Endoscopic hand suturing using a modified through-the-scope needle holder for mucosal closure after colorectal endoscopic submucosal dissection: Prospective multicenter study (with video).

OBJECTIVES: Endoscopic hand suturing (EHS) is a novel technique for closing a mucosal defect after endoscopic submucosal dissection (ESD). We investigated the technical feasibility of colorectal EHS using a modified flexible through-the-scope needle holder. METHODS: This was a prospective multicenter study conducted at two referral centers between June 2022 and April 2023. This study included colorectal neoplasms 20-50 mm in size located in the sigmoid colon or rectum. A modified flexible through-the-scope needle holder, with an increased jaw width to facilitate needle grasping, was used for colorectal EHS. The primary end-points were sustained closure rate on second-look endoscopy (SLE) performed on postoperative days 3-4 and suturing time for colorectal EHS. Secondary end-points included complete closure rate and delayed adverse events. RESULTS: We enrolled 20 colorectal neoplasms in 20 patients, including four patients receiving antithrombotic agents. The tumor location was as follows: lower rectum (n = 8), upper rectum (n = 2), rectosigmoid colon (n = 4), and sigmoid colon (n = 6), and the median mucosal defect size was 37 mm (range, 21-65 mm). The complete closure rate was 90% (18/20 [95% confidence interval (CI) 68.3-98.8%]), and the median suturing time was 49 min (range, 23-92 min [95% CI 35-68 min]). Sustained closure rate on SLE was 85% (17/20 [95% CI 62.1-96.8%]). No delayed adverse events were observed. CONCLUSION: EHS demonstrated a high sustained closure rate. Given the long suturing time and technical difficulty, EHS should be reserved for cases with a high risk of delayed adverse events.

Protocol for a prospective multicenter randomized controlled trial to evaluate the efficacy of texture and color enhancement imaging (TXI) observation in the detection of colorectal lesions (deTXIon study).

Colonoscopy is the gold standard for detecting and resecting adenomas or early stage cancers to reduce the incidence and mortality rates of colorectal cancer. In a recent observational study, texture and color enhancement imaging (TXI) was reported to improve polyp detection during colonoscopy. This randomized controlled trial involving six Japanese institutions aims to confirm the superiority of TXI over standard white-light imaging (WLI) in detecting colorectal lesions during colonoscopy. During the 1-year study period, 960 patients will be enrolled, with 480 patients in the TXI and WLI groups. The primary endpoint is the mean number of adenomas detected per procedure. The secondary endpoints include adenoma detection rate, advanced adenoma detection rate, polyp detection rate, flat polyp detection rate, depressed lesion detection rate, mean polyps detected per procedure, sessile serrated lesion (SSL) detection rate, mean SSLs detected per procedure and adverse events.

Chronological Trend of Opportunistic Endoscopic Screening for Gastric Cancer and Atrophic Gastritis.

BACKGROUND: Opportunistic endoscopic screening for gastric cancer was initiated in 2004 at our institute. We investigated chronological trends in gastric cancer detection rates based on individual characteristics and atrophic gastritis prevalence. METHODS: Overall, 15,081 asymptomatic individuals aged ≥40 years without a medical history of gastric cancer underwent first-time esophagogastroduodenoscopy in our institute between February 2004 and December 2017. We retrospectively investigated individual characteristics and endoscopic diagnoses by period (early period: 2004-2007, middle period: 2008-2012, and late period: 2013-2017), clarified the long-term detection rate and the characteristics of endoscopic screening-detected gastric cancer, and evaluated the relationship between gastric cancer and atrophic gastritis. RESULTS: Gastric cancer detection rates in the early, middle, and late periods were 1.01% (76/7,503, men/women: 4,360/3,143, average age: 59.4 years, prevalence of atrophic gastritis: 72%), 0.69% (40/5,820, men/women: 3,668/2,152, average age: 56.8 years, prevalence of atrophic gastritis: 48%), and 0.46% (8/1,758, men/women: 1,083/675, average age: 58.7 years, prevalence of atrophic gastritis: 37%), respectively. Multivariate analysis revealed that male sex (odds ratio 1.92, 95% confidence interval 1.28-2.95), age ≥75 years (2.73, 95% CI 1.32-5.05), and atrophic gastritis (C1-C3: 2.21, 1.36-3.73, O1-O3: 5.36, 3.17-9.30) were significantly associated with the incidence of gastric cancer. CONCLUSIONS: The gastric cancer detection rate and atrophic gastritis prevalence have decreased over time. However, continuing endoscopic screening is important, especially for those at a high risk of developing gastric cancer complicated by severe atrophic gastritis.

