Risaburo Akasaka

Molecular analysis of gastric differentiated-type intramucosal and submucosal cancers

Identification of the molecular characteristics of intramucosal (IMCs) and submucosal cancers (SMCs) is essential to our understanding of early gastric carcinogenesis. However, little is known regarding the differences between the 2 lesions. One hundred and forty‐eight patients with primary early gastric cancer [IMC, 106; SMC, 42] were characterized for expression of cell cycle‐related proteins and loss of heterozygosity (LOH). We also examined microsatellite instability (MSI) and methylation status. For LOH and methylation studies, we used a panel of 17 microsatellite markers (3p, 4p, 5q, 9p. 13q, 17p, 18q and 22q) and promoter regions of 9 genes (MLH‐1, RUNX3, p16, HPP1, RASSF2A, SFRP1, DKK‐1, ZFP64 and SALL4) that are frequently altered or methylated in gastric cancers. Overexpression of p53 and cyclin D1 was observed in SMC. In addition, low expression of p27 was more frequent in SMC than in IMC. Frequencies of 4p, 9p, 13q and 22q were significantly higher in SMC than in IMC. The SALL4 gene was frequently methylated in SMC compared with IMC. However, other gene methylations were common in both IMC and SMC. The frequency of LOH‐high status/methylation‐low status was significantly higher in SMC than in IMC. However, LOH‐low status/methylation‐high status in SMC was more frequently found in IMC. Our data confirm that methylation of cancer‐related genes plays a major role in the development of IMCs. Importantly, the results also show that gastric submucosal progression is characterized by the accumulation of specific genetic alterations. In addition, changes of cell cycle‐related proteins are associated with cancer progression.

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. Methodology DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. Results A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. Conclusions This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

Colonic Mucosa-Associated Lymphoid Tissue Lymphoma

Colonic mucosa-associated lymphoid tissue (MALT) lymphomas are rare and a definitive treatment has not been established. Solitary or multiple, elevated or polypoid lesions are the usual appearances of MALT lymphoma in the colon and sometimes the surface may reveal abnormal vascularity. In this paper we report our experience with four cases of colonic MALT lymphoma and review the relevant literature. The first patient had a smooth elevated lesion in the rectum and histopathologic examination of the biopsy from the lesion showed centrocyte-like cells infiltrating the lamina propria. Endoscopic ultrasonography (EUS) revealed thickening of the submucosa and muscularis propria. The patient underwent radiation therapy, and 9 months later a repeat colonoscopy showed complete resolution of the lesion. In case 2, colonoscopy showed a polyp in the cecum; the biopsy was diagnostic of MALT lymphoma. EUS detected a hypoechoic lesion confined to the mucosal layer of the colonic wall. The patient underwent endoscopic mucosal resection of the lesion and after 6 years of follow-up there was no evidence of recurrence. The third patient had a sessile elevated lesion in the sigmoid colon for which she underwent sigmoidectomy. Pathological examination of the surgical specimen was suggestive of MALT lymphoma. The last patient had a smooth elevated lesion in the rectum and magnification endoscopy showed irregular vascular pattern. The patient underwent endoscopic submucosal dissection, and biopsy examination showed the tumor to be MALT lymphoma. Although rare, awareness of MALT lymphoma of the colon is important to evaluate the patient appropriately and to plan further management.

The effect of granulocyte and monocyte adsorptive apheresis on serum cytokine levels in patients with ulcerative colitis.

BACKGROUND Granulocyte and monocyte adsorptive apheresis (GMA) with an Adacolumn has been reported to be effective as induction therapy in ulcerative colitis (UC). However, the effects of GMA on serial changes in cytokine levels have not been well characterized. We therefore, investigated cytokine levels in UC patients before and after treatment with GMA. A total of 16 patients with active UC, 10 men, and six women, mean age, 42.6 years were included. Fourteen patients had total colitis and two patients had left-sided colitis. The study included nine patients with a chronic intermittent course, six with a chronic continuous course and one with a single episode. The duration of each GMA session was 60 min at a flow rate of 30 mL/min as per study protocol. Serum levels of 17 cytokines were determined simultaneously using a Bio-Plex suspension array system before and after treatment with GMA. Serum interleukin (IL)-10 and macrophage inflammatory protein-1β levels were increased significantly in UC patients after GMA treatment compared to pre-treatment levels (P < 0.05). In particular, GMA treatment caused a significant increase in serum IL-10 levels compared to pre-treatment in patients with total colitis or with a chronic intermittent UC course. In conclusion, this investigation showed that GMA was associated with a marked increase in serum level of the anti-inflammatory cytokine, IL-10. The rise in circulating IL-10 is interesting, and potentially a significant factor in the efficacy of GMA in patients with inflammatory bowel diseases.

