case Report

Ishiguro Y., Masai K., Sugino K., Yano K., Nomura R., Okubo Y., Kaseda K., Hishida T., Asakura K., Kurebayashi Y., Minami Y.

Japanese Journal of Lung Cancer (Japanese Journal of Lung Cancer)  64 ( 3 ) 208 - 209 2024.06

ISSN  03869628

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Tumor steatosis and glutamine synthetase expression in patients with advanced hepatocellular carcinoma receiving atezolizumab plus bevacizumab therapy.

Kurebayashi Y, Tsujikawa H, Sugimoto K, Yunaiyama D, Araki Y, Saito K, Takahashi H, Kakegawa T, Wada T, Tomita Y, Abe M, Yoshimasu Y, Takeuchi H, Hirata T, Sakamaki K, Kakimi K, Nagao T, Itoi T, Sakamoto M

Hepatology research : the official journal of the Japan Society of Hepatology (Hepatology Research)  53 ( 10 ) 1008 - 1020 2023.10

ISSN  1386-6346

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Aim: The anti-programmed death-ligand 1 antibody atezolizumab and vascular endothelial growth factor-neutralizing antibody bevacizumab in combination (Atezo + Bev) have become the first-line therapy in advanced hepatocellular carcinoma (HCC). Distinct types of tumor immune microenvironment (TIME) and their associations with specific molecular subclasses and driver gene mutations have been identified in HCC; however, these insights are mainly based on surgically resected early-stage tumors. The current study aimed to reveal the biology and TIME of advanced HCC and their significance in predicting clinical outcomes of Atezo + Bev therapy. Methods: Thirty-three patients with advanced HCC who were scheduled for treatment with Atezo + Bev therapy were included in this study. Pretreatment tumor biopsy, pre- and posttreatment diffusion-weighted magnetic resonance imaging (MRI) with nine b values (0–1500 s/mm2), and other clinicopathologic factors were analyzed. Results: Compared with resectable HCC, advanced HCC was characterized by higher proliferative activity, a higher frequency of Wnt/β-catenin-activated HCC, and lower lymphocytic infiltration. Prognostically, two metabolism-related factors, histopathologically determined tumor steatosis and/or glutamine synthetase (GS) expression, and MRI-determined tumor steatosis, were the most significant prognostic indicators for progression-free survival (PFS) and overall survival after Atezo + Bev therapy. Furthermore, changes in the pre- and posttreatment true diffusion coefficients on MRI, which might reflect changes in TIME after treatment, were significantly associated with better PFS. Conclusions: The biology and TIME of HCC were strikingly different in advanced HCC compared with those of surgically resected HCC. Two metabolism-related factors, pathologically determined tumor steatosis and/or GS expression, and MRI-determined tumor steatosis, were found to be the most significant prognostic indicators for Atezo + Bev therapy in advanced HCC.

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IL-15-producing splenic B cells play pathogenic roles in the development of autoimmune hepatitis.

Fujimori S, Chu PS, Teratani T, Harada Y, Suzuki T, Amiya T, Taniki N, Kasuga R, Mikami Y, Koda Y, Ichikawa M, Tabuchi T, Morikawa R, Yamataka K, Noguchi F, Tsujikawa H, Kurebayashi Y, Sakamoto M, Kanai T, Nakamoto N

