Published papers

Uterine tumors are the most common type of gynecologic neoplasm. Uterine leiomyosarcoma (LMS) is rare, accounting for 2% to 5% of tumors of the uterine body. Uterine LMS develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Radiographic evaluation combined with PET/CT can be useless in the diagnosis and surveillance of uterine LMS. Importantly, a diagnostic biomarker, which distinguishes malignant LMS and benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS in order to establish a method of treatment. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of similar to 40% by 14 months of age. It is therefore of interest whether human uterine LMS shows a loss of LMP2 expression. We found LMP2 expression is absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is potentially a diagnostic biomarker for uterine LMS, and gene therapy with LMP2-encording DNA may be a new therapeutic approach.

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC.

Invasive intraductal papillary-mucinous carcinoma (I-IPMC) is a heterogeneous entity with various postoperative outcomes. The aim of this study is to characterize early-stage I-IPMC with nonaggressive characteristics. One hundred and four patients with intraductal papillary-mucinous neoplasm (IPMN) were clinicopathologically investigated. The lesions were classified into 53 noninvasive IPMNs (adenoma, borderline, and,noninvasive IPMC) and 51 I-IPMCs on the basis of the WHO classification. I-IPMCs were divided further into 26 minimally invasive IPMCs (MI-IPMCs) and 25 invasive carcinomas originating in IPMC (IC-IPMCs) by new diagnostic criteria proposed in this study. We examined invasiveness of I-IPMC on 4 patterns, and defined simple and practical diagnostic criteria of minimal invasion for each invasive pattern. The disease-specific survival rates after 3, 5, and 10 years were 100%, 100%, and 100% for both noninvasive IPMN and MI-IPMC, and 51%, 38%, and 0% for IC-IPMC. The overall and disease-specific survival rates for MI-IPMC were both significantly better than those for IC-IPMC (P < 0.001), but there was no significant difference between noninvasive IPMN and MI-IPMC. Multivariate analysis showed that the factors indicative of poor prognosis were a diagnosis of I-IPMC classified as IC-IPMC and a high level of serum carbohydrate antigen 19-9. The prognosis of IC-IPMC was not significantly different from that of pancreatic ductal carcinoma in each of the corresponding tumor-node-metastasis stages. These findings suggest that a category of MI-IPMC provides more accurate and useful information of the stage and the aggressiveness of I-IPMC.

Background: High-resolution computed tomography (HRCT) of lung adenocarcinoma at early stage shows pure ground-glass opacity (GGO) and most cases of pure GGO remain stable during follow-up. There is no consensus on the strategy for follow-up. Identification of the molecular mechanisms that are associated with the natural history of lung adenocarcinoma should provide useful information. Methods: Twenty-three lung adenocarcinomas that were followed-up for more than 6 months pre-operatively by HRCT were included in this study. Patterns of radiological changes during the follow-up period were classified into three groups; type 1, pure GGO without consolidation; type 2, appearance or increase in consolidation within pure GGO; type 3, consolidation without pure GGO. Mutational analysis of the epidermal growth factor receptor (EGFR) and K-ras genes and immunohistochemical staining of p53 protein were performed. Results: EGFR mutations were found in 17 cases (74%), and there was no K-ras mutation. Positive staining of p53 was found in 8 cases (35%). As for radiological findings during the follow-up period, the frequencies of EGFR mutations and positive p53 staining were 67 and 0% in type 1 (n = 9), 89 and 44% in type 2 (n = 9) and 60 and 80% in type 3 (n = 5). Conclusions: EGFR mutations were frequently found in lung adenocarcinoma with GGO on HRCT in this study. Inactivation of p53 may be associated with the appearance of central consolidation within pure GGO on HRCT which reflects invasive features and may be useful as a molecular marker during the follow-up of pure GGO.

