Preclinical evaluation of the versius surgical system, a new robot-assisted surgical device for use in minimal access general and colorectal procedures.

OBJECTIVE: To evaluate the utility of a new robot-assisted surgical system (the Versius Surgical System, CMR Surgical, Cambridge, UK) for use in minimal access general and colorectal surgery, in a preclinical setting. Robot-assisted laparoscopy has been developed to overcome some of the important limitations of conventional laparoscopy. The new system is designed to assist surgeons in performing minimal access surgery and overcome some of the challenges associated with currently available surgical robots. METHODS: Cadaveric sessions were conducted to evaluate the ability of the system to provide adequate surgical access and reach required to complete a range of general and colorectal procedures. Port and bedside unit positions were recorded, and surgical access and reach were evaluated by the lead surgeon using a visual analogue scale. A live animal (porcine) model was used to assess the surgical device's safety in performing cholecystectomy or small bowel enterotomy. RESULTS: Nine types of procedure were performed in cadavers by nine lead surgeons; 35/38 procedures were completed successfully. The positioning of ports and bedside units reflected the lead surgeons' preferred laparoscopic set-up and enabled good surgical access and reach. Cholecystectomy (n = 6) and small bowel enterotomy (n = 5) procedures performed in pigs were all completed successfully by two surgeons. There were no device-related intra-operative complications. CONCLUSIONS: This preclinical study of a new robot-assisted surgical system for minimal access general and colorectal surgery demonstrated the safety and effectiveness of the system in cadaver and porcine models. Further studies are required to assess its clinical utility.

Hereditary diffuse gastric cancer: updated clinical practice guidelines.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

Transcriptomic profiling reveals three molecular phenotypes of adenocarcinoma at the gastroesophageal junction.

Cancers occurring at the gastroesophageal junction (GEJ) are classified as predominantly esophageal or gastric, which is often difficult to decipher. We hypothesized that the transcriptomic profile might reveal molecular subgroups which could help to define the tumor origin and behavior beyond anatomical location. The gene expression profiles of 107 treatment-naïve, intestinal type, gastroesophageal adenocarcinomas were assessed by the Illumina-HTv4.0 beadchip. Differential gene expression (limma), unsupervised subgroup assignment (mclust) and pathway analysis (gage) were undertaken in R statistical computing and results were related to demographic and clinical parameters. Unsupervised assignment of the gene expression profiles revealed three distinct molecular subgroups, which were not associated with anatomical location, tumor stage or grade (p > 0.05). Group 1 was enriched for pathways involved in cell turnover, Group 2 was enriched for metabolic processes and Group 3 for immune-response pathways. Patients in group 1 showed the worst overall survival (p = 0.019). Key genes for the three subtypes were confirmed by immunohistochemistry. The newly defined intrinsic subtypes were analyzed in four independent datasets of gastric and esophageal adenocarcinomas with transcriptomic data available (RNAseq data: OCCAMS cohort, n = 158; gene expression arrays: Belfast, n = 63; Singapore, n = 191; Asian Cancer Research Group, n = 300). The subgroups were represented in the independent cohorts and pooled analysis confirmed the prognostic effect of the new subtypes. In conclusion, adenocarcinomas at the GEJ comprise three distinct molecular phenotypes which do not reflect anatomical location but rather inform our understanding of the key pathways expressed.

Comparative study of endoscopic surveillance in hereditary diffuse gastric cancer according to CDH1 mutation status.

