Masao Miyashita

MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus: Associations with survival

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barretts, is still lacking. Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples. Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barretts were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy. Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

Primary undifferentiated small cell carcinoma of the esophagus

Abstract We histologically examined undifferentiated small cell carcinoma of the esophagus from 21 patients and used immunohistochemical methods for detection of chromogranin A and p53, bc1–2, and Rb oncoproteins. Nine (43%) of the 21 carcinomas consisted solely of undifferentiated cells, but heterogeneous components of in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma were observed in the other 12 (57%) tumors. Squamous cell carcinoma in situ was observed in the mucosa adjacent to the main tumor in 7 (50%) of the 14 resected esophageal specimens. An admixture of invasive squamous cell carcinoma and undifferentiated carcinoma was observed in 4 (19%) of the 21 tumors, and mucoepidermoid carcinoma was noted in one case. Chromogranin A staining yielded a positive reaction in two (10%) undifferentiated components but was negative in all heterogeneous components. Multiple sites of p53 immunopositivity were seen in the undifferentiated component of 17 (81%) of the 21 tumors, as well as in the in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma components of 9 (75%) of 12 tumors. Seven (33%) of the 21 tumors showed positive bc1–2 immunoreactivity in the small cell component, but all of the heterogeneous components were negative. Rb protein immunoreactivity was observed in the small cell component of one (5%) case and in 9 (75%) of the 12 heterogeneous components. Six (86%) of the seven in situ squamous cell carcinoma components were positive for Rb protein. Eighteen (86%) of the 21 patients died within 24 months of diagnosis. Two patients (10%) who survived for more than 24 months had received chemotherapy.

Inflammatory and MicroRNA Gene Expression as Prognostic Classifier of Barrett's-Associated Esophageal Adenocarcinoma

Purpose: Esophageal cancer is one of the most aggressive and deadly forms of cancer; highlighting the need to identify biomarkers for early detection and prognostic classification. Our recent studies have identified inflammatory gene and microRNA signatures derived from tumor and nontumor tissues as prognostic biomarkers of hepatocellular, lung, and colorectal adenocarcinoma. Here, we examine the relationship between expression of these inflammatory genes and micro RNA (miRNA) expression in esophageal adenocarcinoma and patient survival. Experimental Design: We measured the expression of 23 inflammation-associated genes in tumors and adjacent normal tissues from 93 patients (58 Barretts and 35 Sporadic adenocarcinomas) by quantitative reverse transcription-polymerase chain reaction. These data were used to build an inflammatory risk model, based on multivariate Cox regression, to predict survival in a training cohort (n = 47). We then determined whether this model could predict survival in a cohort of 46 patients. Expression data for miRNA-375 were available for these patients and was combined with inflammatory gene expression. Results: IFN-γ, IL-1α, IL-8, IL-21, IL-23, and proteoglycan expression in tumor and nontumor samples were each associated with poor prognosis based on Cox regression [(Z-score)>1.5] and therefore were used to generate an inflammatory risk score (IRS). Patients with a high IRS had poor prognosis compared with those with a low IRS in the training (P = 0.002) and test (P = 0.012) cohorts. This association was stronger in the group with Barretts history. When combining with miRNA-375, the combined IRS/miR signature was an improved prognostic classifier than either one alone. Conclusion: Transcriptional profiling of inflammation-associated genes and miRNA expression in resected esophageal Barretts-associated adenocarcinoma tissues may have clinical utility as predictors of prognosis. Clin Cancer Res; 16(23); 5824–34. ©2010 AACR.

Relationship between altered expression levels of MIR21, MIR143, MIR145, and MIR205 and clinicopathologic features of esophageal squamous cell carcinoma

