Hiroyuki Takamaru

Upregulation of miR-196a and HOTAIR Drive Malignant Character in Gastrointestinal Stromal Tumors

Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs.

A Novel Correlation between LINE-1 Hypomethylation and the Malignancy of Gastrointestinal Stromal Tumors

Purpose: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy. Experimental Design: A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens. Results: LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations. Conclusions: Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations. Clin Cancer Res; 16(21); 5114–23. ©2010 AACR.

Role of DNA methylation in the development of diffuse-type gastric cancer.

Cancer cells exhibit two opposing methylation abnormalities: genome-wide hypomethylation and gene promoter hypermethylation. Downregulation of E-cadherin (CDH1) plays a key role in the development of diffuse-type gastric cancer, and DNA methylation is a major cause of the gene’s silencing. Hereditary diffuse gastric cancer is caused by germline mutation of CDH1 gene, and DNA methylation frequently serves as the second hit completely inactivating the gene. In sporadic diffuse-type gastric cancer, methylation of CDH1 is more prevalent than mutation of the gene. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBV-associated GC) is characterized by concurrent methylation of multiple genes, and diffuse-type gastric cancer is frequently seen among EBV-associated GCs. Patients with pangastritis or enlarged-fold gastritis, which are both caused by Helicobacter pylori infection, reportedly have an increased risk for diffuse-type gastric cancer. Notably, the gastric mucosa of enlarged-fold gastritis patients exhibits CDH1 hypermethylation and genome-wide hypomethylation. These data suggest that aberrant DNA methylation is an essential promoter of carcinogenesis in individuals at high risk for diffuse-type gastric cancer.

Epigenetic Alteration of DNA in Mucosal Wash Fluid Predicts Invasiveness of Colorectal Tumors

Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors. Cancer Prev Res; 4(5); 674–83. ©2011 AACR.

Aberrant Methylation of RASGRF1 Is Associated with an Epigenetic Field Defect and Increased Risk of Gastric Cancer

Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P < 0.001; 8.3% vs. 19.4%, P < 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203–12. ©2012 AACR.

A pilot study of fluorescent imaging of colorectal tumors using a γ-glutamyl-transpeptidase-activatable fluorescent probe.

Backgrounds/Aim: Colorectal laterally spreading tumors (LSTs) are sometimes difficult to visualize even with image-enhanced endoscopy. γ-Glutamyl-transpeptidase (GGT) is a cell surface-associated enzyme that is overexpressed in various types of human cancers. Furthermore, GGT expression is higher in colorectal cancer cells than in normal colorectal mucosa. γ-Glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), an activatable fluorescent probe, is nonfluorescent under a neutral pH and normal cellular environment; however, it turns highly fluorescent upon reaction with GGT. We evaluated ex vivo fluorescent imaging of colorectal LSTs using this GGT-activatable fluorescent probe. Methods: Between March 2013 and March 2014, 30 endoscopically resected colorectal LSTs were prospectively included in this study. Each was analyzed by first taking a baseline image before spraying, then spraying with gGlu-HMRG onto the freshly resected specimen, and finally taking fluorescent images 15 min after spraying with a dedicated imaging machine. Results: Of the LSTs, 67% rapidly showed positive fluorescent activity. These activities were shown in adenoma (54%) and carcinoma in adenoma (76%), and in LST-granular type (80%) and LST-nongranular type (40%). Conclusion: Topically spraying gGlu-HMRG enabled rapid and selective fluorescent imaging of colorectal tumors owing to the upregulated GGT activity in cancer cells.

Long-term clinical outcomes of endoscopic submucosal dissection for colorectal neoplasms in 423 cases: a retrospective study

Background and study aim Endoscopic submucosal dissection (ESD) is known as a curative treatment for colorectal superficial neoplasms. There is however a need for more long-term clinical data to establish the full advantages of colorectal ESD regarding very low recurrence rates. The aim of this retrospective study was to determine long-term clinical outcomes of colorectal ESD. Methods A total of 423 lesions treated by ESD for colorectal adenoma/dysplasia or adenocarcinoma between 1998 and 2008 at a single high volume referral center were included. We conducted a retrospective survey on patients with follow-up and obtained complete 1-, 3-, and 5-year outcome data for 358 (85 %), 292 (69 %), and 209 (49 %) lesions, respectively. Curative resection was defined when the pathological specimen had carcinoma-free resection margins, irrespective of piecemeal or en bloc resection, without submucosal deep invasion (≥ 1000 µm), lymphovascular involvement, or a poorly differentiated adenocarcinoma component. Results After a median 4.9 years of follow-up, the 3-year overall cumulative endoscopic recurrence rate and cancerous recurrence rate were 2.9 % (95 % confidence interval [95 %CI] 1.2 - 4.7) and 1.1 % (0 - 2.1), respectively. The 5-year overall cumulative endoscopic recurrence and cancerous recurrence rates were 3.8 % (1.7 - 5.9) and 1.6 % (0.1 - 3.0), respectively. In 361 lesions eligible for endoscopic follow-up, the 3-year endoscopic recurrence and cancerous recurrence rates were 2.4 % (0.8 - 4.1) and 0.4 % (0 - 1.4), respectively. Multivariate analysis revealed that piecemeal resection and submucosal deep tumor invasion were associated with recurrence. Conclusions The current study demonstrated favorable long-term clinical outcomes of colorectal ESD when en bloc curative resection is achieved.