BACKGROUND AND AIMS: Hereditary diffuse gastric cancer (HDGC) accounts for 1% of gastric cancer cases. For patients with a germline CDH1 mutation, risk-reducing gastrectomy is recommended. However, for those delaying surgery or for families with no causative mutation identified, regular endoscopy is advised. This study aimed to determine the yield of signet ring cell carcinoma (SRCC) foci in individuals with a CDH1 pathogenic variant compared with those without and how this varies with successive endoscopies. METHODS: Patients fulfilling HDGC criteria were recruited to a prospective longitudinal cohort study. Endoscopy was performed according to a strict protocol with visual inspection followed by focal lesion and random biopsy sampling to detect foci of SRCC. Survival analysis determined progression to finding of SRCC according to CDH1 mutation status. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 and 36-item Short Form Health Survey questionnaires assessed quality of life before surveillance and each endoscopy. RESULTS: Eighty-five individuals fulfilling HDGC criteria underwent 201 endoscopies; 54 (63.5%) tested positive for CDH1 mutation. SRCC yield was 61.1% in CDH1 mutation carriers compared with 9.7% in noncarriers, and mutation-positive patients had a 10-fold risk of SRCC on endoscopy compared with those with no mutation detected (P < .0005). Yield of SRCC decreased substantially with subsequent endoscopies. Surveillance was associated with improved psychological health. CONCLUSIONS: SRCC foci are prevalent in CDH1 mutation carriers and can be detected at endoscopy using a standardized, multiple biopsy sampling protocol. Decreasing yield over time suggests that the frequency of endoscopy might be reduced. For patients with no CDH1 pathogenic variant detected, the cost-to-benefit ratio needs to be assessed in view of the low yield.

The Psychosocial Impact of Undergoing Prophylactic Total Gastrectomy (PTG) to Manage the Risk of Hereditary Diffuse Gastric Cancer (HDGC).

Individuals identified as at high risk of developing Hereditary Diffuse Gastric Cancer (HDGC) are advised to undergo prophylactic surgery - have their stomach removed - in their early twenties. Research with (older) cancer patients who undergo gastrectomy for curative reasons suggests that gastric resection has a number of physical and psychosocial sequelae. Because it is difficult to extrapolate the findings of studies of older cancer patients to younger healthy patients who are considering prophylactic total gastrectomy (PTG), the aim of this qualitative interview study was to determine the psychosocial implications of undergoing prophylactic surgery to manage genetic risk. Fourteen men and 13 women from the UK's Familial Gastric Cancer study who had undergone PTG were invited to participate in qualitative interviews. Most reported that undergoing surgery and convalescence was easier than anticipated. There was evidence that age affected experiences of PTG, with younger patients tending to report faster recovery times and more transient aftereffects. All saw the benefits of risk reduction as outweighing the costs of surgery. Surgery was described as having a range of physical impacts (disrupted appetite, weight loss, fatigue, GI symptoms) that had related psychological, social and economic implications. Those considering PTG need to be aware that its impact on quality of life is difficult to predict and negative sequelae may be ongoing for some individuals.

Management of Barrett's high-grade dysplasia: initial results from a population-based national audit.

BACKGROUND AND AIMS: Previous studies reported significant variation in the management of patients with Barrett's esophagus. However, these are based on self-reported clinical practice. The aim of this study was to examine the management of high-grade dysplasia in Barrett's esophagus in England by using patient-level data and to compare practice with guidelines. METHODS: From April 2012 to March 2013, National Health Service (NHS) trusts in England prospectively collected data on patients newly diagnosed with high-grade dysplasia (HGD) of the esophagus as part of the National Oesophago-Gastric Cancer Audit. Data were collected on patient characteristics, diagnosis and endoscopic findings, treatment planning, and therapy. RESULTS: Between April 2012 and March 2013, NHS trusts reported 465 cases of HGD. Diagnosis was confirmed by a second pathologist in 79.4% of cases (270/340), and 86.0% (374/465) had their treatment planned at a multidisciplinary team meeting. A total of 290 patients (62.4%) were managed endoscopically (frequently with endoscopic resection or radiofrequency ablation), whereas 26 patients (5.6%) had esophagectomy. The proportion of patients managed by surveillance varied by age (P < .001), ranging from 19.5% in patients aged <65 years to 63.8% in patients aged ≥85 years. More patients received active treatment if their cases were discussed at a multidisciplinary meeting (73.5% vs 44.3%; P < .001) or managed at higher-volume trusts (87.8% vs 55.4%; P < .001). CONCLUSIONS: There was marked variation in the management of HGD across England, with a third of patients receiving no active treatment. Patients discussed at a specialist multidisciplinary meeting or managed in high-volume trusts were more likely to receive active treatment.

Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers.

Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored.

The impact of prophylactic total gastrectomy on health-related quality of life: a prospective cohort study.

BACKGROUND: The advice to individuals with identified CDH1 mutations is generally to undertake prophylactic total gastrectomy (PTG). This study evaluated the effect of PTG on health-related quality of life (HRQL) in asymptomatic individuals with identified CDH1 mutations at high risk for gastric cancer. METHODS: Individuals with hereditary diffuse gastric cancer (HDGC) were recruited to a prospective, multicenter UK study. Questionnaires, including the European Organization for Research and Treatment for Cancer core Quality-of-Life Questionnaire (EORTC QLQ C30); the gastric cancer specific module (EORTC QLQ STO22); and the 36-item short form health survey version 2.0, were completed before and at regular intervals after surgery. RESULTS: Sixty individuals fulfilled HDGC criteria; 38 (63%) had a CDH1 mutation and 32 (53%) underwent PTG. At baseline, there was no significant difference in mental health depending on CDH1 mutation status and treatment preference. Physical functioning reduced in the first month after surgery but recovered to baseline by 12 months. Similarly mental functioning reduced in the first month after surgery but recovered by 3 to 9 months. However, specific symptoms were identified, such as diarrhoea (70%), fatigue (63%), discomfort when eating (81%), reflux (63%), eating restrictions (45%), and body image (44%), which persisted after PTG. CONCLUSIONS: Patients contemplating prophylactic gastrectomy can be reassured about the long-term HRQL outcomes, but some residual symptoms require adjustment.

Prospective cohort study assessing outcomes of patients from families fulfilling criteria for hereditary diffuse gastric cancer undergoing endoscopic surveillance.

BACKGROUND: Prophylactic total gastrectomy is performed in hereditary diffuse gastric cancer (HDGC) patients carrying the CDH1 mutation because endoscopic surveillance often fails to detect microscopic disease. OBJECTIVE: The aim of this study was to determine the natural history and outcomes of patients with HDGC undergoing endoscopy. DESIGN: Prospective, cohort observational study. SETTINGS: Tertiary referral center. PATIENTS: Patients fulfilling criteria for HDGC who opted to undergo endoscopy. INTERVENTION: Research surveillance program using high-resolution white-light endoscopy with autofluorescence and narrow-band imaging combined with targeted and multiple random biopsies assessed by an expert histopathologist for the presence of signet ring cell carcinoma. MAIN OUTCOME MEASUREMENTS: The primary endpoint was the endoscopic yield of microscopic signet ring cell carcinoma according to patient mutation status and subsequent decision to undergo surgery. The secondary endpoint was the additional yield of targeted biopsies compared with random biopsies. RESULTS: Between September 2007 and March 2013, 29 patients from 17 families underwent 70 surveillance endoscopies. Signet ring cell carcinoma foci were identified in 14 of 22 (63.6%) patients with confirmed CDH1 germline mutations and 2 of 7 (28.6%) with no pathogenic mutation identified. Eleven of 16 (9 CDH1-positive) patients proceeded to gastrectomy in a median 5.7 months. Five patients delayed surgery. In 1 patient, advanced gastric cancer developed 40.2 months after the first endoscopic findings. LIMITATIONS: No control group. CONCLUSIONS: Careful white-light examination with targeted and random biopsies combined with detailed histopathology can identify early lesions and help to inform decision making with regard to gastrectomy. Autofluorescence and narrow-band imaging are of limited utility. Delaying gastrectomy in individuals with signet ring cell carcinoma foci carries a high risk and has to be weighed carefully.

A population-based, retrospective, cohort study of esophageal cancer missed at endoscopy.