In spite of the undisputed importance of altered expression patterns of microRNAs (miRNAs) in various cancers, there is little information on the clinicopathologic significance of cancer-related miRNAs (MIR21, MIR143, MIR144, MIR145, and MIR205) in esophageal squamous cell carcinoma (ESCC). We examined the expression levels of the precursor and mature miRNA genes in ESCC using real-time polymerase chain reaction (PCR). We also investigated the mRNA expression levels of processing elements (RNASEN, DGCR8, and DICER1) that participate in miRNA-biogenesis pathway. Furthermore, we analyzed the relationships between the expression levels of these five miRNAs and the clinicopathologic parameters of ESCC patients. The expression levels of mature MIR21 and mature MIR145 were higher in ESCC than those in normal epithelium (P < 0.05). The mature/pre ratio of MIR21 in ESCC was higher than that in normal epithelium (P < 0.05). With regard to miRNA-processing elements, the expression level of RNASEN was higher in ESCC than in normal epithelium (P < 0.05). Furthermore, altered expression of these miRNAs was related to the clinicopathologic features of ESCC patients. The high expression of mature MIR21 and mature MIR205 was associated with lymph node positivity in ESCC patients (P < 0.05). The high levels of expression of mature MIR143 and mature MIR145 were associated with recurrence of metastasis in ESCC patients (P < 0.05). The findings may imply that miRNA biogenesis is aberrantly accelerated in ESCC. Analysis of the expression levels of miRNAs should provide useful information for evaluation of the staging, prognosis, and treatment of ESCC patients.

Telomerase activity in esophageal carcinoma

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA. Immortalized and carcinoma cells show no loss of telomere length during cell division. Telomerase activity has been demonstrated in carcinomas of various organs, but not in nonneoplastic tissues. In patients with esophageal carcinoma, no data have been reported concerning the relationship between telomerase activity and clinicopathological findings.

Endoscopic dexamethasone injection following balloon dilatation of anastomotic stricture after esophagogastrostomy

BACKGROUND Anastomotic stricture is common after esophagogastrostomy. Recent advances in nonsurgical treatment include the silicon bougie and balloon dilatation. However, simple dilatation alone with a silicon bougie or endoscopic balloon dilator was repeated a mean of 4.7+/-5.4 times to control anastomotic stricture because of its temporary effect. METHODS For 11 patients, endoscopic injection of dexamethasone (8 mg) around the anastomosis was done immediately after balloon dilatation (40 psi for 5 minutes). RESULTS This method significantly reduced the number of the dilatations to 1.1+/-0.3 (P < 0.05). Ten of the 11 patients did not need any further treatment. There were no side effects or complications of dexamethasone injection. CONCLUSION A combination of endoscopic balloon dilatation and dexamethasone injection provided an easy and safe method for preventing the recurrence of anastomotic stricture.

Structures of the normal esophagus and Barrett?s esophagus

This article reviews and discusses several topics, mainly relating to the histology of the normal esophagus and of Barrett’s esophagus, in order to facilitate the understanding of Barrett’s esophagus. The border between the esophagus and stomach is considered in Japan to be the lower limit of longitudinal vessels which are visible in the lower segment of the esophagus at endoscopy. This definition has been authorized by the Japanese Society for Esophageal Diseases. The longitudinal vessels are also visible through the metaplastic columnar epithelium of Barrett’s esophagus. Identification of the esophageal glands proper in biopsy specimens can assist in the histologic diagnosis of Barrett’s esophagus. The histologic diagnosis of Barrett’s esophagus in biopsy specimens, in relation to the presence of esophageal glands proper, is discussed. Ciliated pseudostratified epithelium is discussed in detail, including the fact that it is thought to be an intermediate stage between squamous and columnar epithelium at the esophagogastric junction and at ectopic gastric mucosa in the upper esophagus. The differences in the histopathologic criteria for a diagnosis of Barrett’s adenocarcinoma between Western countries and Japan are also discussed. The four editions of the Comprehensive Registry of Esophageal Cancer in Japan, and the Long Term Results of Esophagectomy in Japan (1988–2000), published by the Japanese Society for Esophageal Diseases and available on its website (http://jsed.umin.ac.jp), are introduced. These editions give detailed information on the pathology, endoscopic features, radiation treatment, and surgery of esophageal cancer in Japan.

