The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.

Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1(+) colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5(+) colonic stem cells; genes upregulated in the Lrig1(+) population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1(+) cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

A method for scoring 4-nitroquinoline 1-oxide-induced murine esophageal squamous neoplasia.

A mouse model for esophageal squamous cell carcinoma (ESCC) is induced by oral administration of the carcinogen 4-nitroquinoline 1-oxide (4-NQO). There is not an objective method for determining histopathologic severity of disease in this model. We aim to create a clearly defined and easily applied scoring system that can quantify the severity of 4-NQO-induced murine ESCC. Fifteen wild-type C57BL/6J mice were treated with 4-NQO for 8 (n = 8) or 16 (n = 7) weeks, while the rest (n = 9) were treated with vehicle, as 8 weeks of 4-NQO typically results in dysplasia and 16 weeks in carcinoma. We identified histologic abnormalities of the esophagus in this model and developed metrics to grade severity of dysplasia, papillomas, and invasion. Scores were then calculated using quantitative digitized image analysis for measuring depth and extent of each feature within the entire sample. Each feature was also assigned a weight based on its relation to cancer severity. Histology scores were significantly different in the three groups, suggesting that this method can discriminate dysplasia from carcinoma. This model can be applied to any mouse treated with 4-NQO.

Diagnosis of digestive system tumours.

The WHO Classification of Tumours provides the international standards for the classification and diagnosis of tumours. It enables direct comparisons to be made between different countries. In the new fifth edition, the series has gone digital with the launch of a website as well as a series of books, known widely as the WHO Blue Books. The first volume to be produced is on the classification of Digestive System tumours, replacing the successful 2010 version. It has been rewritten and updated accordingly. This article summarises the major diagnostic innovations that have occurred over the last decade and that have now been incorporated in the classification. As an example, it incorporates the recently proposed classification of neuroendocrine tumours, based on the recognition that neuroendocrine tumours and carcinomas differ substantially in the genetic abnormalities that drive their growth, findings relevant to treatment selection and outcome prediction. Several themes have emerged during the production process. One is the importance of the progression from hyperplasia to dysplasia to carcinoma in the evolution of the malignant process. Advances in imaging techniques and endoscopy have resulted in enhanced access to precancerous lesions in the gastrointestinal and biliary tract, necessitating both changes in classification schema and clinical practice. Diagnosis of tumours is no longer the sole purview of pathologists, and some patients now receive treatment before tissue is obtained, based on clinical, radiological and liquid biopsy results. This makes the classification relevant to many disciplines involved in the care of patients with tumours of the digestive system.

Key histopathologic features in idiopathic noncirrhotic portal hypertension: an interobserver agreement study and proposal for diagnostic criteria.

Histologic features of idiopathic noncirrhotic portal hypertension (INCPH), loosely termed as obliterative portal venopathy (OPV), are heterogenous, often subtle, and overlap with other entities. To this date, no consensus histopathologic diagnostic criteria have been established for INCPH. For these reasons, rendering a reproducible consensus histologic diagnosis of OPV on a liver biopsy may often be challenging even for experienced hepatopathologists. We report herein a two-phase interobserver agreement study on the diagnosis of OPV and assessed the relative value of histologic features in 104 liver biopsies in distinguishing between INCPH and non-INCPH with the goal to obtain a consensus on specific practical diagnostic criteria. Six hepatopathologists blinded to clinical information and original pathologic diagnosis reviewed internet-based case study sets with high-resolution whole-slide images. The initial interobserver agreement on OPV was expectedly low, but significantly improved (moderate agreement in most categories) upon adopting a consensus view recognizing portal vein sclerosis as the only strong independent histologic predictor for INCPH, and that contrary to the conventional view, aberrant portal/periportal vessels does not significantly contribute to the positive assignment of OPV status. We propose a three-tiered classification with diagnostic criteria to facilitate the histologic assignment of OPV status for the evaluation of INCPH. Furthermore, we have validated the performance of the proposed criteria either based on histology alone or coupled with clinicopathologic correlation. This classification may aid in practical histologic assessment of liver biopsies with or without portal hypertension and help to improve diagnostic consistency and accuracy.

Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic ß-catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling is unknown. Here, we confirm that BVES loss increases ß-catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modeling using a Wnt-driven genetic model and a chemically induced model of colorectal carcinogenesis demonstrate that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.

BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.

OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.

Factors Associated With Persistent Increase in Level of Alanine Aminotransferase in Patients With Chronic Hepatitis B Receiving Oral Antiviral Therapy.