BACKGROUND AND STUDY AIMS: Several studies have suggested that a significant minority of esophageal cancers are missed at endoscopy The aim of this study was to estimate the proportion of esophageal cancers missed at endoscopy on a national level, and to investigate the relationship between miss rates and patient and tumor characteristics. PATIENTS AND METHODS: This retrospective, population-based, cohort study identified patients diagnosed with esophageal cancer between April 2011 and March 2012 in England, using two linked databases (National Oesophago-Gastric Cancer Audit and Hospital Episode Statistics). The main outcome was the rate of previous endoscopy within 3 - 36 months of cancer diagnosis. This was calculated for the overall cohort and by patient characteristics, including tumor site and disease stage. RESULTS: A total of 6943 new cases of esophageal cancer were identified, of which 7.8 % (95 % confidence interval 7.1 - 8.4) had undergone endoscopy in the 3 - 36 months preceding diagnosis. Of patients with stage 0/1 cancers, 34.0 % had undergone endoscopy in the 3 - 36 months before diagnosis compared with 10.0 % of stage 2 cancers and 4.5 % of stage 3/4 cancers. Of patients with stage 0/1 cancers, 22.1 % were diagnosed after ≥ 3 endoscopies in the previous 3 years. Patients diagnosed with an upper esophageal lesion were more likely to have had an endoscopy in the previous 3 - 12 months (P = 0.040). The most common diagnosis at previous endoscopy was an esophageal ulcer (48.2 % of investigations). CONCLUSION: Esophageal cancer may be missed at endoscopy in up to 7.8 % of patients who are subsequently diagnosed with cancer. Endoscopists should make a detailed examination of the whole esophageal mucosa to avoid missing subtle early cancers and lesions in the proximal esophagus. Patients with an esophageal cancer may be misdiagnosed as having a benign esophageal ulcer.

Location of lymph node involvement in patients with esophageal adenocarcinoma predicts survival.

BACKGROUND: The location of positive lymph nodes has been abandoned in the seventh classification of the TNM staging system for esophageal adenocarcinoma. The present study evaluates whether distribution of involved nodes relative to the diaphragm in addition to TNM 7 further refines prediction. METHODS: Pathology reports of patients who underwent esophagectomy between 2000 and 2008 for adenocarcinoma of the esophagus were reviewed and staging was performed according to the seventh UICC-AJCC staging system. In addition, lymph node involvement of nodal stations above and below the diaphragm was investigated by endoscopic ultrasonography (EUS) in a separate cohort of patients who were scheduled for esophagectomy between 2008 and 2009 at two institutions. Survival was calculated by the Kaplan-Meier method, and multivariate analysis was performed with a Cox regression model. RESULTS: Some 327 patients who had undergone esophagectomy for cancer were included. Multivariate analysis revealed that patients with from three to six involved lymph nodes in the resection specimen on both sides of the diaphragm had a twofold higher chance of dying compared to patients with the same number of involved lymph nodes on one side of the diaphragm. EUS assessment of lymph node metastases relative to the diaphragm in 102 patients showed that nodal involvement on both sides of the diaphragm was associated with worse survival than when nodes on one side or no nodes are involved [HR (95 % CI) 2.38 (1.15-4.90)]. CONCLUSIONS: A combined staging system that incorporates distribution of lymph nodes relative to the diaphragm refines prognostication after esophagectomy as assessed in the resected specimen and pretreatment as assessed by EUS. This improved staging has the potential to have a great impact on clinical decision making as to whether to embark upon potentially curative or palliative treatments.

Incidence and survival of oesophageal and gastric cancer in England between 1998 and 2007, a population-based study.

