Gastric epithelial neoplasm of fundic-gland mucosa lineage: proposal for a new classification in association with gastric adenocarcinoma of fundic-gland type.

BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a rare variant of gastric neoplasia. However, the etiology, classification, and clinicopathological features of gastric epithelial neoplasm of fundic-gland mucosa lineage (GEN-FGML; generic term of GA-FG related neoplasm) are not fully elucidated. We performed a large, multicenter, retrospective study to establish a new classification and clarify the clinicopathological features of GEN-FGML. METHODS: One hundred GEN-FGML lesions in 94 patients were collected from 35 institutions between 2008 and 2019. We designed a new histopathological classification of GEN-FGML using immunohistochemical analysis and analyzed via clinicopathological, immunohistochemical, and genetic evaluation. RESULTS: GEN-FGML was classified into 3 major types; oxyntic gland adenoma (OGA), GA-FG, and gastric adenocarcinoma of fundic-gland mucosa type (GA-FGM). In addition, GA-FGM was classified into 3 subtypes; Type 1 (organized with exposure type), Type 2 (disorganized with exposure type), and Type 3 (disorganized with non-exposure type). OGA and GA-FG demonstrated low-grade epithelial neoplasm, and GA-FGM should be categorized as an aggressive variant of GEN-FGML that demonstrated high-grade epithelial neoplasm (Type 2 > 1, 3). The frequent presence of GNAS mutation was a characteristic genetic feature of GEN-FGML (7/34, 20.6%; OGA 1/3, 33.3%; GA-FG 3/24, 12.5%; GA-FGM 3/7, 42.9%) in mutation analysis using next-generation sequencing. CONCLUSIONS: We have established a new histopathological classification of GEN-FGML and propose a new lineage of gastric epithelial neoplasm that harbors recurrent GNAS mutation. This classification will be useful to estimate the malignant potential of GEN-FGML and establish an appropriate standard therapeutic approach.

Artificial Intelligence System to Determine Risk of T1 Colorectal Cancer Metastasis to Lymph Node.

BACKGROUND & AIMS: In accordance with guidelines, most patients with T1 colorectal cancers (CRC) undergo surgical resection with lymph node dissection, despite the low incidence (∼10%) of metastasis to lymph nodes. To reduce unnecessary surgical resections, we used artificial intelligence to build a model to identify T1 colorectal tumors at risk for metastasis to lymph node and validated the model in a separate set of patients. METHODS: We collected data from 3134 patients with T1 CRC treated at 6 hospitals in Japan from April 1997 through September 2017 (training cohort). We developed a machine-learning artificial neural network (ANN) using data on patients' age and sex, as well as tumor size, location, morphology, lymphatic and vascular invasion, and histologic grade. We then conducted the external validation on the ANN model using independent 939 patients at another hospital during the same period (validation cohort). We calculated areas under the receiver operator characteristics curves (AUCs) for the ability of the model and US guidelines to identify patients with lymph node metastases. RESULTS: Lymph node metastases were found in 319 (10.2%) of 3134 patients in the training cohort and 79 (8.4%) of /939 patients in the validation cohort. In the validation cohort, the ANN model identified patients with lymph node metastases with an AUC of 0.83, whereas the guidelines identified patients with lymph node metastases with an AUC of 0.73 (P < .001). When the analysis was limited to patients with initial endoscopic resection (n = 517), the ANN model identified patients with lymph node metastases with an AUC of 0.84 and the guidelines identified these patients with an AUC of 0.77 (P = .005). CONCLUSIONS: The ANN model outperformed guidelines in identifying patients with T1 CRCs who had lymph node metastases. This model might be used to determine which patients require additional surgery after endoscopic resection of T1 CRCs. UMIN Clinical Trials Registry no: UMIN000038609.

Indolent T cell lymphoproliferative disorder with villous atrophy in small intestine diagnosed by single-balloon enteroscopy.