JHEP reports : innovation in hepatology (JHEP Reports)  5 ( 7 ) 100757 2023.07

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Background & Aims: B-cell depletion therapy with an anti-CD20 is an effective treatment strategy for patients with refractory autoimmune hepatitis (AIH). However, the mechanisms underlying B-cell action are unclear. Methods: Herein, we used the adeno-associated virus IL-12 model, in which hepatic IL-12 expression triggers liver injuries characteristic of AIH. We also analysed the clinical samples of patients with AIH. Results: B-cell depletion using anti-CD20 or splenectomy was found to improve liver functions and decrease the cytotoxic CD8+ T-cell (cytotoxic T lymphocyte [CTL]) count in the liver. This improvement was reversed by the adoptive transfer of splenic B cells derived from AAV IL-12-treated mice to splenectomised mice as it caused the hepatic CTL count to increase. RNA-sequencing analysis identified IL-15 as a key factor in pathogenic B cells, which promotes CTL expansion and subsequent migration to the liver via the CXCL9/CXCR3 axis. Indeed, IL-15 neutralisation ameliorated hepatitis by suppressing splenic and hepatic CTLs in vivo. The close distribution of B220+ B cells and CD8+ T cells in the spleen of AIH mice suggested mutual interactions. Mechanistically, IFNγ and CD40L/CD40 signalling were indispensable for the expression of IL-15 in B cells, and in vitro co-culture experiments revealed that splenic CD40L+CD8+ T cells promoted IL-15 production in B cells, which led to CTL expansion. In patients with AIH, high serum IL-15 concentration and IL-15+ B-cell counts, positively correlating with serum alanine aminotransferase levels, support translation and potential therapeutic targeting in human AIH. Conclusions: This investigation elucidated the roles of IL-15-producing splenic B cells that occur in concert with pathogenic CD8+ T cells during the development of AIH. Impact and Implications: IL-15-producing B cells were shown to exacerbate experimental AIH via cytotoxic T lymphocyte expansion. CD40L+CD8+ T cells promoted IL-15 expression in B cells, indicating the mutual interaction of both cells. High serum IL-15 concentrations, IL-15+ B-cell counts, and CD40L+IL-15Rα+CD8+ T-cell counts were confirmed in the blood of patients with AIH.

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Immunovascular microenvironment in relation to prognostic heterogeneity of WNT/β-catenin-activated hepatocellular carcinoma.

Matsuda K, Kurebayashi Y, Masugi Y, Yamazaki K, Ueno A, Tsujikawa H, Ojima H, Kitago M, Itano O, Shinoda M, Abe Y, Sakamoto M

Hepatology research : the official journal of the Japan Society of Hepatology (Hepatology Research)  53 ( 4 ) 344 - 356 2023.04

ISSN  1386-6346

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Aim: WNT/β-catenin-activated hepatocellular carcinoma (W/B subclass HCC) is considered a molecularly homogeneous entity and has been linked to resistance to immunotherapy. However, recent studies have indicated possible heterogeneity in the immunovascular microenvironment in this subclass. We set out to test the hypothesis that specific immunovascular features might stratify W/B subclass HCCs into tumors having distinct aggressive natures. Methods: In this study, we analyzed 352 resected HCCs including 78 immunohistochemically defined W/B subclass HCCs. The density of tumor-infiltrating CD3+ T cells and the area ratio of vessels encapsulating tumor clusters (VETC) were calculated on tissue specimens. The gene expressions of angiogenic factors were measured by quantitative reverse transcription–polymerase chain reaction. Disease-free survival (DFS) was assessed using multivariable Cox regression analyses. Results: The T-cell density of W/B subclass HCCs was regionally heterogenous within tumor tissues, and focally reduced T-cell density was observed in areas with VETC. VETC-positivity (defined as VETC area ratio greater than 1%) was inversely associated with T-cell infiltration in both W/B subclass and non-W/B subclass HCCs. Fibroblast growth factor 2 (FGF2) gene expression was higher in W/B subclass than in non-W/B subclass HCCs. The VETC-positivity and low T-cell density correlated with increased expression of FGF2 in W/B subclass HCCs. Additionally, VETC-positive HCCs showed significantly shorter DFS in W/B subclass HCCs. Conclusions: In conclusion, the immune and vascular microenvironments are interrelated and are also correlated with clinicopathological heterogeneity in W/B subclass HCC. These results could inform clinical practice and translational research on the development of therapeutic stratification of HCCs.