BACKGROUND: Major changes in the incidence of oesophageal and gastric cancers have been reported internationally. This study describes recent trends in incidence and survival of subgroups of oesophageal and gastric cancer in England between 1998 and 2007 and considers the implications for cancer services and policy. METHODS: Data on 133,804 English patients diagnosed with oesophageal and gastric cancer between 1998 and 2007 were extracted from the National Cancer Data Repository. Using information on anatomical site and tumour morphology, data were divided into six groups; upper and middle oesophagus, lower oesophagus, oesophagus with an unspecified anatomical site, cardia, non-cardia stomach, and stomach with an unspecified anatomical site. Age-standardised incidence rates (per 100,000 European standard population) were calculated for each group by year of diagnosis and by socioeconomic deprivation. Survival was estimated using the Kaplan-Meier method. RESULTS: The majority of oesophageal cancers were in the lower third of the oesophagus (58%). Stomach with an unspecified anatomical site was the largest gastric cancer group (53%). The incidence of lower oesophageal cancer increased between 1998 and 2002 and remained stable thereafter. The incidence of cancer of the cardia, non-cardia stomach, and stomach with an unspecified anatomical site declined over the 10 year period. Both lower oesophageal and cardia cancers had a much higher incidence in males compared with females (M:F 4:1). The incidence was also higher in the most deprived quintiles for all six cancer groups. Survival was poor in all sub-groups with 1 year survival ranging from 14.8-40.8% and 5 year survival ranging from 3.7-15.6%. CONCLUSIONS: An increased focus on prevention and early diagnosis, especially in deprived areas and in males, is required to improve outcomes for these cancers. Improved recording of tumour site, stage and morphology and the evaluation of focused early diagnosis programmes are also needed. The poor long-term survival reinforces the need for early detection and multidisciplinary care.

A 4-gene signature predicts survival of patients with resected adenocarcinoma of the esophagus, junction, and gastric cardia.

BACKGROUND & AIMS: The incidence of esophageal and junctional adenocarcinoma has increased 6-fold in the past 30 years and 5-year survival remains approximately 20%. Current staging is limited in its ability to predict survival which has ramifications for treatment choices. The aim of this study was to generate and validate a molecular prognostic signature for esophageal adenocarcinoma. METHODS: Gene expression profiling was performed and the resulting 42,000 gene signatures correlated with clinical and pathologic features for 75 snap-frozen esophageal and junctional resection specimens. External validation of selected targets was performed on 371 independent cases using immunohistochemistry to maximize clinical applicability. RESULTS: A total of 119 genes were associated significantly with survival and 270 genes with the number of involved lymph nodes. Filtering of these lists resulted in a shortlist of 10 genes taken forward to validation. Four genes proved to be prognostic at the protein level (deoxycytidine kinase [DCK], 3'-phosphoadenosine 5'-phosphosulfate synthase 2 [PAPSS2], sirtuin 2 [SIRT2], and tripartite motif-containing 44 [TRIM44]) and were combined to create a molecular prognostic signature. This 4-gene signature was highly predictive of survival in the independent external validation cohort (0/4 genes dysregulated 5-year survival, 58%; 95% confidence interval [CI], 36%-80%; 1-2/4 genes dysregulated 5-year survival, 26%; 95% CI, 20%-32%; and 3-4/4 genes dysregulated 5-year survival, 14%; 95% CI, 4%-24% (P = .001). Furthermore, this 4-gene signature was independently prognostic in a multivariable model together with the existing clinical TNM staging system (P = .013). CONCLUSIONS: This study has generated a clinically applicable prognostic gene signature that independently predicts survival in an external validation cohort and may inform management decisions.

Acute appendicitis management during the COVID-19 pandemic: A prospective cohort study from a large UK centre.

BACKGROUND: During the Covid-19 pandemic, non-operative management for acute appendicitis (AA) was implemented in the UK. The aim of this study was to determine the efficacy and outcomes of conservative versus surgical management of AA during the pandemic. MATERIALS & METHODS: We conducted an observational study in a tertiary referral centre. Data was collected from all patients (≥16 years) with a diagnosis of AA between November 1, 2019 to March 10, 2020 (pre-COVID period) and March 10, 2020 to July 5, 2020 (COVID period). RESULTS: A total of 116 patients in the pre-COVID period were included versus 91 in the COVID period. 43.1% (n = 50) of patients pre-COVID were classified as ASA 2 compared to 26.4% (n = 24) during the COVID period (p-value = 0.042). 72.5% (n = 66) of the patients during the COVID period scored as high risk using the Alvarado score compared to 24.1% (n = 28) in the pre-COVID period (p-value<0.001). We observed a significant increase in radiological evaluation, 69.8% versus 87.5% of patients had a CT in the pre-COVID and COVID periods respectively (p-value = 0.008). 94.9% of patients were managed operatively in the pre-COVID period compared to 60.4% in the COVID period (p-value<0.001). We observed more open appendicectomies (37.3% versus 0.9%; p-value<0.001) during the COVID period compared to the pre-COVID period. More abscess formation and free fluid were found intraoperatively in the COVID period (p-value = 0.021 and 0.023 respectively). Re-attendance rate due to appendicitis-related issues was significantly higher in the COVID period (p = 0.027). CONCLUSION: Radiological diagnosis of AA was more frequent during the COVID period. More conservative management for AA was employed during the COVID-19 pandemic, and for those managed operatively an open approach was preferred. Intra-operative findings were suggestive of delayed presentation during the COVID period without this affecting the length of hospital stay.

