Published papers

© 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed “The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research” based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about −180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fi

© 2018 Elsevier Inc. We investigated the clinicopathological and molecular characteristics of scirrhous hepatocellular carcinoma (HCC) to elucidate its uniqueness. Samples from 120 resected HCC cases underwent immunohistochemical analysis. Tumor area containing fibrous stroma and the percentage of steatotic cells within the tumor were evaluated. In our previous report, tumors were immunohistochemically subclassified as biliary/stem cell markers–positive (B/S) (cytokeratin 19 and/or sal-like protein 4 and/or epithelial cell adhesion molecule positive), Wnt/β-catenin signaling–related markers–positive (W/B) (β-catenin and/or glutamine synthetase positive), or all markers–negative (−/−) groups. Thirty-seven cases (31%) with fibrous stroma making up ≥50% of the largest tumor area were defined as scirrhous HCC (sHCC); the other 83 cases (69%) were categorized as common HCC (cHCC). Clinicopathologically, sHCC had fewer poorly differentiated tumors (P =.037) and a higher percentage of cases with steatosis (P =.025) than cHCC. sHCC cases were further divided into two subgroups: those with ≥5% steatotic cells (steatotic sHCC) and those with <5% steatotic cells (nonsteatot

Muscle injury is one of the most common traumas in orthopedic and sports medicine. However, there are only a few treatment options with marginal clinical benefits for this condition. Muscle repair after injury involves multiple and complex processes, such as the inflammation phase, regeneration phase, and remodeling phase. To develop a treatment modality and to examine the efficacy of novel interventions and agents for patients with muscle injuries, it is essential to establish a reliable and sensitive method to monitor the changes in muscle structure and status during muscle repair. Diffusion-weighted magnetic resonance imaging has been widely used to assess the diffusivity of water molecules in tissue. When it is used in combination with diffusion tensor imaging (DTI), the microstructure of muscle tissue can be indirectly depicted. In the present study, we evaluated the time-course changes in the diffusivity and anisotropy in muscles by DTI and histology after injury in mice. We found that the diffusivity and anisotropy exhibit distinct kinetics during muscle repair and that these kinetics were significantly altered in mutant mice with a defect in muscle regeneration. Our data show that muscle repair processes can be readily evaluated and monitored by DTI technique and suggest that DTI can be clinically applied for assessing muscle injury and repair in humans. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Purpose: Homologous recombination deficiency (HRD), which influences the efficacy of PARP inhibitor- and platinum agent-based therapies, is a prevalent phenotype of breast cancer in adolescents and young adults (AYAs; 15–39 years old). However, HRD score, indicating HRD status, is not routinely assessed in the breast oncology clinic, particularly in patients without germline BRCA1/2 mutations. Hence, we sought to develop a model for determining HRD status based on genetic and clinicopathological factors. Methods: Subjects were our own cohort of 46 Japanese AYA breast cancer patients and two existing breast cancer cohorts of US and European patients. Models for prediction of the HRD-high phenotype, defined as HRD score ≥ 42, were constructed by logistic regression analysis, using as explanatory variables genetic and clinicopathological factors assessable in the clinical setting. Results: In all three cohorts, the HRD-high phenotype was associated with germline BRCA1/2 mutation, somatic TP53 mutation, triple-negative subtype, and higher tumor grade. A model based on these four factors, developed using the US cohort, was validated in the Japanese and European AYA cases: area under the receiver operating characteristic curve [AUC] was 0.90 and 0.96, respectively. A model based on three factors excluding germline BRCA1/2 mutation also yielded high-predictive power in cases from these two cohorts without germline BRCA1/2 mutations: AUC was 0.92 and 0.90, respectively. Conclusions: The HRD-high phenotype of AYA breast cancer patients can be deduced from genomic and pathological factors that are routinely examined in the oncology clinic, irrespective of germline BRCA1/2 mutations.

© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Proteolytic balance is crucial for the maintenance of tissue homeostasis. In cancer, dysregulated proteolysis is involved in unregulated tissue remodeling and inflammation, leading to the promotion of tumor growth, local invasion, and metastasis. Metalloproteinases, which were first identified as collagen cleaving enzymes, have been shown to extensively degrade extracellular matrix proteins or selectively release cell surface-bound cytokines, growth factors, or their receptors, thereby impacting extracellular matrix integrity, immune cell recruitment and tissue turnover. Although tumor cells produce various metalloproteinases, the major source is thought to be stromal cells infiltrating the tumor. Different types of stromal cells express specific sets of metalloproteinases and their inhibitors, which specifically alter the milieu within the tumor. In this review, recent findings and knowledge regarding metalloproteinases derived from stromal cells during the creation of the tumor microenvironment are described and their contribution to the tumor progression and metastasis discussed.

Background: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH. Results: Normal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort. Conclusions: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

CD4+ T-cell immunity helps clonal proliferation, migration, and cancer cell killing activity of CD8+ T cells and is essential in antitumor immune responses. To identify CD4+ T-cell clusters responsible for antitumor immunity, we simultaneously analyzed the naïve-effector state, Th polarization, and T-cell receptor clonotype based on single-cell RNA-sequencing data. Unsupervised clustering analysis uncovered the presence of a new CD4+ T-cell metacluster in the CD62Llow CD4+ T-cell subpopulation, which contained multicellular clonotypes associated with efficacy of programmed death-ligand 1 (PD-1) blockade therapy. The CD4+ T-cell metacluster consisted of CXCR3+CCR4-CCR6+ and CXCR3-CCR4-CCR6+ cells and was characterized by high expression of IL7 receptor and TCF7. The frequency of these cells in the peripheral blood significantly correlated with progression-free survival and overall survival of patients with lung cancer after PD-1 blockade therapy. In addition, the CD4+ metacluster in the peripheral blood correlated with CD4+ T-cell infiltration in the tumor microenvironment, whereas peripheral Th1 correlated with local CD8+ T-cell infiltration. Together, these findings suggest that CD62Llow CCR4-CCR6+ CD4+ T cells form a novel metacluster with predictive potential of the immune status and sensitivity to PD-1 blockade, which may pave the way for personalized antitumor immunotherapy strategies for patients. SIGNIFICANCE: The identification of a new CD4+ T-cell metacluster that corresponds with immune status could guide effective tumor treatment by predicting response to immunotherapy using peripheral blood samples from patients.

Resistance to immune checkpoint blockade remains challenging in patients with non–small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell–dominant, and CD8+ T cell–dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction–related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

Purpose: It is difficult to predict the biological behavior of pancreatic endocrine tumors (PETs). Our aim was to evaluate the prognostic significance of certain variables in PETs. Experimental Design: The following variables were examined in 37 patients with PETs and then compared with other clinicopathologic characteristics: histologic tumor structure; microvessel density (MVD) measured by three different methods, including a unique method involving calculation of solid area MVD; endothelial proliferation; and the immunohistochemical expression of vascular endothelial growth factor-A and CXC chemokine CXCL-12. Intratumoral vascular structures were analyzed by double immunofluorescence using 30-mu m-thick sections. Results: The presence of focal and intensive solid growth of tumor cells (large solid nests; P=0.003), low solid area MVD (P=0.002), a high endothelial cell proliferation index (EPI; P=0.005), and high expression of CXCL-12 in PET cells (P=0.018) were significant unfavorable prognostic indicators. The predominant structure of the overall tumor histology and the expression of vascular endothelial growth factor-A did not separate aggressive PETs. In areas of focal solid growth, tumor-associated blood vessels had obviously low MVD and high EPI, and their structures were poorly formed with highly abnormal features, in comparison with other areas. High expression of CXCL-12 in tumor cells was significantly associated with variables representing tumor growth, hematogenous tumor spread, low MVD, high EPI, and the presence of large solid nests. Conclusions: This study has provided novel findings on the prognostic features of tumor architecture and tumor-associated angiogenesis in PETs. CXCL-12 is the first candidate molecule in association with neoangiogenesis in PETs.