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Two Distinct Characteristics of Immune Microenvironment in Human Hepatocellular Carcinoma with Wnt/β-Catenin Mutations

Aoki T., Nishida N., Kurebayashi Y., Sakai K., Morita M., Chishina H., Takita M., Hagiwara S., Ida H., Ueshima K., Minami Y., Tsurusaki M., Nakai T., Sakamoto M., Nishio K., Kudo M.

Liver Cancer (Liver Cancer)  13 ( 3 ) 285 - 305 2023

ISSN  22351795

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Introduction: Immunotherapy is becoming a promising approach for unresectable-hepatocellular carcinoma (HCC); the anti-tumor response is affected by the tumor microenvironment (TME). Although Wnt/β-catenin mutations are reported to cause non-inflamed phenotype, their role on TME remains controversial. We aimed to clarify the heterogeneity of immunophenotype in HCC with Wnt/β-catenin mutations. Methods: This study includes 152 resected HCCs; mutations in the catenin beta-1, adenomatous polyposis coli, or AXIN1, or AXIN2 genes were defined as Wnt/βcatenin mutations. With hierarchical cluster analyses, TME was classified into inflamed or non-inflamed classes based on the gene expressions associated with T-cell activation. Expression profiles of molecules related to cell differentiation and biliary-stem cell markers were compared between the TME classes to investigate whether differences in tumor traits were associated with TME. Results: Forty of 152 (26.3%) HCCs carried the Wnt/β-catenin mutations. Of these, 33 were classified as non-inflamed (33/40, 82.5%) and 7 as inflamed (7/40, 17.5%). Non-inflamed class was characterized by low number of CD3+, CD4+, and CD8+ cells on immunostaining, and high mRNA expressions of AXIN2 and GLUL, which are involved in the canonical Wnt/β-catenin signaling and hepatocyte differentiation, respectively. Non-inflamed tumors showed higher enhancement on the hepatobiliary-phase of gadolinium-ethoxybenzyl-diethylenetriamine (Gd-EOB-DTPA)enhanced magnetic resonance imaging (MRI) compared to inflamed tumors. HCCs classified as inflamed class are revealed to have high numbers of CD3+, CD4+, and CD8+ tumor infiltrating lymphocytes on immunostaining. This class is associated with increased expression of anti-epithelial cell adhesion molecule and FOXM1 accompanied by upregulation of genes related to interferon-gamma signaling, dendritic cell migration, regulatory T cells, and myeloid-derived suppressor cell activation and recognized as low enhancement nodule Tomoko Aoki and Naoshi Nishida contributed equally to this manuscript. on Gd-EOB-DTPA-enhanced MRI. Conclusion: Heterogeneity of tumor traits and TME was observed in HCC with Wnt/βcatenin mutation. The potential was indicated that tumor traits and TME are determined not only by the activation of the HNF4A but also by FOXM1, both of which are downstream transcription factor of the Wnt/β-catenin signaling pathway.

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Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Iriki H, Takahashi H, Wada N, Nomura H, Mukai M, Kamata A, Ito H, Yamagami J, Matsui T, Kurebayashi Y, Mise-Omata S, Nishimasu H, Nureki O, Yoshimura A, Hori S, Amagai M

Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America)  118 ( 49 )  2021.12

ISSN  0027-8424

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Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor–transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg32/2 mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

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The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group

El Bairi K., Haynes H.R., Blackley E., Fineberg S., Shear J., Turner S., de Freitas J.R., Sur D., Amendola L.C., Gharib M., Kallala A., Arun I., Azmoudeh-Ardalan F., Fujimoto L., Sua L.F., Liu S.W., Lien H.C., Kirtani P., Balancin M., El Attar H., Guleria P., Yang W., Shash E., Chen I.C., Bautista V., Do Prado Moura J.F., Rapoport B.L., Castaneda C., Spengler E., Acosta-Haab G., Frahm I., Sanchez J., Castillo M., Bouchmaa N., Md Zin R.R., Shui R., Onyuma T., Yang W., Husain Z., Willard-Gallo K., Coosemans A., Perez E.A., Provenzano E., Ericsson P.G., Richardet E., Mehrotra R., Sarancone S., Ehinger A., Rimm D.L., Bartlett J.M.S., Viale G., Denkert C., Hida A.I., Sotiriou C., Loibl S., Hewitt S.M., Badve S., Symmans W.F., Kim R.S., Pruneri G., Goel S., Francis P.A., Inurrigarro G., Yamaguchi R., Garcia-Rivello H., Horlings H., Afqir S., Salgado R., Adams S., Kok M., Dieci M.V., Michiels S., Demaria S., Loi S., Haynes H.R., Fineberg S., Shear J., Turner S., de Freitas J.R., Sur D., Amendola L.C., Gharib M., Arun I., Fujimoto L., Liu S.W., Lien H.C., Yang W., Chen I.C., Bautista V.