Genomic evidence supports a clonal diaspora model for metastases of esophageal adenocarcinoma.

The poor outcomes in esophageal adenocarcinoma (EAC) prompted us to interrogate the pattern and timing of metastatic spread. Whole-genome sequencing and phylogenetic analysis of 388 samples across 18 individuals with EAC showed, in 90% of patients, that multiple subclones from the primary tumor spread very rapidly from the primary site to form multiple metastases, including lymph nodes and distant tissues-a mode of dissemination that we term 'clonal diaspora'. Metastatic subclones at autopsy were present in tissue and blood samples from earlier time points. These findings have implications for our understanding and clinical evaluation of EAC.

Re-organisation of oesophago-gastric cancer care in England: progress and remaining challenges.

BACKGROUND: Oesophago-gastric cancer services in England have been extensively reorganised since 2001 to deliver a centralised, specialist-led service. Our aim was to assess how well the National Health Service (NHS) in England met organisational standards for oesophago-gastric cancer care. METHODS: Questionnaires that asked about the provision of staging investigations, curative and palliative treatments and key personnel were sent in September 2007 to the lead clinician for oesophago-gastric cancer at all 30 cancer networks and 156 NHS acute trusts in England. RESULTS: Responses were received from all networks and 81% of NHS trusts. All networks provided essential staging investigations and a range of endoscopic palliative therapies. Only 16 of the 30 cancer networks discussed all patients at the specialist multi-disciplinary team meeting and 11 networks had not fully centralised curative surgery. There was also variation between NHS trusts in the integration of the palliative care team, the availability of nurse specialists and the use of dietitians to provide nutritional support. CONCLUSION: There has been considerable progress in reforming oesophago-gastric cancer services but the process of reorganisation is still incomplete and regional differences in service provision exist that may lead to variation in patient outcomes.

Important Role of the GLP-1 Axis for Glucose Homeostasis after Bariatric Surgery.

Bariatric surgery is widely used to treat obesity and improves type 2 diabetes beyond expectations from the degree of weight loss. Elevated post-prandial concentrations of glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and insulin are widely reported, but the importance of GLP-1 in post-bariatric physiology remains debated. Here, we show that GLP-1 is a major driver of insulin secretion after bariatric surgery, as demonstrated by blocking GLP-1 receptors (GLP1Rs) post-gastrectomy in lean humans using Exendin-9 or in mice using an anti-GLP1R antibody. Transcriptomics and peptidomics analyses revealed that human and mouse enteroendocrine cells were unaltered post-surgery; instead, we found that elevated plasma GLP-1 and PYY correlated with increased nutrient delivery to the distal gut in mice. We conclude that increased GLP-1 secretion after bariatric surgery arises from rapid nutrient delivery to the distal gut and is a key driver of enhanced insulin secretion.

Characterisation of proguanylin expressing cells in the intestine - evidence for constitutive luminal secretion.