As macroscopic appearance represents tumor microenvironment, it may also reflect the biological and clinicopathological characteristics of a cancer. The aim of the study was to evaluate the clinicopathological significance of the gross appearance of pancreatic ductal adenocarcinoma (PDA). We investigated fresh macroscopic features in 352 cases of PDA and their clinicopathological significance. Three unique gross features were found: a honeycomb-like appearance (diffusely distributed microcysts and interstitial fibrotic thickening), macroscopic necrosis, and a tube/branching structure (apparent small cylindrical or linear structure). A honeycomb-like appearance was present in 24 cases (6.8 %) and significantly associated with low serum CA19-9 level and well-differentiated adenocarcinoma. Macroscopic necrosis was present in 235 cases (66.8 %) and significantly correlated with tumor size, nodal metastasis, nerve plexus invasion, no adjuvant chemotherapy, and distant recurrence. The presence of macroscopic necrosis was significantly associated with shorter disease-specific survival (DSS) and disease-free survival (DFS). The presence of larger areas of necrosis (ae2 mm) was closely associated with shorter survival. A tube/branching structure was found in 179 cases (50.9 %), which was correlated with larger tumor size and no adjuvant chemotherapy and macroscopic necrosis. The presence of a tube/branching structure was significantly associated with shorter DSS and DFS. Multivariate survival analyses showed that the presence of tube/branching structures was an independent negative prognostic factor in patients having PDA. We suggest that the gross appearance of PDA reflects clinicopathological characteristics and may be useful in predicting prognosis.

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. BNIP3 and NIX are critical mediators for the Mieap-regulated mitochondrial quality control. Mieap suppresses murine intestinal tumor via its mitochondrial quality control function. To explore the role of the Mieap-regulated mitochondria quality control function in colorectal cancer patients, we examined the statuses of p53, Mieap, BNIP3 and NIX in 57 primary colorectal cancer tissues. Promoter methylation of the Mieap and BNIP3 genes was found in 9% and 47% of colorectal cancer cases, respectively, whereas p53 mutation was found in more than 50% of colorectal cancer tissues lacking methylation of the Mieap and BNIP3 promoters, implying that the p53/Mieap/BNIP3-regulated mitochondria quality control pathway is inactivated in more than 70% of colorectal cancer patients. In LS174T colorectal cancer cells, hypoxia activated the Mieap-regulated mitochondria quality control function. Knockdown of p53, Mieap or BNIP3 in LS174T cells severely impaired the hypoxia-activated function, leading to the accumulation of unhealthy mitochondria and increase of mitochondrial reactive oxygen species generation. The mitochondrial reactive oxygen species generated by unhealthy mitochondria in the p53/Mieap/BNIP3-deficient cells remarkably enhanced cancer cell migration and invasion under hypoxic condition. These results suggest that the Mieap-regulated mitochondria quality control has a critical role in colorectal cancer suppression in the in vivo hypoxic tumor microenvironment.

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass, with atypical presentations of hypercalcemia and eosinophilia also being reported. Radiographic evaluation with combined positron-emission tomography/computed tomography may assist in diagnosis and surveillance in women with uterine LMS; these are commonly used with stage and tumour grade as prognostic indicators and a recently developed risk-assessment index to predict disease-specific survival. Recent studies have shown that the addition of adjuvant therapy after surgical management does not seem to improve survival, and ovarian preservation does not appear to negatively impact outcome. Experimentally, it is noteworthy that proteasome subunit beta 9 (PSMB9)/beta 1i-deficient mice exhibit spontaneous development of uterine LMS, with a disease prevalence of similar to 37% by 12 months of age. Furthermore, a recent report showed the loss of ability to induce PSMB9/beta 1i expression, that is up-regulated by interferon-gamma (IFN gamma), in human uterine LMS tissues. Here, we reviewed human uterine LMS for genetic mutations in the IFN gamma signal cascade, and found serious mutations in three genes, Janus activated kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and PSMB9/beta 1i promoter regions. Moreover, molecular experiments demonstrated differential expression of cyclin E and P27/KIP1, that regulate cell-cycle G1 arrest via PSMB9/beta 1i expression. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

The hallmark of most cancer cells is the metabolic shift from mitochondrial to glycolytic metabolism for adapting to the surrounding environment. Although epigenetic modification is intimately linked to cancer, the molecular mechanism, by which epigenetic factors regulate cancer metabolism, is poorly understood. Here, we show that lysine-specific demethylase-1 (LSD1, KDM1A) has an essential role in maintaining the metabolic shift in human hepatocellular carcinoma cells. Inhibition of LSD1 reduced glucose uptake and glycolytic activity, with a concurrent activation of mitochondrial respiration. These metabolic changes coexisted with the inactivation of the hypoxia-inducible factor HIF1 alpha, resulting in a decreased expression of GLUT1 and glycolytic enzymes. In contrast, during LSD1 inhibition, a set of mitochondrial metabolism genes was activated with the concomitant increase of methylated histone H3 at lysine 4 in the promoter regions. Consistently, both LSD1 and GLUT1 were significantly overexpressed in carcinoma tissues. These findings demonstrate the epigenetic plasticity of cancer cell metabolism, which involves an LSD1-mediated mechanism. (C)2015 AACR.