npj Breast Cancer (npj Breast Cancer)  7 ( 1 )  2021.12

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The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

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Immunovascular classification of HCC reflects reciprocal interaction between immune and angiogenic tumor microenvironments.

Kurebayashi Y, Matsuda K, Ueno A, Tsujikawa H, Yamazaki K, Masugi Y, Kwa WT, Effendi K, Hasegawa Y, Yagi H, Abe Y, Kitago M, Ojima H, Sakamoto M

Hepatology (Baltimore, Md.) (Hepatology)  75 ( 5 ) 1139 - 1153 2021.10

ISSN  0270-9139

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Background and Aims: Immune cells and tumor vessels constitute important elements in tumor tissue; however, their detailed relationship in human tumors, including HCC, is still largely unknown. Consequently, we expanded our previous study on the immune microenvironment of HCC and analyzed the relationship among the immune microenvironment, inflammatory/angiostatic factor expression, angiogenic factor expression, and tumor vessel findings, including vessels encapsulating tumor clusters (VETC) and macrotrabecular-massive (MTM) patterns. Approach and Results: We classified HCC into four distinct immunovascular subtypes (immune-high/angiostatic [IH/AS], immune-mid/angio-mid [IM/AM], immune-low/angiogenic [IL/AG], and immune-low/angio-low [IL/AL]). IH/AS, IM/AM, and IL/AG subtypes were associated with decreasing lymphocytic infiltration and increasing angiogenic factor expression and VETC/MTM positivity, reflecting their reciprocal interaction in the tumor microenvironment of HCC. IL/AG subtype was further characterized by CTNNB1 mutation and activation of Wnt/β-catenin pathway. IL/AL subtype was not associated with increased lymphocyte infiltration or angiogenic factor expression. Prognostically, IH/AS subtype and VETC/MTM positivity were independently significant in two independent cohorts. Increased angiogenic factor expression was not necessarily associated with VETC/MTM positivity and poor prognosis, especially when inflammatory/angiostatic milieu coexisted around tumor vessels. These results may provide insights on the therapeutic effects of immunotherapy, antiangiogenic therapies, and their combinations. The potential of evaluating the immunovascular microenvironment in predicting the clinical effect of these therapies in nonresectable HCC needs to be analyzed in the future study. Conclusions: HCC can be classified into four distinct immunovascular subtypes (IH/AS, IM/AM, IL/AG, and IL/AL) that reflect the reciprocal interaction between the antitumor immune microenvironment and tumor angiogenesis. In addition to its clinicopathological significance, immunovascular classification may also provide pathological insights on the therapeutic effect of immunotherapy, antiangiogenic therapy, and their combination.

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Rapid Depletion of Intratumoral Regulatory T Cells Induces Synchronized CD8 T- and NK-cell Activation and IFNγ-Dependent Tumor Vessel Regression.

Kurebayashi Y, Olkowski CP, Lane KC, Vasalatiy OV, Xu BC, Okada R, Furusawa A, Choyke PL, Kobayashi H, Sato N

Cancer research (Cancer Research)  81 ( 11 ) 3092 - 3104 2021.06

ISSN  0008-5472

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Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab0)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNg expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNg produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNg receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNg, providing insight into the mechanism of Treg-targeting therapies.