Proportions and characteristics of interval cancer in annual fecal immunochemical test screening and postcolonoscopy colorectal cancer: Results from a Japanese multicenter prospective study using questionnaires, the C-DETECT study.

OBJECTIVES: There are several types of colorectal cancer (CRC) according to the detection methods and intervals, including interval CRC (iCRC) and postcolonoscopy CRC (PCCRC). We aimed to examine their proportions and characteristics. METHODS: We conducted a multicenter prospective study using questionnaires in Japan ("C-DETECT study"), in which differences in CRC characteristics according to detection methods and intervals were examined from consecutive adult patients. Because the annual fecal immunochemical test (FIT) was used in population-based screening, the annual FIT-iCRC was assessed. RESULTS: In total, 1241 CRC patients (1064 with invasive CRC) were included. Annual FIT-iCRC (a), 3-year PCCRC (b), and CRC detected within 1 year after a positive FIT with noncompliance to colonoscopy (c) accounted for 4.5%, 7.0%, and 3.9% of all CRCs, respectively, and for 3.9%, 5.4%, and 4.3% of invasive CRCs, respectively. The comparison among these (a, b, c) and other CRCs (d) demonstrated differences in the proportions of ≥T2 invasion ([a] 58.9%, [b] 44.8%, [c] 87.5%, [d] 73.0%), metastasis ([a] 33.9%, [b] 21.8%, [c] 54.2%, [d] 43.9%), right-sided CRC ([a] 42.9%, [b] 40.2%, [c] 18.8%, [d] 28.6%), and female sex ([a] 53.6%, [b] 49.4%, [c] 27.1%, [d] 41.6%). In metastatic CRC, (a) and (b) showed a higher proportions of BRAF mutations ([a] [b] 12.0%, [c] [d] 3.1%). CONCLUSIONS: Annual FIT-iCRC and 3-year PCCRC existed in nonnegligible proportions. They were characterized by higher proportions of right-sided tumors, female sex, and BRAF mutations. These findings suggest that annual FIT-iCRC and 3-year PCCRC may have biological features different from those of other CRCs.

Efficacy of endoscopic submucosal resection with a ligation device for small rectal neuroendocrine tumor: study protocol of a multicenter open-label randomized control trial (BANDIT trial).

BACKGROUND: Endoscopic resection is widely accepted as a local treatment for rectal neuroendocrine tumors sized ≤ 10 mm. However, there is no consensus on the best method for the endoscopic resection of rectal neuroendocrine tumors. As a simplified endoscopic procedure, endoscopic submucosal resection with a ligation device (ESMR-L) indicates a histologically complete resection rate comparable to that of endoscopic submucosal dissection (ESD). We hypothesized that ESMR-L than ESD would be preferred for rectal neuroendocrine tumors. Hence, this trial aimed to verify whether ESMR-L is non-inferior to ESD in terms of histologically complete resection rate. METHODS: This is a prospective, open-label, multicenter, non-inferiority, randomized controlled trial of two parallel groups, conducted at the Shizuoka Cancer Center and 31 other institutions in Japan. Patients with a lesion endoscopically diagnosed as a rectal neuroendocrine tumor ≤ 10 mm are eligible for inclusion. A total of 266 patients will be recruited and randomized to undergo either ESD or ESMR-L. The primary endpoint is the rate of en bloc resection with histologically tumor-free margins (R0 resection). Secondary endpoints include en bloc resection rate, procedure time, adverse events, hospitalization days, total devices and agents cost, adverse event rate between groups with and without resection site closure, outcomes between expert and non-expert endoscopists, and factors associated with R0 resection failure. The sample size is determined based on the assumption that the R0 resection rate will be 95.2% in the ESD group and 95.3% in the ESMR-L group, with a non-inferiority margin of 8%. With a one-sided significance level of 0.05 and a power of 80%, 226 participants are required. Assuming a dropout rate of 15%, 266 patients will be included in this study. DISCUSSION: This is the first multicenter randomized controlled trial comparing ESD and ESMR-L for the R0 resection of rectal neuroendocrine tumors ≤ 10 mm. This will provide valuable information for standardizing endoscopic resection methods for rectal neuroendocrine tumors. TRIAL REGISTRATION: Japan Registry of Clinical Trials, jRCTs042210124. Registered on Jan 6, 2022.