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass. Typical presentations with hypercalcemia or eosinophilia have been reported. Radiographic evaluation with combined positron emission tomography/computed tomography may assist in the diagnosis and surveillance of women with uterine LMS. A recently developed risk-assessment index is highly predictive of disease-specific survival. Ovarian preservation does not appear to negatively impact outcome, and the addition of adjuvant therapy after surgical treatment does not seem to improve survival. It is noteworthy that LMP2-deficient mice exhibit spontaneous development of uterine LMS with a disease prevalence of approximately 37% by 12 months of age. The LMP2 gene is transcribed from a promoter containing an interferon (IFN)-gamma-response factor element; thus, the IFN-gamma-signal strongly induces LMP2 expression. Furthermore, a recent report demonstrated the loss of ability to induce LMP2 expression, which is an interferon (IFN)-gamma-inducible factor, in human uterine LMS tissues and cell lines. Analysis of human uterine LMS shows somatic mutations in the IFN gamma signalling pathway, thus the loss of LMP2 induction is attributable to a defect in the earliest steps of the IFN-gamma signalling pathway. The discovery of an impaired key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

Peritoneal metastasis, a hallmark of incurable advanced gastric cancer (GC), presently has no curative therapy and its molecular features have not been examined extensively. Here we present a comprehensive multi-omic analysis of malignant ascitic fluid samples and their corresponding tumor cell lines from 98 patients, including whole-genome sequencing, RNA sequencing, DNA methylation and enhancer landscape. We identify a higher frequency of receptor tyrosine kinase and mitogen-activated protein kinase pathway alterations compared to primary GC; moreover, approximately half of the gene alterations are potentially treatable with targeted therapy. Our analyses also stratify ascites-disseminated GC into two distinct molecular subtypes: one displaying active super enhancers (SEs) at the ELF3, KLF5 and EHF loci, and a second subtype bearing transforming growth factor-β (TGF-β) pathway activation through SMAD3 SE activation and high expression of transcriptional enhancer factor TEF-1 (TEAD1). In the TGF-β subtype, inhibition of the TEAD pathway circumvents therapy resistance, suggesting a potential molecular-guided therapeutic strategy for this subtype of intractable GC.

Background: Gastric cancer (GC) has been classified based on molecular profiling like The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG), and attempts have been made to establish therapeutic strategies based on these classifications. However, it is difficult to predict the survival according to these classifications especially in radically resected patients. We aimed to establish a new molecular classification of GC which predicts the survival in patients undergoing radical gastrectomy. Methods: The present study included 499 Japanese patients with advanced GC undergoing radical (R0/R1) gastrectomy. Whole-exome sequencing, panel sequencing, and gene expression profiling were conducted (High-tech Omics-based Patient Evaluation [Project HOPE]). We classified patients according to TCGA and ACRG subtypes, and evaluated the clinicopathologic features and survival. Then, we attempted to classify patients according to their molecular profiles associated with biological features and survival (HOPE classification). Results: TCGA and ACRG classifications failed to predict the survival. In HOPE classification, hypermutated (HMT) tumors were selected first as a distinctive feature, and T-cell-inflamed expression signature-high (TCI) tumors were then extracted. Finally, the remaining tumors were divided by the epithelial-mesenchymal transition (EMT) expression signature. HOPE classification significantly predicted the disease-specific and overall survival (p < 0.001 and 0.020, respectively). HMT + TCI showed the best survival, while EMT-high showed the worst survival. The HOPE classification was successfully validated in the TCGA cohort. Conclusions: We established a new molecular classification of gastric cancer that predicts the survival in patients undergoing radical surgery.