Guanylin, a peptide implicated in regulation of intestinal fluid secretion, is expressed in the mucosa, but the exact cellular origin remains controversial. In a new transgenic mouse model fluorescent reporter protein expression driven by the proguanylin promoter was observed throughout the small intestine and colon in goblet and Paneth(-like) cells and, except in duodenum, in mature enterocytes. In Ussing chamber experiments employing both human and mouse intestinal tissue, proguanylin was released predominantly in the luminal direction. Measurements of proguanylin expression and secretion in cell lines and organoids indicated that secretion is largely constitutive and requires ER to Golgi transport but was not acutely regulated by salt or other stimuli. Using a newly-developed proguanylin assay, we found plasma levels to be raised in humans after total gastrectomy or intestinal transplantation, but largely unresponsive to nutrient ingestion. By LC-MS/MS we identified processed forms in tissue and luminal extracts, but in plasma we only detected full-length proguanylin. Our transgenic approach provides information about the cellular origins of proguanylin, complementing previous immunohistochemical and in-situ hybridisation results. The identification of processed forms of proguanylin in the intestinal lumen but not in plasma supports the notion that the primary site of action is the gut itself.

Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling.

Enteroendocrine cells (EECs) produce hormones such as glucagon-like peptide 1 and peptide YY that regulate food absorption, insulin secretion, and appetite. Based on the success of glucagon-like peptide 1-based therapies for type 2 diabetes and obesity, EECs are themselves the focus of drug discovery programs to enhance gut hormone secretion. The aim of this study was to identify the transcriptome and peptidome of human EECs and to provide a cross-species comparison between humans and mice. By RNA sequencing of human EECs purified by flow cytometry after cell fixation and staining, we present a first transcriptomic analysis of human EEC populations and demonstrate a strong correlation with murine counterparts. RNA sequencing was deep enough to enable identification of low-abundance transcripts such as G-protein-coupled receptors and ion channels, revealing expression in human EECs of G-protein-coupled receptors previously found to play roles in postprandial nutrient detection. With liquid chromatography-tandem mass spectrometry, we profiled the gradients of peptide hormones along the human and mouse gut, including their sequences and posttranslational modifications. The transcriptomic and peptidomic profiles of human and mouse EECs and cross-species comparison will be valuable tools for drug discovery programs and for understanding human metabolism and the endocrine impacts of bariatric surgery.

Gastrectomy with Roux-en-Y reconstruction as a lean model of bariatric surgery.

BACKGROUND: Altered enteroendocrine hormone responses are widely believed to underlie the beneficial effects of bariatric surgery in type 2 diabetes. While elevated postprandial glucagon-like peptide-1 (GLP-1) is considered one of the mediators, increased postprandial glucagon levels have recently been implicated. OBJECTIVES: We investigated hormonal responses in lean patients after prophylactic total gastrectomy (PTG), as a model of Roux-en-Y gastric bypass without the confounding effects of obesity or massive weight loss. SETTING: University hospital, United Kingdom. METHODS: Ten participants after PTG and 9 healthy volunteers were recruited for oral glucose tolerance tests. Plasma glucose, insulin, GLP-1, peptide YY, glucose-dependent insulinotropic-polypeptide, glucagon, oxyntomodulin, glucagon(1-61), and glicentin levels were assessed using immunoassays and/or mass spectrometry. RESULTS: PTG participants exhibited accelerated plasma glucose appearance, followed, in 3 of 10 cases, by hypoglycemia (<3 mM glucose). Plasma GLP-1, peptide YY, glucose-dependent insulinotropic-polypeptide, glicentin, and oxyntomodulin responses were elevated, and glucagon appeared to rise in PTG participants when measured with a glucagon-specific enzyme-linked immunosorbent assay. We revisited the specificity of this assay, and demonstrated significant cross-reactivity with glicentin and oxyntomodulin at concentrations observed in PTG plasma. Reassessment of glucagon with the same assay using a modified protocol, and by liquid chromatography-mass spectrometry, demonstrated suppression of glucagon secretion after oral glucose tolerance tests in both PTG and control cohorts. CONCLUSIONS: Care should be taken when assessing glucagon levels in the presence of elevated plasma levels of other proglucagon products. Substantial elevation of GLP-1 and insulin responses after PTG likely contribute to the observed hypoglycemia, and mirror similar hormone levels and complications observed in bariatric weight loss patients.