Endoscopist-related factors affecting adenoma detection during colonoscopy: Data from the J-SCOUT study.

OBJECTIVES: Colonoscopy withdrawal times are associated with the adenoma detection rate (ADR). However, the relationship between ADR and cecal insertion time has been inadequately characterized. We aimed to evaluate endoscopist-related factors involved in the ADR, including the average individual colonoscopy insertion and withdrawal times. METHODS: This observational study used a colonoscopy database with pathology data from routine clinical practice in Japanese institutions. The odds ratios (OR) of endoscopist-related factors related to ADRs were examined using a generalized linear mixed model. RESULTS: Of the 186,293 colonoscopies performed during the study period, 47,705 colonoscopies by 189 endoscopists in four hospitals were analyzed for ADR. The overall ADR was 38.3% (95% confidence interval [CI] 37.8, 38.7). Compared to endoscopists with mean cecal insertion times of <5 min, the OR of ADR for those with mean cecal insertion times of 5-9, 10-14, and ≥15 min were 0.84 (95% CI 0.71, 0.99), 0.68 (95% CI 0.52, 0.90), and 0.45 (95% CI 0.25, 0.78), respectively. Compared to endoscopists with mean withdrawal times of <6 min, the OR of ADR for those with mean withdrawal times of 6-9, 10-14, and ≥15 min were 1.38 (95% CI 1.03, 1.85), 1.48 (95% CI 1.09, 2.02), and 1.68 (95% CI 1.04, 2.61), respectively. There were no significant differences in ADRs by endoscopist specialty, gender, or the total number of examinations performed. CONCLUSION: Individual mean colonoscopy insertion time was associated with ADR and might be considered as a colonoscopy quality indicator as well as withdrawal time.

"Endoscopic" adenoma detection rate as a quality indicator of colonoscopy: First report from the J-SCOUT study.

OBJECTIVES: To examine whether reasonable detection rate of endoscopically diagnosed lesions as adenoma ("endoscopic" adenoma detection rate [ADR]) could be calculated with a database generated from colonoscopy reports and whether it could be used as a surrogate colonoscopy quality indicator of "pathological" ADR. METHODS: A lesion-by-lesion database of colonoscopies performed between 2010 and 2020 at eight Japanese endoscopy centers and corresponding pathology database were integrated. Differences in numbers of detected polyps, "endoscopic" and "pathological" adenomas, and what these differences could be attributed to were examined. Polyp detection rate (PDR), "endoscopic" and "pathological" ADRs, and correlation coefficients between "pathological" ADR and PDR or "endoscopic" ADR by each endoscopist were calculated. RESULTS: Overall, 129,065 colonoscopy reports were analyzed. Among a total of 146,854 polyps, more "endoscopic" adenomas (n = 117,359) were observed than "pathological" adenomas (n = 70,076), primarily because adenomas were not resected on site, rather than because of a misdiagnosis. In all patients analyzed, PDR, "endoscopic" and "pathological" ADRs were 56.4% (95% confidence interval [CI] 56.2-56.7), 48.0% (95% CI 47.7-48.3), and 32.7% (95% CI 32.5-33.0), respectively. "Endoscopic" and "pathological" ADRs from each endoscopist showed a high correlation in hospitals where adenomas were usually resected at the time of examination. CONCLUSIONS: By appropriately describing endoscopically diagnosed lesions as "adenomas" in endoscopy reports, "endoscopic" ADR might be used as a surrogate colonoscopy quality indicator of "pathological" ADR (UMIN000040690).