Use of the dye-guided sentinel lymph node biopsy method alone for breast cancer metastasis to avoid unnecessary axillary lymph node dissection

For sentinel lymph node biopsy (SLNB), a combination of dye-guided and γ-probe-guided methods is the most commonly used technique. However, the number of institutes in which the γ-probe-guided method is able to be performed is limited, since special equipment is required for the method. In this study, SLNB with the dye-guided method alone was evaluated, and the clinicopathological characteristics were analyzed to identify any factors that were predictive of whether the follow-up axillary lymph node dissection (ALND) was able to be omitted. A total of 374 patients who underwent SLNB between 1999 and 2009 were studied. The SLN identification rate was analyzed, in addition to the false-positive and false-negative rates and the correlation between the clinicopathological characteristics and axillary lymph node metastases. The SLN was identified in 96.8% of cases, and, out of the patients who had SLN metastasis, 63.0% did not exhibit metastasis elsewhere. The sensitivity was 96.4% and the specificity was 100%. The false-negative rate was 3.6%. Univariate analyses revealed significant differences in the lymph vessel invasion (ly) status, nuclear grade (NG), maximum tumor size and the percentage of the area occupied by the tumor cells in the SLN (SLN occupation ratio) between the patients with and without non-SLN metastasis, indicating that these factors may be predictive of axillary lymph node metastasis. Multivariate analysis revealed that ly status was an independent risk factor for non-SLN metastasis. In conclusion, SLN with the dye-guided method alone provided a high detection rate. The study identified a predictive factor for axillary lymph node metastasis that may improve the patients’ quality of life.

Endoscopic submucosal dissection for colorectal neoplasms

Endoscopic submucosal dissection (ESD) is an established therapeutic technique for the treatment of gastrointestinal neoplasms. Because it is typically completed as en bloc resection, this technique provides a complete specimen for precise pathological evaluation. On the other hand, ESD is not as widely applied in treating colorectal neoplasms as with gastric cancers, due to its technical difficulty, longer procedure time, and increased risk of perforation. However, some devices that facilitate ESD and improve the safety of the procedure have been recently reported, and the use of the technique has gradually spread worldwide. Endoscopists who begin to perform ESD need to recognize the indications of ESD, the technical issue involved in this procedure, and its associated complications. This review outlines the methods and certain types of devices used for colorectal ESD.

Desmoplastic malignant mesothelioma originating from the peritoneum

To the Editor, Malignant mesothelioma is an uncommon tumor arising in body cavities lined by mesothelium. Primary malignant peritoneal mesotheliomas make up 20–30 % of all mesotheliomas as represented by the total of pleural origin and are categorized into an epitheloid type. Desmoplastic mesothelioma categorized as a specific subtype of sarcomatoid mesothelioma is very rare and has a poor prognosis. Consequently, a peritoneal origin combined with desmoplastic histology is an extremely rare disease entity. A 74-year-old man presented to our hospital in February 2008 complaining of progressive abdominal distension due to an enlarging abdominal mass. Soluble interleukin-2 receptor was slightly elevated (452 U/mL) while the tumor markers examined were within the normal range. The tumor was rich in lipid content with an ill-defined border by contrast-enhanced abdominal CT scan (Fig. 1a) and magnetic resonance imaging (MRI) (Fig. 1b) the tumor was not enhanced but appeared to be originating from small bowel mesentery. It was difficult to make a definitive diagnosis of malignant lymphoma or mesenteric panniculitis without histological examination. Exploratory laparotomy revealed disseminated small white nodules in the mesentery (Fig. 1c) and the histology led to a pathologic diagnosis of desmoplastic mesothelioma, with a spindlelike shape based on collagenous fibers forming a storiform pattern (Fig. 1d). On immunohistochemistry, the tumor cells expressed vimentin, a smooth muscle actin, and D2-40 but tests for the following epithelial markers, calretinin, WT-1, desmin, CD34, and S-100 were negative (Fig. 1e–g). FISH analysis excluded a p16 deletion. Combination therapy based on either irinotecan (CPT-11) or gemcitabine (GEM) with cisplatin (CDDP) [1] was ineffective. We then gave CDDP intraperitoneally for 15 courses until we judged him to have unresponsive progressive disease. He was placed on optimal supportive care and survived for 49 months after the initial diagnosis. This is the first report on desmoplastic malignant mesothelioma of the peritoneum, since the disease entity was defined by the WHO in 2004. In an extensive review of the literature, we have identified only one previous report of this rare disease, published in 1982 [2]. The present patient is categorized as a diffuse mesothelioma based on the type of tumor spread. Generally, malignant peritoneal mesothelioma is considered to have a poor prognosis. Risk factors for malignant mesothelioma tumorigenesis are known to be an exposure to SV40 virus or to asbestos. The association of peritoneal mesothelioma with asbestos exposure is limited to a few cases and the associations authenticity is controversial. Regarding molecular mechanisms, somatic mutations of CDKN2A or NF2 or epigenetic alterations of RASSF1 gene promoter have been reported to be involved in tumorigenesis [3]. Currently, there are no established standard treatments for malignant peritoneal mesothelioma. Multidisciplinary strategies involving cyto-reductive surgery, systemic chemotherapy, and/or intraperitoneal chemotherapy as the present patient received have improved patient survival. Early diagnosis by exploratory laparotomy followed by combination chemotherapy may have contributed to longer survival for our patient than those of previous reports (median overall survival time for 20.1–26.8 months [1]). In conclusion, our patient highlights two important lessons; first, earlier intervention may contribute to longer H. Takamaru (&) Y. Arimura Y. Shinomura First Department of Internal Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo 060-8543, Japan e-mail: h.takamaru@gmail.com