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Lipofibroadenoma arising in hyperplastic thymic tissue: Possible perivascular origin of lipofibroadenoma.

Kurebayashi Y, Hayashi Y, Emoto K, Kaseda K, Asamura H, Mukai K, Sakamoto M

Pathology international (Pathology International)  71 ( 4 ) 275 - 277 2021.04

ISSN  1320-5463

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<i>TNFRSF13B</i> c.226G&gt;A (p.Gly76Ser) as a Novel Causative Mutation for Pulmonary Arterial Hypertension.

Shinya Y, Hiraide T, Momoi M, Goto S, Suzuki H, Katsumata Y, Kurebayashi Y, Endo J, Sano M, Fukuda K, Kosaki K, Kataoka M

Journal of the American Heart Association (Journal of the American Heart Association)  10 ( 5 ) e019245 - 4 2021.02

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BACKGROUND: Recently, some studies reported the pulmonary artery hypertension (PAH)–associated genes. However, a majority of patients with familial or sporadic PAH lack variants in the known pathogenic genes. In this study, we investigated the new causative gene variants associated with PAH. METHODS AND RESULTS: Whole-exome sequencing in 242 Japanese patients with familial or sporadic PAH identified a heterozygous substitution change involving c.226G>A (p.Gly76Ser) in tumor necrotic factor receptor superfamily 13B gene (TNFRSF13B) in 6 (2.5%) patients. TNFRSF13B controls the differentiation of B cell and secretion of inflammatory cytokines and may be involved in vascular inflammation. In silico structural analysis simulation demonstrated the structural instability of the N-terminal region of the protein synthesized from TNFRSF13B p.Gly76Ser variant. These suggest that the TNFRSF13B p.Gly76Ser variant may be involved in the development of PAH via aberrant inflammation in pulmonary vessels. CONCLUSIONS: TNFRSF13B p.Gly76Ser variant is a candidate of novel causative gene variant for PAH.

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Imaging of cell-based therapy using⁸⁹ zr-oxine ex vivo cell labeling for positron emission tomography

Kurebayashi Y., Choyke P.L., Sato N.

Nanotheranostics (Nanotheranostics)  5 ( 1 ) 27 - 35 2021

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© The author(s). With the rapid development of anti-cancer cell-based therapies, such as adoptive T cell therapies using tumor-infiltrating T cells, T cell receptor transduced T cells, and chimeric antigen receptor T cells, there has been a growing interest in imaging technologies to non-invasively track transferred cells in vivo. Cell tracking using ex vivo cell labeling with positron emitting radioisotopes for positron emission tomography (PET) imaging has potential advantages over single-photon emitting radioisotopes. These advantages include intrinsically higher resolution, higher sensitivity, and higher signal-to-background ratios. Here, we review the current status of recently developed Zirconium-89 (89Zr)-oxine ex vivo cell labeling with PET imaging focusing on its applications and future perspectives. Labeling of cells with89Zr-oxine is completed in a series of relatively simple steps, and its low radioactivity doses required for imaging does not interfere with the proliferation or function of the labeled immune cells. Preclinical studies have revealed that 89Zr-oxine PET allows high-resolution in vivo tracking of labeled cells for 1-2 weeks after cell transfer both in mice and non-human primates. These results provide a strong rationale for the clinical translation of 89Zr-oxine PET-based imaging of cell-based therapy.

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Cholesterol 25-hydroxylase is a metabolic switch to constrain T cell–mediated inflammation in the skin

Takahashi H., Nomura H., Iriki H., Kubo A., Isami K., Mikami Y., Mukai M., Sasaki T., Yamagami J., Kudoh J., Ito H., Kamata A., Kurebayashi Y., Yoshida H., Yoshimura A., Sun H.W., Suematsu M., O’Shea J.J., Kanno Y., Amagai M.