Dabigatran‐induced esophagitis: The prevalence and endoscopic characteristics

There have been some descriptions of dabigatran‐induced esophagitis in the literature. The aim of this study was to examine the prevalence and endoscopic characteristics of the disease.

Characterization of Follicular Lymphoma in the Small Intestine Using Double-Balloon Endoscopy

Follicular lymphomas occur rarely in the gastrointestinal tract, representing only 1–3% of all gastrointestinal tract B-cell non-Hodgkin lymphomas. We describe endoscopic analysis of 3 cases of follicular lymphoma in the small intestine using double-balloon endoscopy. Double-balloon endoscopy revealed multiple nodular lesions and elevated white patches, multiple polypoid lesions, and scattered white polypoid and nodular lesions in the duodenum and small intestine. Fuji Intelligent Chromo Endoscopy demonstrated small, whitish nodules, and narrow-band imaging showed a coiled, elongated vascular pattern within the elevated lesions. These cases are the first follicular lymphomas in the small intestine evaluated using narrow-band imaging or Fuji Intelligent Chromo Endoscopy to be reported.

Genome-wide analysis of DNA copy number alterations in early and advanced gastric cancers

To better understand progressive changes in gastric cancer (GC), early and advanced GCs (EGC and AGC, respectively) were examined for copy number alterations (CNAs). A crypt isolation method was used to isolate DNA from tumors and normal glands in 20 AGCs, and fresh tumor samples were obtained from 45 EGCs. We assessed CNAs for differentiated‐type GCs using an Infinium HumanCytoSNP‐12v2.1 BeadChip in EGCs and AGCs. The most frequent aberrations in EGC were gains at 8q23.3 (42.2%) and 8q23.2 (40%), and loss of heterozygosity (LOH) at 3p14.2 (24.2%), suggesting that these CNAs were involved in the development of EGC. On the other hand, the highest frequencies of gains in AGC were found at 8q24.21 (65%) and 8q24.3 (60%). The most frequent LOHs in AGC were at 11q24.3‐25, 11q23.2‐24.1, 11q14.1, and 12p11.21‐13.33, whereas that in EGC was at 3p14.2. In addition, regions of copy‐neutral LOHs in AGC were detected at 11q21, 11q13.3‐14.3, 11q11, 11p13‐15.3, 12q21.1, 12q12‐13.3 and 5q33.3‐35.1. Comparisons of gains in EGC and AGC showed significant differences at 12q22‐q23.2, 12q21.33, 11p12, 11p14.1, 12q21.31‐32.32, 3p12.3, 3p14.1, 10p15.1, 1q24.2 and 2q12.1. Copy neutral LOHs were significantly higher in AGC than in EGC at 14q32.11‐32.33, 14q21.3, 14q11.2, 5q11.2, 5q 13.3, 14q21.1‐23.2, 14q13.2‐13.3, 5q12.1‐12.3, 5q11.1, and 17p13.3. The total lengths of the CNAs were significantly greater in AGC than in EGC. We found that the pattern of CNAs in AGC was quite different from that in EGC. We suggest that increasing numbers of CNAs are associated with disease progression from EGC to AGC.

Clinicopathological Features and Magnifying Chromoendoscopic Findings of Non-Ampullary Duodenal Epithelial Tumors