Detection of colorectal adenomas with texture and color enhancement imaging: Multicenter observational study.

OBJECTIVES: We aimed to evaluate the efficacy of texture and color enhancement imaging (TXI), which allows the acquisition of brighter images with enhanced color and surface structure in colorectal polyp detection compared to white light imaging. METHODS: Patients who underwent colonoscopy with repeated ascending colon observation using TXI and white light imaging between August 2020 and January 2021 were identified in three institutions. The outcomes included the mean number of adenomas detected per procedure (MAP), adenoma detection rate (ADR), and ascending colonic adenoma miss rate (Ac-AMR). Logistic regression was used to determine the effects of the variables on the outcomes. RESULTS: We included 1043 lesions from 470 patients in the analysis. The MAP, ADR, flat polyp detection rate, and Ac-AMR in TXI and white light imaging were 1.5% (95% confidence interval 1.3-1.6%) vs. 1.0% (0.9-1.1%), 58.2% (51.7-64.6%) vs. 46.8% (40.2-53.4%), 66.2% (59.8-72.2%) vs. 49.8% (43.2-56.4%), and 17.9% (12.1-25.2%) vs. 28.2% (20.0-37.6%), respectively. TXI, age, withdrawal time, and endoscopy type were identified as significant factors affecting the MAP and the ADR using multivariate regression analysis. CONCLUSIONS: Our study indicates that TXI improve the detection of colorectal neoplastic lesions. However, prospective randomized trials are required to confirm these findings.

Conditional inference tree models to perceive depth of invasion in T1 colorectal cancer.

BACKGROUND AND AIM: Accurate diagnosis of invasion depth for T1 colorectal cancer is of critical importance as it decides optimal resection technique. Few reports have previously covered the effects of endoscopic morphology on depth assessment. We developed and validated a novel diagnostic algorithm that accurately predicts the depth of early colorectal cancer. METHODS: We examined large pathological and endoscopic databases compiled between Jan 2015 and Dec 2018. Training and validation data cohorts were derived and real-world diagnostic performance of two conditional interference tree algorithms (Models 1 and 2) was evaluated against that of the Japan NBI-Expert Team (JNET) classification used by both expert and non-expert endoscopists. RESULTS: Model 1 had higher sensitivity in deep submucosal invasion than that of JNET alone in both training (45.1% vs. 28.6%, p < 0.01) and validation sets (52.3% vs. 40.0%, p < 0.01). Model 2 demonstrated higher sensitivity than Model 1 (66.2% vs. 52.3%, p < 0.01) in excluding deeper invasion of suspected Tis/T1a lesions. CONCLUSION: We discovered that machine-learning classifiers, including JNET and macroscopic features, provide the best non-invasive screen to exclude deeper invasion for suspected Tis/T1 lesions. Adding this algorithm improves depth diagnosis of T1 colorectal lesions for both expert and non-expert endoscopists.

Diagnosis and treatment of colorectal tumors: Differences between Japan and the West and future prospects.

Dye-based chromoendoscopy has long been used routinely for endoscopic diagnosis of gastrointestinal tumors including colorectal tumors in Japan. In the West, on the other hand, dye-based chromoendoscopy was not so commonly used. However, with the development of narrow band imaging (NBI), image-enhanced endoscopy diagnosis has rapidly increased in the West. The most critical difference between Japan and the West is the histopathological evaluation of the lesions, which determines a major cause of differences in diagnostic and treatment strategies. In the West, intramucosal adenocarcinoma is not diagnosed until the cancer has invaded submucosal layer. In Japan, on the other hand, cancer is mainly diagnosed based on nuclear and structural atypia, and thus intramucosal adenocarcinoma is diagnosed in lesions that correspond to high-grade adenoma in the West. In the West, since intramucosal carcinoma is not diagnosed by pathology, all benign adenomas are treated by piecemeal endoscopic resection, and only cancer invading the superficial submucosal layer is indicated for endoscopic submucosal dissection (ESD). Because of the risk of lymph node metastasis in the deep submucosal invasion, the European Society of Gastrointestinal Endoscopy and American Society for Gastrointestinal Endoscopy guidelines state that only superficial submucosal cancer is an indication for ESD. Unfortunately, it is impossible to selectively extract only superficial submucosal invasive cancer even with the use of magnified NBI and pit pattern observation. Therefore, we think that pathologists need to diagnose intramucosal adenocarcinoma with the potential to invade the submucosal layer based on the nuclear and structural atypia. Consequently, intramucosal adenocarcinoma and superficial submucosal cancers should be considered for en-bloc ESD.