Science Immunology (Science Immunology)  6 ( 64 ) eabb6444 2021

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Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4+ T cells by IL-27, enhanced by transforming growth factor–β (TGF-β), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4+ T cells that received IL-27 and TGF-β signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27–treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

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Quantification of intratumoral collagen and elastin fibers within hepatocellular carcinoma tissues finds correlations with clinico-patho-radiological features

Maehara J., Masugi Y., Abe T., Tsujikawa H., Kurebayashi Y., Ueno A., Ojima H., Okuda S., Jinzaki M., Shinoda M., Kitagawa Y., Oda Y., Honda H., Sakamoto M.

Hepatology Research (Hepatology Research)  50 ( 5 ) 607 - 619 2020.05

ISSN  13866346

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© 2019 The Japan Society of Hepatology Aim: Emerging evidence suggests a promising role for tumor stromal factors in characterizing patients with various types of malignancies, including hepatocellular carcinoma (HCC). We quantified the amount of collagen and elastin fibers in HCC samples with the aim of clarifying the clinico-patho-radiological significance of fiber deposition in HCC. Methods: We computed the amount of collagen and elastin fibers using digital image analysis of whole-slide images of Elastica van Gieson-stained tissues from 156 surgically resected HCCs. Furthermore, we assessed the correlations between the fiber content of HCC samples and clinical, pathological, and radiological features, including immunohistochemistry-based molecular subtypes and immunosubtypes. Results: The intratumoral area ratio of collagen in HCC tissues (median 3.4%, range 0.1–22.2%) was more than threefold that of elastin (median 0.9%, range 0.1–9.0%); there was a strong positive correlation between the amounts of collagen and elastin. Higher levels of combined collagen and elastin were significantly associated with the confluent multinodular macroscopic tumor type, the absence of a fibrous capsule, intratumoral steatosis, scirrhous tumor stroma, dense inflammatory-cell infiltrates, and the biliary/stem cell markers-positive HCC subtype. The associations of higher collagen levels with radiological findings, including heterogeneous enhancement and persistent enhancement on dynamic computed tomography, were significant. In contrast, the associations of radiological findings with elastin fibers were not significant. Intratumoral fibrous stroma in HCC comprised septum-like and perisinusoidal fibrosis; these two forms represented distinct distribution patterns of fibers and fibroblasts. Conclusion: Quantitative analysis suggested that stromal fiber-rich HCCs likely represent a distinct clinico-patho-radiological entity.

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Clinicopathological features of hepatocellular carcinoma with fatty change: Tumors with macrovesicular steatosis have better prognosis and aberrant expression patterns of perilipin and adipophilin

Kubota N., Ojima H., Hatano M., Yamazaki K., Masugi Y., Tsujikawa H., Fujii-Nishimura Y., Ueno A., Kurebayashi Y., Shinoda M., Kitago M., Abe Y., Kitagawa Y., Sakamoto M.

Pathology International (Pathology International)  70 ( 4 ) 199 - 209 2020.04

ISSN  13205463

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© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd The clinicopathological characteristics of steatosis in hepatocellular carcinoma (HCC) remain unclear. Here, we elucidate the features of macrovesicular steatosis (MaS) and microvesicular steatosis (MiS) in HCC and their relationships with background liver steatosis. A total of 165 HCC lesions were classified as MaS-HCC, MiS-HCC, or conventional HCC (cHCC) according to the cutoff value of 30% MaS or MiS in tumor cells. We analyzed the clinicopathological differences among these groups. MaS-HCC had less portal vein invasion, a higher proportion of HCC with intratumoral fibrosis, and a lower cumulative risk of recurrence than MiS-HCC or cHCC. Moreover, both MaS-HCC and MiS-HCC had lower incidences of hepatitis virus infection and higher levels of HbA1c than cHCC. Background liver steatosis was also higher in MaS-HCC than in cHCC. Immunohistochemical expression of perilipin (Plin1) and adipophilin (ADRP), major proteins expressed on lipid droplet membranes, revealed that almost all lipid droplets in HCC were Plin1 negative, whereas those in background liver were positive. In contrast, ADRP was expressed on lipid droplets in both HCC and background liver. We concluded that MaS-HCC and MiS-HCC were associated with metabolic abnormalities but exhibited different biologic behaviors. Furthermore, lipid droplets in HCC were pathophysiologically different from those in background liver.

