Kohei Shomori

Cancer‐associated fibroblasts and CD163‐positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance

BACKGROUNDnStromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer-associated fibroblasts (CAFs) and the incidence of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion.nnnMETHODSnThe study included 108 patients with OSCC. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated.nnnRESULTSnThe cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low- and high-grade groups on the basis of the number of CD68-positive and CD163-positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan-Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163-positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163-positive macrophage level was a significant and an independent prognostic factor (P = 0.045).nnnCONCLUSIONSnCancer-associated fibroblasts and CD163-positive macrophages may be potential prognostic predictors of OSCC.

Clinical usefulness of serum telomerase reverse transcriptase (hTERT) mRNA and epidermal growth factor receptor (EGFR) mRNA as a novel tumor marker for lung cancer

Using a newly developed assay of telomerase reverse transcriptase (hTERT) mRNA in serum by real‐time RT‐PCR, we previously reported this assay to be superior to other tumor markers for hepatoma. In this study, we aimed to clarify its clinical significance as a biomarker for lung cancer. In 112 patients with lung tumor and 80 individuals without cancer, we measured serum hTERT mRNA and epidermal growth factor receptor (EGFR) mRNA levels, using a quantitative one‐step real‐time RT‐PCR assay. We examined its sensitivity and specificity in lung cancer diagnosis, its clinical significance in comparison with other tumor markers, and its correlation with the clinical parameters using multivariate analyses and correlation relative tests. The copy number of serum hTERT mRNA was independently correlated with tumor size, tumor number, presence of metastasis and recurrence, and smoking (all P < 0.05). EGFR mRNA correlated with tumor number and clinical stage (both P < 0.05). The sensitivity and specificity in lung cancer diagnosis were 89.0% and 72.7% for hTERT mRNA, and 71.3% and 80.0% for EGFR mRNA, respectively. hTERT mRNA was superior to other tumor markers in lung cancer diagnosis. For both mRNAs, serum levels were significantly correlated with levels in lung cancer tissues (both P < 0.05). The copy number of hTERT mRNA significantly decreased after the surgical treatment. The data suggest that hTERT mRNA, especially when combined with EGFR mRNA, is a novel and excellent biomarker for pulmonary malignancies to diagnose and assess the clinical stage. (Cancer Sci 2006; 97: 1366–1373)

High expression of EZH2 is associated with tumor proliferation and prognosis in human oral squamous cell carcinomas

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.

Expression of Minichromosome Maintenance 2 (MCM2), Ki-67, and Cell-Cycle-Related Molecules, and Apoptosis in the Normal-Dysplasia-Carcinoma Sequence of the Oral Mucosa

Objective: We examined cell cycle and cell death biomarker trends with the normal-dysplasia-carcinoma sequence of the oral epithelia analyzing the pathological significance of a new biomarker, minichromosome maintenance 2 (MCM2). Methods: This study analyzed 12 patients with normal oral epithelia, 69 with dysplasia, and 35 with squamous cell carcinoma (SCC); in 13 patients, SCCs were preceded by dysplasia. The sections were immunostained for MCM2, Ki-67, P53, P27Kip1 and P21CIP1/WAF1, and conducted by TUNEL methods. Western blot analysis of MCM2 was performed in the 4 human cultured oral SCCs, all of which showed the expression. Results: Significantly higher labeling indices (LI; %) of MCM2, Ki-67, and P53, as well as lower LI of TUNEL indices (TI; %), P27, and P21 were noted in the SCCs than in the dysplasias. The 13 dysplasias developed SCC with significantly higher LI of MCM2 and P53, and lower LI of P21 than the other dysplasias (each p < 0.05). The LI of MCM2, P21 and the TI were not correlated with P53 expression. Conclusions: Oral dysplasia was characterized by lower cell proliferation and a higher frequency of cell death compared to SCCs. The higher LI of MCM2 and P53 and the lower LI of P21 might predict malignant transformation of oral dysplasia. MCM2 is regulated via a P53-independent pathway, and a useful biomarker of proliferating cells.

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2‐40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF‐C), VEGF‐D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF‐D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF‐C or VEGF‐D expression. High LVD, induced by VEGF‐C or VEGF‐D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.

Expression of minichromosome maintenance 7 (MCM7) in small lung adenocarcinomas (pT1): Prognostic implication.