To clarify the significance of GNAS mutations in cervical tumorigenesis, we performed mutational analyses in a total of 154 lesions and in 22 normal tissues of the uterine cervix. Activating GNAS mutations were found in 8 of the 19 lobular endocervical glandular hyperplasias (LEGH; 42%) and 4 of the 79 endocervical-type mucinous adenocarcinomas (5%) but were never seen in the normal endocervical tissue, minimal deviation adenocarcinomas, endometrioid adenocarcinomas, or squamous cell carcinomas. We further examined the presence of human papillomavirus (HPV) DNA and p16 expression to probe the relationship between GNAS mutations and HPV infection in LEGHs and carcinomas. All the GNAS-mutated LEGHs were negative for HPV DNA and p16 expression, whereas all the GNAS-mutated adenocarcinomas were positive for HPV DNA and/or p16 expression, implicating GNAS mutations in the development of LEGH and a minor subset of HPV-related cervical adenocarcinomas. Additional mutational analyses of LEGH identified KRAS and STK11 mutations in 1 and 2 cases, respectively. The GNAS, KRAS, and STK11 mutations were mutually exclusive; thus, a total of 11 LEGHs (58%) had 1 of these genetic alterations. Although LEGH has been regarded as a metaplastic lesion, the frequent presence of genetic alterations suggests a neoplastic nature.

It has been speculated that intraductal dissemination, via the pancreatic duct, bile duct, or mammary duct, is a unique form of cancer cell spread. However, clinical evidence to confirm this form of dissemination has been lacking. Here we report a case of papillary adenocarcinoma of the ampulla of Vater in which retrograde dissemination to the pancreatic duct was strongly suggested. A 79-year-old woman underwent pancreatoduodenectomy for a 22 mm microinvasive papillary adenocarcinoma of the ampulla. Multiple carcinomas in situ were found in the pancreatic duct distant from the ampulla. Seven months later, she underwent a second operation for a recurrent papillary adenocarcinoma at the pancreato-jejunal anastomosis showing exophytic and expansive growth into the jejunal lumen that connected to an intraductal adenocarcinoma in the pancreatic body. None of these tumors showed invasive growth, or vascular or neural invasion, being separate from each other but sharing identical histological, immunohistochemical, and molecular features; papillary growth, a pancreatobiliary phenotype, the same pattern of genomic loss of heterozygosity, and no mutation of the KRAS, TP53, and GNAS genes. These results imply that this papillary adenocarcinoma of the ampulla of Vater had disseminated to the pancreatic duct in a retrograde manner and recurred in the remnant pancreas.

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.

Background: The host immune reaction is represented by immune/inflammatory cell infiltrates. Here we systematically analysed tumour-infiltrating immune/inflammatory cells in pancreatic ductal carcinoma (PDC) and evaluated their clinicopathological impact. Methods: Using immunohistochemistry, we examined tumour-infiltrating CD68(+) pan-macrophages, HLA-DR(+)CD68(+) M1 macrophages (M1), CD163(+) or CD204(+) M2 macrophages (M2), CD66b(+) neutrophils (Neu), CD4(+) T cells (CD4(+) T), CD8(+) T cells (CD8(+)T), and FOXP3(+)CD4(+) regulatory T cells (Treg) in 212 cases of PDC, and conducted correlation and survival analyses using the Kaplan Meier method and Cox proportional hazards model. Results: Higher levels of tumour-infiltrating pan-macrophages, M2, Neu, or the ratio of Tregs to CD4(+)T (%Treg) were significantly associated with shorter survival, whereas higher levels of tumour-infiltrating CD4(+)T, CD8(+)T, or the ratio of M1 to pan-macrophages (%M1) were significantly associated with longer survival. Survival analysis of pairs of these variables revealed that some of the resulting patient groups had exclusively longer survival. We then connected the apparently related factors, and two significant variables emerged: tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and tumour-infiltrating %M1(high)/M2(low). Multivariate survival analysis revealed that these variables were significantly correlated with longer survival and had a higher hazard ratio. Conclusion: Tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and %M1(high)/M2(low) are independent prognosticators useful for evaluating the immune microenvironment of PDC.

Background: Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone however, the development of human uterine leiomyosarcoma is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine leiomyosarcoma from benign tumor leiomyoma is yet to be established. Aims: It is necessary to analyze risk factors associated with human uterine leiomyosarcoma, in order to establish a diagnostic-biomarker and a clinical treatment method. Patients and Methods: Histology and Immunofluorescence Staining: Uteri obtained from LMP2-/- mice or its parental mice (C57BL/6 mice) were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections (5 μm) were prepared and stained with H& E for routine histological examination or were processed further for immunofluorescence staining with appropriate antidodies. Furthermore, a total of 101 patients between 32 and 83 years of age and diagnosed as having smooth muscle tumors of the uterus were selected from pathological files. Immunohistochemistry staining for LMP2 was performed on serial human uterine leiomyosarcoma, leiomyoma and myometrium sections. Results: Homozygous deficient mice for a proteasome β1i subunit, LMP2 spontaneously develop uterine leiomyosarcoma, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine leiomyosarcoma was demonstrated, but present in human leiomyoma and myometrium. Conclusions: Loss in LMP2 expression may be one of the risk factors for human uterine leiomyosarcoma. LMP2 may be a potential diagnostic-biomarker and targeted-molecule for a new therapeutic approach.