Endoscopic features of isolated and traditional serrated adenoma-associated superficially serrated adenomas of the colorectum.

BACKGROUND AND AIM: Superficially serrated adenoma (SuSA) is a recently proposed subtype of colorectal serrated lesions. It is characterized by distinct clinicopathological and molecular features, including mixed serrated and adenomatous histology and frequent genetic alterations involving KRAS and RSPO. This study aimed to characterize the endoscopic features of isolated and traditional serrated adenoma (TSA)-associated SuSAs. METHODS: We retrospectively evaluated the endoscopic findings of 25 isolated SuSAs and 21 TSA-associated SuSAs that were histologically and molecularly characterized. RESULTS: SuSAs appeared as a sessile polyp or slightly elevated lesion located mostly in the sigmoid colon and rectum (88%). The size was between 3 and 20 mm (median, 6 mm). Most of them exhibited KRAS mutations (96%) and RSPO fusions/overexpression (92%). Endoscopically, many lesions had a whitish color (84%), a distinct border (96%), an irregular border (76%), and a lobulated surface (72%). However, diminutive lesions exhibited overlapping features with hyperplastic polyps. On narrow-band imaging, vessel patterns were invisible or appeared as lacy microvessels in most lesions (80%). Chromoendoscopy invariably showed stellar or elongated/branched stellar pits, indicating a serrated microarchitecture. Most TSA-associated SuSAs typically presented as polyps with a two-tier raised appearance, consisting of whitish lower and reddish higher components corresponding to a SuSA and a TSA, respectively. CONCLUSIONS: SuSAs exhibit several characteristic endoscopic features on white-light and image-enhanced endoscopy. Diminutive lesions exhibit endoscopic features overlapping with hyperplastic polyps. Nonetheless, the endoscopic diagnosis of larger and TSA-associated SuSAs may be feasible.

Cost-effectiveness analysis of endoscopic resection for colorectal laterally spreading tumors: Endoscopic submucosal dissection versus piecemeal endoscopic mucosal resection.

OBJECTIVES: The cost-effectiveness of endoscopic submucosal dissection (ESD) and piecemeal endoscopic mucosal resection (pEMR) for colorectal laterally spreading tumors (LSTs) remains unclear. We examined the cost-effectiveness of these procedures for cases of colon/rectal LST-non-granular-type ≥2 cm and LST-granular-mixed-type ≥3 cm. METHODS: We performed a simulation model analysis using parameters based on clinical data from the National Cancer Center Hospital, Tokyo, and previous literature. The number of recurrences and surgeries and the required costs for 5 years following ESD and pEMR were assessed. Japanese cost data were used in the base-case analysis, and probabilistic sensitivity analysis (PSA) was performed. The Swedish cost data were used in the scenario analysis. RESULTS: Endoscopic submucosal dissection yielded a considerably lower number of recurrences and surgeries but required a higher cost than pEMR. The recurrence rates following ESD and pEMR were 0.9-1.3% and 21.1-25.9%, respectively. The incremental cost-effectiveness ratios for an avoided recurrence and surgery for ESD against pEMR were 376,796-476,496 JPY (3575-4521 USD) and 7,335,436-8,187,476 JPY (69,604-77,689 USD), respectively. PSA demonstrated that the probability of ESD being chosen as a more cost-effective option than pEMR was >50% at willingness-to-pay values of ≥400,000-500,000 JPY (3795-4744 USD) for avoiding a recurrence and ≥9,500,000-10,500,000 JPY (90,143-99,631 USD) for avoiding a surgery. In the scenario analysis, the required cost was also lower for ESD. CONCLUSIONS: Our findings suggest potentially favorable cost-effectiveness of ESD, depending on cost settings and the willingness-to-pay value for avoiding recurrence/surgery.