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Precision pathology analysis of the development and progression of hepatocellular carcinoma: Implication for precision diagnosis of hepatocellular carcinoma

Ueno A., Masugi Y., Yamazaki K., Kurebayashi Y., Tsujikawa H., Effendi K., Ojima H., Sakamoto M.

Pathology International (Pathology International)  70 ( 3 ) 140 - 154 2020.03

ISSN  13205463

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© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Outcomes for patients with hepatocellular carcinoma (HCC) remain poor because the condition is often unresponsive to the available treatments. Consequently, the early and precise diagnosis of HCC is crucial to achieve improvements in prognosis. For patients with chronic liver disease, the assessment of liver fibrosis is also important to ascertain both the staging of fibrosis and the risk of HCC occurrence. Early HCC was first described in 1991 in Japan and was defined internationally in 2009. As the concept of early HCC spread, the multistage hepatocarcinogenesis process became accepted. Consequently, improvements in imaging technology made the early diagnosis of HCC possible. At present, the most appropriate therapeutic strategy for HCC is determined using an integrated staging system that assesses the tumor burden, the degree of liver dysfunction and the patient performance status; however, pathological and molecular features are not taken into account. The recent introduction of several new therapeutic agents will change the treatment strategy for HCC. Against this background, HCC subclassification based on tumor cellular and microenvironmental characteristics will become increasingly important. In this review, we give an overview of how pathological analysis contributes to understanding the development and progression of HCC and establishing a precision diagnosis of HCC.

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Immune microenvironment of hepatocellular carcinoma, intrahepatic cholangiocarcinoma and liver metastasis of colorectal adenocarcinoma: Relationship with histopathological and molecular classifications

Kurebayashi Y., Kubota N., Sakamoto M.

Hepatology Research (Hepatology Research)  51 ( 1 ) 5 - 18 2020

ISSN  13866346

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© 2020 The Japan Society of Hepatology Tumor tissue is composed of tumor cells and tumor stroma. Tumor stroma contains various immune cells and non-immune stromal cells, forming a complex tumor microenvironment which plays pivotal roles in regulating tumor growth. Recent successes in immunotherapies against tumors, including immune checkpoint inhibitors, have further raised interests in the immune microenvironment of liver carcinoma. The immune microenvironment of tumors is formed because of interactions among tumor cells, immune cells and non-immune stromal cells, including fibroblasts and endothelial cells. Different patterns of immune microenvironment are observed among different tumor subtypes, and their clinicopathological significance and intertumor/intratumor heterogeneity are being intensively studied. Here, we review the immune microenvironment of hepatocellular carcinoma, intrahepatic cholangiocarcinoma and liver metastasis of colorectal adenocarcinoma, focusing on its histopathological appearance, clinicopathological significance, and relationship with histological and molecular classifications. Understanding the comprehensive histopathological picture of a tumor immune microenvironment, in addition to molecular and genetic approaches, will further potentiate the effort for precision medicine in the era of tumor-targeting immunotherapy.

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Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis

Morikawa R., Nakamoto N., Amiya T., Chu P.s., Koda Y., Teratani T., Suzuki T., Kurebayashi Y., Ueno A., Taniki N., Miyamoto K., Yamaguchi A., Shiba S., Katayama T., Yoshida K., Takada Y., Ishihara R., Ebinuma H., Sakamoto M., Kanai T.

Journal of Hepatology (Journal of Hepatology)  74 ( 3 ) 511 - 521 2020

ISSN  01688278

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© 2020 European Association for the Study of the Liver Background & Aims: The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. Methods: Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9−/− mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. Results: Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9−/− mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9−/− mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. Conclusions: These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. Lay summary: Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.

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