We report a case of primary adenocarcinoma of the rete testis in a 55-year-old man with pulmonary metastases that were detected 11 months prior to the diagnosis of the primary lesion. Primary adenocarcinoma of the rete testis is an extremely rare malignant tumor with a poor outcome. The most common primary symptom is a scrotal mass, often accompanied by hydrocele and chronic epididymitis. The diagnosis is often delayed because of non-specific clinical presentation and symptoms. We cannot forget that rete testis is a possible primary site for a primary, unknown metastatic adenocarcinoma.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion-exchange column for high-performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time-consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylat

Copyright © 2018 by the American Thoracic Society. Lung cancer and chronic obstructive pulmonary disease are leading causes of morbidity and mortality worldwide, and cigarette smoking is a main risk factor for both. The presence of emphysema, an irreversible lung disease, further raises the risk of lung cancer in patients with chronic obstructive pulmonary disease. The mechanisms involved in smoke-induced tumorigenesis and emphysema are not fully understood, attributable to a lack of appropriate animal models. Here, we optimized a model of cigarette smoke (CS)–induced lung cancer and emphysema in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen. We investigated whether variations in CS exposure patterns with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Continuous CS exposure for 3 months significantly suppressed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone–induced development of adenomas and adenocarcinomas; however, emphysema independently developed during this period. Surprisingly, intermittent CS exposure increased the severity of emphysema and resulted in a higher incidence o

Copyright © 2019 The Author(s). Lynch syndrome (LS) is an autosomal dominant disease caused by a germline mutation in DNA mismatch repair genes which increases the risk of several cancers such as endometrial and colorectal cancers. However, there are only a few reports of peritoneal malignancies in patients with LS. Herein, we report the first case of a primary peritoneal lowgrade serous carcinoma in a woman with LS and provide a literature review of peritoneal malignancies in patients with LS. The patient was a 72-yr-old gravid 2 para 2 Japanese woman with a germline mutation in MLH1. She had a history of colon cancer and endometrial cancer and was treated with total hysterectomy and bilateral salpingo-oophorectomy 14 yr ago. During the follow-up, peritoneal nodules were detected by abdominal computed tomography which were surgically resected. Pathologic examination revealed a low-grade serous carcinoma with cells positive for BerEP4, MOC31, CEA, and WT-1 and negative for BAP1, PAX8, MLH1, and PMS2, by immunohistochemistry. This case report and literature review show that peritoneal low-grade serous carcinoma can occur in patients with LS and that LS-related cancers usually prece

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. INTRODUCTION: Renal tubular injury contributes to the decline in kidney function in patients with diabetes. Cell type-specific DNA methylation patterns have been used to calculate proportions of particular cell types. In this study, we developed a method to detect renal tubular injury in patients with diabetes by detecting exfoliated tubular cells shed into the urine based on tubular cell-specific DNA methylation patterns. RESEARCH DESIGN AND METHODS: We identified DNA methylation patterns specific for human renal proximal tubular cells through compartment-specific methylome analysis. We next determined the methylation levels of proximal tubule-specific loci in urine sediment of patients with diabetes and analyzed correlation with clinical variables. RESULTS: We identified genomic loci in SMTNL2 and G6PC to be selectively unmethylated in human proximal tubular cells. The methylation levels of SMTNL2 and G6PC in urine sediment, deemed to reflect the proportion of exfoliated proximal tubular cells due to injury, correlated well with each other. Methylation levels of SMTNL2 in urine sediment significantly correlated with the annual decline in estimated glomerular filtration rate. Moreover, addition of urinary SMTNL2 methylation to a model containing known risk factors significantly improved discrimination of patients with diabetes with faster estimated glomerular filtration rate decline. CONCLUSIONS: This study demonstrates that patients with diabetes with continual loss in kidney function may be stratified by a specific DNA methylation signature through epigenetic urinalysis and provides further evidence at the level of exfoliated cells in the urine that injury of proximal tubular cells may contribute to pathogenesis of diabetic kidney disease.