Comparison of postpolypectomy bleeding events between cold snare polypectomy and hot snare polypectomy for small colorectal lesions: a large-scale propensity score-matched analysis.

BACKGROUND AND AIMS: Cold snare polypectomy (CSP), a safe procedure for removing colon polyps, has a low prevalence of postpolypectomy bleeding (PPB). Previous studies have failed to demonstrate differences in PPB rates between CSP and hot snare polypectomy (HSP), possibly because of their small sample sizes. This study analyzed PPB rates after CSP and HSP. METHODS: This was a retrospective analysis of colorectal lesions (diameter <10 mm) treated using endoscopic resection at our institution between January 2015 and December 2019. Resections were performed using CSP or HSP, depending on the endoscopist's preference. Endoscopic and histologic findings were recorded in the endoscopic database at our institution. Propensity score (PS) matching was performed to match patient age, lesion size, macroscopic features, location of the lesions, clipping after resection, and antithrombotic agent use. The CSP and HSP groups were compared to determine the adverse event (PPB) rates. RESULTS: The CSP and HSP groups included 12,928 and 2408 lesions (total of 5371 patients), respectively. Univariate analysis revealed that the overall prevalence of PPB after HSP was higher than that after CSP (odds ratio [OR], 5.39; 95% confidence interval [CI], 2.50-11.60). After PS matching (2135 lesions per group), the prevalence of PPB after HSP remained higher than that after CSP (OR, 6.0; 95% CI, 1.34-26.8). CONCLUSIONS: For colorectal lesions <10 mm in diameter, the risk of PPB after CSP is significantly lower than that after HSP, after PS matching. CSP for lesions <10 mm could be safely performed compared with HSP.

Prevalence of serrated lesions, risk factors, and their association with synchronous advanced colorectal neoplasia in asymptomatic screened individuals.

BACKGROUND AND AIM: Serrated lesions (SLs) have attracted attention as precursors of colorectal cancer (CRC). However, their prevalence, risk factors, and clinical significance have not been satisfactorily elucidated. This study used high-quality colonoscopy data to determine the prevalence of SLs and to identify their risk factors and relationship with synchronous advanced colorectal neoplasia (ACN) in asymptomatic screened individuals. METHODS: This study included data for 5218 individuals who underwent first-time screening colonoscopy by highly experienced endoscopists. The relationships between baseline characteristics and the presence of SLs and those between the presence of SLs and synchronous ACN were assessed using the chi-squared test and multivariate logistic regression. RESULTS: The proportions of individuals with SLs and right-sided SLs were 23.3% and 7.6%, respectively. Age, sex, family history of CRC, smoking, and body mass index were significantly related with the presence of SLs, and current smoking was most strongly associated with SLs (adjusted odds ratio [aOR] 2.6, 95% confidence interval [CI] 2.1-3.2). The aOR (95% CI) of the presence of SLs, SLs sized ≥ 10 mm, and right-sided SLs ≥ 5 mm for synchronous ACN was 1.4 (1.1-1.9), 3.5 (1.3-9.6), and 1.9 (1.0-3.8), respectively. The presence of left-sided SLs ≥ 10 mm (without right-sided SLs) was also significantly associated with ACN (aOR 8.1, 95% CI 2.0-33.7). CONCLUSIONS: The relatively high prevalence of SLs and risk factors in screened individuals were elucidated and the significant relationship between SLs, particularly SLs ≥ 10 mm and right-sided SLs ≥ 5 mm, and synchronous ACN was confirmed.

Identification of a novel PRR15L-RSPO2 fusion transcript in a sigmoid colon cancer derived from superficially serrated adenoma.

