Katsutaka Sannomiya

B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P

Background:Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients.Methods:ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR–restriction fragment length polymorphism (RFLP) and PCR–SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method.Results:B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF<SSA/P<cancer. The most commonly methylated genes in SSA/P were PQLC1, HDHD3, RASL10B, FLI1, GJA3, and SLC26A2. Some of these genes were methylated in ACF, whereas all genes were methylated in cancers. Immunohistochemistry revealed their silenced expression. Microsatellite instability and MLH1 methylation were observed only in cancer. The prevalence and number of ACF were significantly higher in SSA/P patients than in normal subjects. A significant correlation was seen between the numbers of SSA/P and ACF in SSA/P patients.Conclusions:Our results suggest that ACF are precursor lesions of the SSA/P-cancer sequence in patients with SSA/P, where ACF arise by B-RAF mutation and methylation of some of the six identified genes and develop into SSA/Ps through accumulated methylation of these genes.

High antitumor activity of pladienolide B and its derivative in gastric cancer

The antitumor activity of pladienolide B, a novel splicing inhibitor, against gastric cancer is totally unknown and no predictive biomarker of pladienolide B efficacy has been reported. We investigated the antitumor activity of pladienolide B and its derivative on gastric cancer cell lines and primary cultured cancer cells from carcinomatous ascites of gastric cancer patients. The effect of pladienolide B and its derivative on six gastric cancer cell lines was investigated using a MTT assay and the mean IC50 values determined to be 1.6 ± 1.2 (range, 0.6–4.0) and 1.2 ± 1.1 (range, 0.4–3.4) nM, respectively, suggesting strong antitumor activity against gastric cancer. The mean IC50 value of pladienolide B derivative against primary cultured cells from 12 gastric cancer patients was 4.9 ± 4.7 nM, indicative of high antitumor activity. When 18 SCID mice xenografted with primary cultured cells from three patients were administered the pladienolide B derivative intraperitoneally, all tumors completely disappeared within 2 weeks after treatment. Histological examination revealed a pathological complete response for all tumors. In the xenograft tumors after treatment with pladienolide B derivative, immature mRNA were detected and apoptotic cells were observed. When the expressions of cell‐cycle proteins p16 and cyclin E in biopsied gastric cancer specimens were examined using immunohisctochemistry, positivities for p16 and cyclin E were significantly and marginally higher, respectively, in the low‐IC50 group compared with the high‐IC50 group, suggesting the possibility that they might be useful as predictive biomarkers for pladienolide B. In conclusion, pladienolide B was very active against gastric cancer via a mechanism involving splicing impairment and apoptosis induction.

Reducing Effect of Feeding Powdered Nacre of Pinctada maxima on the Visceral Fat of Rats

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.

Novel des-γ-carboxy prothrombin in serum for the diagnosis of hepatocellular carcinoma.

Serum des‐γ‐carboxy prothrombin (DCP) levels using a newly developed electrochemiluminescence immunoassay (ECLIA, novel DCP [NX‐DCP]) were measured, and the utility of NX‐DCP and DCP/NX‐DCP ratio for the diagnosis of hepatocellular carcinoma (HCC) was investigated. Antigenic differences in DCP between HCC and non‐HCC patients were elucidated.

Suppressive effect of RAS inhibitor manumycin A on aberrant crypt foci formation in the azoxymethane-induced rat colorectal carcinogenesis model.

The chemopreventive effect of RAS inhibitors on colorectal cancer is unknown. Because aberrant crypt foci (ACF), earliest preneoplastic lesions, are highly positive for K‐RAS mutation, RAS inhibitors are likely to be effective for chemoprevention. Therefore, in the present study, the suppressive effect of a RAS inhibitor, manumycin A, on ACF formation in an azoxymethane (AOM)‐induced rat colorectal carcinogenesis model was investigated.

Molecular imaging of aberrant crypt foci in the human colon targeting glutathione S-transferase P1-1

Aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancers (CRCs), are a good surrogate marker for CRC risk stratification and chemoprevention. However, the conventional ACF detection method with dye-spraying by magnifying colonoscopy is labor- and skill-intensive. We sought to identify rat and human ACF using a fluorescent imaging technique that targets a molecule specific for ACF. We found that glutathione S-transferase (GST) P1-1 was overexpressed in ACF tissues in a screening experiment. We then synthesized the fluorogenic probe, DNAT-Me, which is fluorescently quenched but is activated by GSTP1-1. A CRC cell line incubated with DNAT-Me showed strong fluorescence in the cytosol. Fluorescence intensities correlated significantly with GST activities in cancer cell lines. When we sprayed DNAT-Me onto colorectal mucosa excised from azoxymethane-treated rats and surgically resected from CRC patients, ACF with strong fluorescent signals were clearly observed. The ACF number determined by postoperative DNAT-Me imaging was almost identical to that determined by preoperative methylene blue staining. The signal-to-noise ratio for ACF in DNAT-Me images was significantly higher than that in methylene blue staining. Thus, we sensitively visualized ACF on rat and human colorectal mucosa by using a GST-activated fluorogenic probe without dye-spraying and magnifying colonoscopy.

Radiofrequency Ablation Using a Balloon Catheter for Hepatocellular Carcinoma Adjacent to the Gastrointestinal Tract: Experimental and Clinical Study

Purpose: Radiofrequency ablation (RFA) is a safe and effective technique for hepatocellular carcinoma and it has minimal morbidity and mortality. One of the most important major complications of RFA is perforation of the gastrointestinal tract, which occurs when the tumor is adjacent to the digestive tract. In particular, the risk is much higher in patients with lesions located within 1 cm from the liver surface in proximity to the digestive tract. In such cases, an artificial ascites technique has been employed. However, the separation of adjacent digestive organ from the liver is insufficient in this method. Therefore, in this study, to overcome this problem, we devised a novel RFA technique using a double-balloon catheter. In the first step, an animal experiment was performed to evaluate the safety and feasibility of the balloon RFA method. In the second step, the human pilot trial was carried out to evaluate the safety, feasibility and effectiveness of this method. Materials and Methods: We produced an 8 Fr silicone catheter equipped with 2 balloons of 2.5 cm diameter. In experiments using pigs, we first inserted this balloon catheter percutaneously into the peritoneal space between the liver and gastrointestinal tracts, filled it with cooled water, and performed RFA for in normal liver 1 cm from the liver surface. Then, heat damage to the excised liver and gastrointestinal tract was evaluated macroscopically andmicroscopically. In a human pilot study, balloon catheter RFA was performed in 4 patients with HCC (1.5 ± 0.7 cm) abutting the gastrointestinal tract. Results: In pigs, we performed each 6 RFA sessions with or without balloon catheter. It was technically easy to place the balloon catheter between liver and gastrointestinal tracts to separate them. Heat damage reached the liver surface in all lesions. In the groupwith balloon catheter, no heat damage of the gastrointestinal tracts was observed (0%, 0/6). In contrast, in the group without balloon catheter, heat damage was observed in 5/6 (83.3%): stomach (2/6), small intestine (2/6), and omentum (1/6). The coolant temperature in the balloon was significantly increased after RFA, suggesting that the heat generated by RFA was absorbed by the coolant. In the human pilot study, balloon catheter RFA was easily performed in all patients without associated complications. CT confirmed complete ablation with an appreciable safety margin in all patients, without recurrence for 20.3 ± 4.5 months. Conclusions: RFA with our balloon catheter is safe and effective for the treatment of HCC abutting the gastrointestinal tract, suggesting an expanded indication of these lesions for RFA.