Quantitative Detection of p53 Mutations in Plasma DNA from Tobacco Smokers

In lung tumors, the p53 tumor suppressor gene is commonly mutated with a characteristic mutation spectrum. The amount of and alterations in plasma DNA, such as mutations in p53, were associated with several cancers. Few studies used quantitative methods of high sensitivity. Previously, we observed p53 mutations in the noncancerous tissue that differed from those in lung tumors using the highly sensitive p53 mutation load assay. Based on our observation of an increased p53 mutation load in nontumorous lung tissue in smokers, we hypothesized that plasma DNA may contain mutant p53 indicative of tobacco smoke exposure and will be an effective biomarker of lung cancer or smoking exposure. We modified the p53 mutation load assay to detect mutations at p53 codons 248 and 249, common mutations in lung cancer, in plasma DNA samples with a sensitivity of 1:5,000. The assay was applied to a set of lung cancer cases (n = 39), hospital controls (n = 21), and population controls (n = 20) from a larger study. Controls were selected to consist of equal numbers of both ever and never smokers. The p53 mutation load (mutated p53 copies per total number of p53 copies) was associated with smoking (P = 0.06), but not with lung cancer (P = 0.59). Most of the individuals with p53 mutations observed in plasma DNA were ever smokers and the p53 mutation load was higher in those who smoked for longer durations (P = 0.04). In summary, we were able to detect p53 mutations in plasma DNA from healthy individuals and our data suggest that p53 mutations in plasma DNA may be a marker of carcinogen exposure from tobacco smoke.

Combination of protein coding and noncoding gene expression as a robust prognostic classifier in stage I lung adenocarcinoma.

Prognostic tests for patients with early-stage lung cancer may provide needed guidance on postoperative surveillance and therapeutic decisions. We used a novel strategy to develop and validate a prognostic classifier for early-stage lung cancer. Specifically, we focused on 42 genes with roles in lung cancer or cancer prognosis. Expression of these biologically relevant genes and their association with relapse-free survival (RFS) were evaluated using microarray data from 148 patients with stage I lung adenocarcinoma. Seven genes associated with RFS were further examined by quantitative reverse transcription PCR in 291 lung adenocarcinoma tissues from Japan, the United States, and Norway. Only BRCA1, HIF1A, DLC1, and XPO1 were each significantly associated with prognosis in the Japan and US/Norway cohorts. A Cox regression-based classifier was developed using these four genes on the Japan cohort and validated in stage I lung adenocarcinoma from the US/Norway cohort and three publicly available lung adenocarcinoma expression profiling datasets. The results suggest that the classifier is robust across ethnically and geographically diverse populations regardless of the technology used to measure gene expression. We evaluated the combination of the four-gene classifier with miRNA miR-21 (MIR21) expression and found that the combination improved associations with prognosis, which were significant in stratified analyses on stage IA and stage IB patients. Thus, the four coding gene classifier, alone or with miR-21 expression, may provide a clinically useful tool to identify high-risk patients and guide recommendations regarding adjuvant therapy and postoperative surveillance of patients with stage I lung adenocarcinoma.

Esophageal motility in Japanese patients with Barrett's esophagus

BackgroundThe prevalence of gastroesophageal reflux disease has been increasing in Japan as it has in Western countries, but Barrett’s esophagus (BE) is less common in Japan than in Western countries. The aim of this study, therefore, was to investigate esophageal motility and clinical characteristics in Japanese patients with BE.MethodsTen patients with BE were compared with ten patients with mild reflux esophagitis (RE), ten patients with severe RE, and ten healthy subjects of comparable age and sex. The prevalence of Helicobacter pylori was investigated in the patients with BE. The intraluminal microtransducer method was used to test for esophageal motility. Basal lower esophageal sphincter (LES) pressure was assessed by the rapid pull-through method. The esophageal wave after ten repeated 5-ml water swallowings at 30-s intervals was measured at 3, 8, 13, and 18 cm above the LES.ResultsThe basal LES pressure, the amplitude of the esophageal wave at 3 and 8 cm above the LES, and the frequency of primary peristalsis in the severe RE group and BE group were significantly lower than the values in the healthy subjects and the mild RE group. The amplitude of the esophageal wave 13 cm above the LES in the BE group was significantly lower than that in the healthy subjects and the mild RE group. There was no difference between the severe RE group and the BE group in the basal LES pressure and the amplitude of the esophageal wave. The frequency of primary peristalsis in the BE group, however, was significantly lower than that in the severe RE group. Nine of the ten patients with BE were H. pylori-negative.ConclusionsOur conclusions are that esophageal dysmotility in Japanese patients with BE represents an advanced stage of severe RE, and that most Japanese patients with BE are H. pylori-negative.