Background and Aims: We aimed to investigate an association between clinicopathological features, including immunohistochemical mucin phenotypes, and magnifying chromoendoscopic findings with crystal violet staining (ME-CV) in non-ampullary duodenal epithelial tumors (NADETs). Methods: A total of 55 patients with NADET were divided into 3 groups by mucin phenotype: intestinal, gastrointestinal, or gastric. ME-CV findings were classified into 4 patterns: convoluted, leaf-like, reticular/sulciolar, and pinecone. The clinicopathological features and ME-CV findings were compared among the mucin phenotypes. Results: Tumors of the gastric type were located in the duodenal bulb (p < 0.001), and contained pyloric gland adenoma (p < 0.001) more frequently than the other types. White-light endoscopy indicated that milk-white mucosa was less frequent in tumors of the gastric type than in those of the gastrointestinal type (p = 0.006) and the intestinal type (p < 0.001). ME-CV findings were significantly different between the gastric type and the other type (p = 0.028). Totally, 5 of 8 tumors of the gastric type manifested a pinecone pattern, 4 of which were compatible with pyloric gland adenoma. Conclusions: The endoscopic findings of NADETs differ according to mucin phenotype. A pinecone pattern under ME-CV may be characteristic of NADETs of the gastric type, especially pyloric gland adenoma.

Molecular subtypes of colorectal cancers determined by PCR-based analysis

Tumor tissue consists of a heterogeneous cell population. The allelic imbalance (AI) ratio, determined in isolated tumor glands, is a good index of tumor heterogeneity. However, associations of the patterns of AI and microsatellite instability (MSI) development, observed in most cases of colorectal cancer (CRC), with tumor progression have not been reported previously. In this study, we examined whether CRC genetic profiles stratified by a combination of the AI ratio and MSI facilitate categorization of CRC, and whether these genetic profiles are associated with specific molecular alterations in CRC. A crypt isolation method was used to isolate DNA from tumors and normal glands obtained from 147 sporadic CRCs. AI and MSI statuses were determined using PCR‐based microsatellite analysis and stratified based on AI ratio and MSI status. DNA methylation status (high methylation, intermediate methylation and low methylation status and mutations in KRAS, BRAF, and TP53 were examined. In addition, mucin markers were immunostained. Based on this analysis, four subgroups were categorized. Subgroup 1 was characterized by a high MSI status and BRAF mutation; subgroup 2 was closely associated with a high AI ratio, which accumulated during the early phases of colorectal carcinogenesis, and TP53 mutation; subgroup 3 was associated with a low AI ratio, seen during the later phases of colorectal carcinogenesis, and KRAS mutation; and subgroup 4 was defined as a minor subgroup. These results confirmed that classification of distinct molecular profiles provides important insights into colorectal carcinogenesis.

Practical fecal calprotectin cut-off value for Japanese patients with ulcerative colitis

AIM To determine appropriate fecal calprotectin cut-off values for the prediction of endoscopic and histologic remission in Japanese patients with ulcerative colitis (UC). METHODS We performed a cross-sectional observational study of 131 Japanese patients with UC and measured fecal calprotectin levels by fluorescence enzyme immunoassay. The clinical activity of UC was assessed with the partial Mayo score (PMS). Relapse was defined as increase of PMS by 2 points or more in stool frequency or rectal bleeding subscore. The endoscopic and histologic activities of UC were evaluated in 50 patients within a 2-mo period from fecal sampling. Endoscopic activity was determined by Mayo endoscopic subscore, Rachmilewitz endoscopic index, and ulcerative colitis endoscopic index of severity. The histologic grade of inflammation was evaluated with biopsy specimens obtained from the endoscopically most severely inflamed site, according to the scheme by Matts grade and Riley’s score. RESULTS Fecal calprotectin levels varied from 1-20783 μg/g. There was a significant correlation between the partial Mayo score and fecal calprotectin levels (r = 0.548, P < 0.001). In 50 patients who underwent colonoscopy with biopsy, levels were significantly correlated with the Mayo endoscopic subscore (r = 0.574, P < 0.001), Rachmilewitz endoscopic index (r = 0.628, P < 0.001), ulcerative colitis endoscopic index of severity (r = 0.613, P < 0.001), Riley’s histologic score (r = 0.400, P = 0.006), and Matts grade (r = 0.586, P < 0.001). Receiver-operating characteristic analyses identified the best cut-off value for the prediction of endoscopic remission as 288 μg/g, with an area under the curve of 0.777 or 0.823, while that for histologic remission was 123 or 125 μg/g, with an AUC of 0.881 or 0918, respectively. Of the 131 study patients, 88 patients in clinical remission were followed up 6 mo. During the follow-up period, 19 patients relapsed. The best fecal calprotectin cut-off value for predicting relapse was 175 μg/g. CONCLUSION Fecal calprotectin is a predictive biomarker for endoscopic and histologic remission in Japanese patients with UC.