A 72-year-old man was found to have a 40 mm mass in liver segment VIII during follow-up abdominal ultrasonography for type C viral hepatitis. Abdominal ultrasound showed a well-defined mass containing a cystic component, and computed tomographic hepatic arteriography showed heterogeneous enhancement except for cystic necrosis. Under a pre-operative diagnosis of atypical hepatocellular carcinoma (HCC), partial resection of liver segment VIII was performed. The encapsulated tumor consisted of a peripheral solid component with a central necrotic area. Histologically, the solid component had a two-layer structure, an HCC component in the external area and a sarcomatous component with neoplastic osteoid formation in the internal area, showing histological transition. Immunohistochemically, the HCC component was positive for hepatocyte antigen and negative for vimentin. The Ki-67 labeling index was found to increase from 5% to 58% with increasing histologic atypia. The sarcomatous component was positive for vimentin and negative for pan-keratin and hepatocyte antigen, with a Ki-67 labeling index of > 90%. These findings led to a diagnosis of primary hepatic carcinosarcoma. Although previously reported patients with hepatic carcinosarcoma showed early metastasis with a very poor outcome, this patient has remained free of recurrence for 30 months, which is the longest recurrence-free survival time recorded for this type of cancer. Since relatively early-stage hepatic carcinosarcoma rarely seems to present as a small tumor showing a concentric growth pattern, we report this case with a review of the literature.

Carcinosarcoma of the pancreas is extremely rare and its histogenesis is still unclear. This is a report on a 64-year-old female patient with an intraductal carcinosarcoma arising from intraductal papillary-mucinous carcinoma (IPMC) in the pancreas tail. The carcinosarcoma grew as a polypoid mass within the main pancreatic duct. Histologically, the tumour consisted of adenocarcinoma covering the luminal surface of the lesion with minimal stromal invasion, and osteosarcoma occupying the stroma. Immunohistochemical and gene mutation analyses revealed that both the carcinomatous and sarcomatous tumour cells of the carcinosarcoma, as well as the IPMC cells, expressed TP53 and had identical mutations in KRAS and TP53 genes, indicating that these two neoplastic components of the carcinosarcoma shared a common tumorigenesis and arose from the IPMC. This is the first report of a carcinosarcoma originating in IPMC. These findings imply that carcinosarcoma with a heterologous mesenchymal component is of ductal origin.

Aberrant DNA methylation has been shown to play important roles during multistage carcinogenesis in various human organs. The aim of this study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during cervical carcinogenesis. We carried out an immunohistochemical examination for DNMT1 in 34 samples of histologically normal squamous epithelium, 36 samples of low-grade cervical intraepithelial neoplasia (CIN), 61 samples of higher-grade CIN and 30 samples of squamous cell carcinoma of the uterine cervix. The DNMT1 protein expression score, reflecting the intensity and incidence of DNMT1 nuclear immunoreactivity, was increased even in low-grade CIN (P < 0.0001) in comparison with histologically normal squamous epithelium and was further increased in higher-grade CIN (P < 0.0001 compared to low-grade CIN). The DNMT1 protein expression score remained at a plateau in microinvasive carcinoma (Stage IA, P = 0.0690 compared to higher-grade CIN) and then decreased with cancer invasion (Stage IB or more, P = 0.0 176 compared to Stage IA), whereas the proliferating cell nuclear antigen (PCNA) labeling index did not decrease with cancer invasion (P = 0.8259 between Stage IA and Stage IB or more). Thus, the DNMT1 protein expression score and the PCNA labeling index were not mutually correlated in squamous cell carcinoma of the uterine cervix (P = 0.2304). These data suggest that progressively increasing expression of DNMT1 protein is not entirely a secondary result of increased cell proliferative activity, but is associated with an early step of multistage cervical carcinogenesis. (C) 2006 Elsevier Ireland Ltd. All rights reserved.