Minichromosome maintenance (MCM) proteins, essential molecules in the initiation and elongation of DNA replication, have been considered to be good indicators of cell proliferation. We examined the expressions of MCM7 and Ki-67 in lung adenocarcinomas (ACs) with a diameter less than 3cm (pT1), to clarify their pathobiological significance. Immunohistochemistry was conducted to obtain labeling indices (LIs%) for MCM7, MCM2 and Ki-67 in 100 surgically removed pT1 ACs. The LIs were compared with clinicopathological profiles and overall survival rates. The mean LIs of MCM7 and Ki-67 were 20.2+/-15.2% and 13.7+/-11.2%, the value being higher in the former than in the latter (P<0.01). MCM7 LIs were significantly correlated with sex, histological grade, histological subtype, tumor size, LIs of Ki-67, MCM2 and P53 (P<0.05). LIs of MCM7 and Ki-67 were significantly higher in the 84 non-bronchioloalveolar carcinomas than in the 16 bronchioloalveolar carcinomas (P<0.01). Kaplan-Meier survival curves showed that patients with higher MCM7 LIs had poorer prognosis in the 100 pT1 ACs as well as in the 73 stage I ACs. Multivariate Cox regression analysis confirmed that the LIs of MCM7, but not the LIs of MCM2 and Ki-67, was an independent prognostic marker in the 73 stage I ACs. These results suggest that MCM7 is an independent prognostic marker, being more reliable than MCM2 or Ki-67 in human pT1 ACs as well as in human stage I ACs.

Genetic analysis of expression profile involved in retinoid metabolism in non-alcoholic fatty liver disease

Aim:u2002 The patients with non‐alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation.

Frequent occurrence of apoptosis is an early event in the oncogenesis of human gastric carcinoma.

Abstractu2002We examined the relationship between apoptosis and the progression of human gastric carcinoma. Studies were conducted on a total of 88 surgically removed stomachs, comprising 26 minute (less than 5xa0mm in diameter), 29 early (limited to the mucosal and submucosal layer) and 33 advanced carcinomas. Apoptotic cells were visualized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL). Serial sections were immunostained for p53 and Ki-67. The mean apoptotic indices (AI: percentage of TUNEL signal positive cells) of minute, early, and advanced carcinomas were 4.1±0.6, 3.8±1.2, and 4.0±1.2 in 46 well differentiated carcinomas, and 2.1±0.5, 2.7±0.9, and 2.2±1.1 in 42 poorly differentiated carcinomas, respectively. Similarly, the mean Ki-67 labelling indices (KI) were 39.2±7.8, 47.2±12.8, 52.6±13.1 in the former, and 35.0±9.3, 36.9±10.3, and 40.0±9.2 in the latter, respectively. Both mean AI and mean KI were significantly higher in well differentiated than in poorly differentiated carcinomas (P<0.05). However, the value of mean AI did not differ among minute, early, and advanced carcinomas in either histological type, while KI increased gradually with tumour progression. The frequency of nuclear p53 expression did not differ among the three categories, implying that the gene mutation is an early event in gastric carcinogenesis. There was no statistical significance between nuclear p53 expression and mean AI. These results suggest that the progression of gastric cancer is defined by a gradual increase of proliferative activity and constant occurrence of apoptosis and that naturally occurring apoptosis is induced predominantly via a p53-gene-independent pathway.

Retinoids ameliorate insulin resistance in a leptin-dependent manner in mice†

Transgenic mice expressing dominant‐negative retinoic acid receptor (RAR) α specifically in the liver exhibit steatohepatitis, which leads to the development of liver tumors. Although the cause of steatohepatitis in these mice is unknown, diminished hepatic expression of insulin‐like growth factor‐1 suggests that insulin resistance may be involved. In the present study, we examined the effects of retinoids on insulin resistance in mice to gain further insight into the mechanisms responsible for this condition. Dietary administration of all‐trans‐retinoic acid (ATRA) significantly improved insulin sensitivity in C57BL/6J mice, which served as a model for high‐fat, high‐fructose diet–induced nonalcoholic fatty liver disease (NAFLD). The same effect was observed in genetically insulin‐resistant KK‐Ay mice, occurring in concert with activation of leptin‐signaling pathway proteins, including signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2. However, such an effect was not observed in leptin‐deficient ob/ob mice. ATRA treatment significantly up‐regulated leptin receptor (LEPR) expression in the livers of NAFLD mice. In agreement with these observations, in vitro experiments showed that in the presence of leptin, ATRA directly induced LEPR gene expression through RARα, resulting in enhancement of STAT3 and insulin‐induced insulin receptor substrate 1 phosphorylation. A selective RARα/β agonist, Am80, also enhanced hepatic LEPR expression and STAT3 phosphorylation and ameliorated insulin resistance in KK‐Ay mice. Conclusion: We discovered an unrecognized mechanism of retinoid action for the activation of hepatic leptin signaling, which resulted in enhanced insulin sensitivity in two mouse models of insulin resistance. Our data suggest that retinoids might have potential for treating NAFLD associated with insulin resistance. (HEPATOLOGY 2012)

Nicorandil ameliorates ischaemia-reperfusion injury in the rat kidney

Nicorandil, an ATP‐sensitive potassium (KATP) channel opener and nitric oxide donor, is used in the treatment of angina and acute heart failure. Here we investigated the effects of two KATP channel openers, nicorandil and cromakalim on ischaemia reperfusion (I‐R) injury in the kidney.