Superficially serrated adenoma (SuSA) is a recently proposed subtype of colorectal serrated lesion. We here report a sigmoid colon cancer derived from SuSA, which exhibited aggressive clinical behavior. Endoscopically, the tumor appeared as a superficial elevated lesion with a large nodule. Histological examination of the surgically resected material showed tubular adenocarcinoma associated with SuSA. Although tumor invasion was limited to the submucosal layer, lymph node and extranodal metastases were detected. The patient subsequently developed peritoneal metastases and died 15 months after surgery. Molecular analyses identified a KRAS mutation and a novel PRR15L-RSPO2 fusion, which retains the entire coding region of RSPO2, in both SuSA and adenocarcinoma components. The present study demonstrates the malignant potential of SuSA and expands the spectrum of RSPO fusions in colorectal neoplasms.

A Novel SDHB IVS2-2A>C Mutation Is Responsible for Hereditary Pheochromocytoma/Paraganglioma Syndrome.

Pheochromocytomas and paragangliomas are neuroendocrine tumors which arise from adrenal medulla, and sympathetic or parasympathetic nerves, respectively. Hereditary cases afflicted by both or either pheochromocytomas and paragangliomas have been reported: these are called hereditary pheochromocytoma/paraganglioma syndromes (HPPS). Many cases of HPPS are caused by mutations of one of the succinate dehydrogenase (SDH) genes; mainly SDHB and SDHD that encode subunits for the mitochondrial respiratory chain complex II. In this study, we investigated mutations of SDH genes in six HPPS patients from four Japanese pedigrees using peripheral blood lymphocytes (from one patient with pheochromocytoma and five patients with neck paraganglioma) and tumor tissues (from two patients with paraganglioma). Results showed that all of these pedigrees harbor germline mutations in one of the SDH genes. In two pedigrees, a novel IVS2-2A>C mutation in SDHB, at the acceptor-site in intron 2, was found, and the tumor RNA of the patient clearly showed frameshift caused by exon skipping. Each of the remaining two pedigrees harbors a reported missense mutation, R242H in SDHB or G106D in SDHD. Importantly, all these mutations are heterozygous in constitutional DNAs, and two-hit mutations were evident in tumor DNAs. We thus conclude that the newly identified IVS2-2A>C mutation in SDHB is responsible for HPPS. The novel mutation revealed by our study may contribute to improvement of clinical management for patients with HPPS.

NDRG2, suppressed expression associates with poor prognosis in pancreatic cancer, is hypermethylated in the second promoter in human gastrointestinal cancers.

Although N-myc downstream regulated gene 2 (NDRG2) is frequently downregulated in various cancers and is considered to be a candidate tumor suppressor gene, molecular mechanisms of the expressional suppression that lead to cancers are largely unknown. Recent studies indicated that epigenetic suppression of NDRG2 involved carcinogenesis and progression in several tumor types, and we demonstrated positive association with NDRG2 suppression and poor prognosis in pancreatic cancer. In this study, we analyzed mRNA and protein expressions of NDRG2 in 26 cancer cell lines (20 colorectal and 6 gastric cancers) and found that many cell lines showed variously reduced NDRG2 expressions. Furthermore, NDRG2 expressions were significantly reduced in primary resected cancer tissues compared to corresponding normal tissues immunohistochemically (19 of 20 colorectal and 14 of 17 gastric cancers). Treatment with 5-Aza-2' deoxycytidine predominantly upregulated NDRG2 expressions in NDRG2 low-expressing cell lines. Bisulfite sequencing analyses and methylation specific PCR revealed that methylation status at one of the two promoters (around exon 2) correlated well with the suppressed expression, and this is the major promoter in colorectal and gastric cancer cell lines. Our present results suggest that hypermethylation in promoter around exon 2 is functioning as essential factors of NDRG2 silencing in gastrointestinal cancers.

Targeted TET oxidase activity through methyl-CpG-binding domain extensively suppresses cancer cell proliferation.

DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl-CpG-binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD-TET1-CDwt were also reactivated after treatment with a DNMT inhibitor, 5-azacytidine (Aza-CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD-TET1-CDwt in a prostate cancer cell line, LNCaP. The induction of MBD-TET1-CDwt demethylated and upregulated glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.