Chronic diarrhea is one of the major symptoms in gastroenterology. However, this may be caused by pathologic conditions for which the diagnosis is critical. Villous atrophy, as an endoscopic lesion, accompanied by chronic diarrhea can occasionally be observed in the patients with inflammatory diseases of the gastrointestinal (GI) tract. Herein, we present a case with persistent diarrhea accompanied by intestinal wall thickening without any other significant endoscopic features other than villous atrophy in the jejunum and the ileum, where we diagnosed as an indolent T cell lymphoproliferative disorder (T-LPD) of the GI tract, defined in the 2016-2017 revised World Health Organization classification, via single-balloon enteroscopy (SBE). Interestingly, we found the same lymphocyte infiltration from the distal third portion of the duodenum, where gastroscopy could not reach, via SBE, even though no endoscopic findings were observed such as villous atrophy. Since infiltrating cells in the intestinal tissues were CCR4+, mogamulizumab was administered with resulting durable symptomatic remission for more than 2 years. Patients with persistent diarrhea may have serious small intestinal disorder including not only chronic inflammatory diseases but also lymphoid neoplasmic conditions including T-LPD of GI tract.

Efficacy of Endocuff-assisted colonoscopy in the detection of colorectal polyps.

BACKGROUND AND STUDY AIMS: Colonoscopy is the gold standard for detecting colorectal adenomas and cancers. Endoscopic surveillance has been shown to be effective for preventing colorectal cancer. Although detection of colorectal polyps at an early stage is important, endoscopic visualization of early neoplasia can be difficult. The Endocuff is a new device that can be attached to the tip of the colonoscope to hold the colonic folds away from the field of view during withdrawal. The aim of this study was to compare the adenoma detection rate (ADR) and the mean number of adenomas detected per patient (MAP) achieved using Endocuff-assisted colonoscopy (EAC) and standard colonoscopy (SC). PATIENTS AND METHODS: This randomized prospective study was conducted at two academic endoscopy departments in Japan. A total of 447 patients underwent a complete colonoscopic examination between April 2015 and September 2015. The EAC group included 239 patients. The cecal intubation rate, insertion time, withdrawal time, pain score, complications, polyp detection rate (PDR), ADR, the mean number of polyps detected per patient (MPP), and the MAP were assessed. RESULTS: There were no differences between the EAC and SC groups in terms of cecal intubation rate, insertion time, withdrawal time, or pain scores. The PDR in patients increased by about 12 % (61.9 % vs. 49.2 %, P  = 0.013) and ADR increased by 15 % (52.5 % vs. 39.2 %, P  = 0.001) with the use of the Endocuff. The advanced ADR was higher in the EAC group but no statistically significant difference was found (7.7 % vs. 4.6 %, P  = 0.17). Both MPP and MAP were also higher in the EAC group (mean ±â€ŠSD: 1.33 ±â€Š1.43 vs. 0.83 ±â€Š0.99 per patient; P  < 0.01, 1.11 ±â€Š1.41 vs. 0.66 ±â€Š0.99 per patient; P  < 0.01, respectively). No major complications occurred. CONCLUSIONS: EAC not only enabled a higher ADR but also significantly increased the mean number of adenomas identified per patient, as compared with SC.

Round ligament varicosities diagnosed as inguinal hernia during pregnancy: A case report and series from two regional hospitals in Japan.

INTRODUCTION: Round ligament varicosities (RLV) are not well-known and they are usually caused by pregnancy. Although the groin swelling of RLV mimics an inguinal hernia, it is difficult to distinguish between them through clinical examination alone, and there have been few published reports on this topic, especially from Asia. CASE PRESENTATION: A 37-year-old Japanese woman complained of left groin swelling for 2 weeks at her 28th week of gestation of her first pregnancy. According to a physical examination, she had a soft, painless swelling in the superficial inguinal ring of the left groin. An inguinal hernia was suspected and she was thus scheduled to undergo herniorrhaphy. However, since she had varicosities in the left labia majora, she first underwent color Doppler ultrasonography and a final diagnosis of round ligament varicosities was this made. Her symptoms resolved after delivery of her baby. DISCUSSION: We herein report 10 cases of RLV in pregnant Japanese women who were initially suspected of having an inguinal hernia. All of them were suspected to suffer from inguinal hernias after a clinical examination by their attending gynecologist. All of them were diagnosed using gray scale and color Doppler ultrasonography, treated with conservative management, and the symptoms resolved in all cases after the delivery. CONCLUSION: This is the largest report from Asia regarding RLV. To avoid unnecessary surgery in pregnant women, surgeons must be aware of this entity make an accurate diagnosis based of the findings of Doppler ultrasonography.

Retinol Promotes In Vitro Growth of Proximal Colon Organoids through a Retinoic Acid-Independent Mechanism.

Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance.

Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes.

BACKGROUND: Intraepithelial lymphocytes (IELs) in the intestine play important roles in the regulation of local immune responses. Although their functions have been studied in a variety of animal experiments, in vitro studies on spatiotemporal behaviors of IELs and their interaction with intestinal epithelial cells (IECs) have been hampered due to the lack of a suitable culture system. In this study, we aimed at developing a novel co-culture system of IELs with IECs to investigate dynamic interaction between these two populations of cells in vitro. METHODS: We optimized experimental conditions under which murine IELs can be efficiently maintained with IECs cultured as three-dimensional organoids. We then tested the effect of IL-2, IL-7, and IL-15 on the maintenance of IELs in this co-culture system. By time-lapse imaging, we also examined the dynamic behaviors of IELs. RESULTS: IELs can be expanded with epithelial organoids in the presence of IL-2, IL-7, and IL-15. IELs were efficiently maintained within and outside of organoids showing a ~four-fold increase in both αßT and γδT IELs for a period of 2 weeks. Four-dimensional fluorescent imaging revealed an active, multi-directional movement of IELs along the basolateral surface of IECs, and also their inward or outward migration relative to organoid structures. Cell tracking analysis showed that αßT and γδT IELs shared indistinguishable features with regard to their dynamics. CONCLUSIONS: This novel co-culture method could serve as a unique tool to investigate the motility dynamics of IELs and their temporal and spatial interaction with IECs in vitro.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.

BACKGROUND AND AIMS: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon.

To develop stem cell therapy for small intestinal (SI) diseases, it is essential to determine whether SI stem cells in culture retain their tissue regeneration capabilities. By using a heterotopic transplantation approach, we show that cultured murine SI epithelial organoids are able to reconstitute self-renewing epithelia in the colon. When stably integrated, the SI-derived grafts show many features unique only to the SI but distinct from the colonic epithelium. Our study provides evidence that cultured adult SI stem cells could be a source for cell therapy of intestinal diseases, maintaining their identity along the gastrointestinal tract through an epithelium-intrinsic mechanism.

Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro.

P-glycoprotein (P-gp) is an efflux transporter that regulates bioavailability of orally administered drugs at the intestinal epithelium. To develop an in vitro experimental model that mimics P-gp-mediated intestinal drug transport in vivo, we employed normal intestinal epithelium three-dimensionally cultured. Physiological expression of P-gp mRNA and the expression of its protein at the apical membrane were observed in the small intestinal epithelium grown as cystic organoids. Rhodamine123 (Rh123), a substrate for P-gp, was actively transported in the basoapical direction and accumulated in the luminal space, while the epithelial integrity was kept intact. Furthermore, we were able to monitor the whole process of Rh123 transport and its inhibition by verapamil in real-time, from which kinetic parameters for Rh123 transport could be estimated by a mathematical modeling. The method here described to evaluate the dynamics of P-gp-mediated transport in primary intestinal epithelial cells would be instrumental in investigating the physiological function of P-gp and its inhibitors/inducers in vitro.

[Four cases of anal squamous cell carcinoma treated by chemoradiotherapy].

We reviewed clinical records of 4 cases with squamous cell carcinoma in anus to evaluate the clinical effectiveness of the chemoradiotherapy. The radiation therapy consisted of 40 Gy was delivered to pelvis and bilateral inguinal lesion, and perianal booster dose of 20 Gy, in fractions of 2.0 Gy per day, was given five days a week. On the first day of radiation therapy, 750 mg/m2 of 5-FU in the form of a continuous 24-hour infusion for 5 days was given. On the first day of chemotherapy, 10 mg/m2 of MMC was also given as a single bolus infusion. 5-FU and MMC were administered 4 times every 4 weeks. Three patients had T2 tumor, and one patient had T1 tumor. One patient had metastases in para-aortic and Virchow lymph node. No patients had hematogenous metastases. Grade 1 or 2 adverse effects occurred in 3 patients during chemoradiotherapy, but the completion of chemoradiotherapy was achieved in all of the 4 patients. All patients had complete response in the anal lesion after chemoradiotherapy. No patients had any sign of recurrence in anal lesion. Chemoradiotherapy was expected to be a safe and effective treatment to improve prognosis for anal squamous carcinoma.