BACKGROUND & AIMS: Despite complete suppression of viral DNA with antiviral agents, in some patients with chronic hepatitis B (CHB), serum levels of alanine aminotransferase (ALT) do not normalize. We investigated factors associated with persistent increases in ALT level in patients with CHB given long-term tenofovir disoproxil fumarate. METHODS: We analyzed data from 471 hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients with CHB participating in 2 phase 3 trials. We identified patients with an increased level of ALT (above the upper limit of normal range) after 5 years (240 weeks) of tenofovir disoproxil fumarate therapy. We analyzed findings from liver biopsy specimens collected from 467 patients (99%) at baseline and 339 patients (72%) at year 5 of treatment; biopsy specimens were evaluated by an independent pathologist. We performed stepwise, forward, multivariate regression analyses of specified baseline characteristics and on-treatment response parameters to identify factors associated with persistent increases in ALT level. RESULTS: Of the 471 patients, 87 (18%) still had an increased ALT level at year 5 of treatment. Factors associated significantly with a persistent increase in ALT level were a steatosis score of 5% or greater (grade 1 or more) at baseline (odds ratio [OR], 2.236; 95% confidence interval [CI], 1.031-4.852; P = .042) and at year 5 (OR, 3.392; 95% CI, 1.560 ≥ 7.375; P = .002), HBeAg seropositivity at baseline (OR, 3.297; 95% CI, 1.653-6.576; P < .001), and age 40 years or older (OR, 2.099; 95% CI, 1.014-4.342; P = .046). Of the 42 HBeAg-positive patients with steatosis at baseline, 21 (50%) had an increased ALT level at year 5 of treatment. Patients with persistent increases in ALT level were more likely to have an increase in steatosis at year 5 than those with a normal ALT level. CONCLUSIONS: HBeAg seropositivity and hepatic steatosis contribute to persistent increases in ALT level in patients with CHB receiving suppressive antiviral treatment. ClinicalTrials.gov registration numbers: NCT00117676 and NCT00116805.

Prevention of DNA damage in Barrett's esophageal cells exposed to acidic bile salts.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

The impact of stage, grade, and mucinous histology on the efficacy of systemic chemotherapy in adenocarcinomas of the appendix: Analysis of the National Cancer Data Base.

BACKGROUND: Adenocarcinomas of the appendix represent a heterogeneous disease depending on the presence of mucinous histology, histologic grade, and stage. In the current study, the authors sought to explore the interplay of these factors with systemic chemotherapy in a large population data set. METHODS: Patients in the National Cancer Data Base (NCDB) who were diagnosed with mucinous, nonmucinous, and signet ring cell-type appendiceal neoplasms from 1985 through 2006 were selected. Multivariable Cox proportional hazards regression models were developed. RESULTS: A total of 11,871 patients met the inclusion criteria for the current study: 50.3% had mucinous neoplasms, 40.5% had nonmucinous neoplasms, and 9.2% had signet ring cell-type neoplasms. The 5-year overall survival (OS) stratified by grade was similar among patients with American Joint Committee on Cancer stage I to stage III disease but not for those with stage IV disease. The median OS for patients with stage IV mucinous and nonmucinous tumors was 6.4 years and 2.3 years, respectively, for those with well differentiated histology (P<.0001) and was 1.5 years and 0.8 years, respectively, for those with poorly differentiated histology (P<.0001). In multivariable modeling for stage I to III disease, adjuvant chemotherapy improved OS for both mucinous and nonmucinous histologies, with hazard ratios (HRs) of 0.78 (95% confidence interval [95% CI], 0.68-0.89 [P = .0002]) and 0.83 (95% CI, 0.74-0.94 [P = .002]), respectively. For patients with stage IV disease, systemic chemotherapy significantly improved OS for those with nonmucinous (HR, 0.72; 95% CI, 0.64-0.82 [P<.0001]) but not mucinous (HR, 0.95; 95% CI, 0.86-1.04 [P = .2) histologies, although this was grade-dependent. The median OS for chemotherapy versus no chemotherapy was 6.4 years versus 6.5 years (P value not significant) for patients with mucinous, well-differentiated tumors and 1.6 years versus 1.0 years (P = .0007) for patients with mucinous, poorly differentiated tumors. CONCLUSIONS: Adjuvant chemotherapy demonstrated a significant OS benefit regardless of histology. However, for patients with stage IV disease, the benefit of systemic chemotherapy varied by tumor histology and grade, with patients with well-differentiated, mucinous, appendiceal adenocarcinomas deriving no survival benefit from systemic chemotherapy. Cancer 2016;122:213-221. © 2015 American Cancer Society.

Adenoma-like adenocarcinoma: a subtype of colorectal carcinoma with good prognosis, deceptive appearance on biopsy and frequent KRAS mutation.

AIMS: A subset of colorectal carcinomas (CRCs) architecturally and cytologically resembles adenomatous change, making them difficult to diagnose on biopsy. This subset has not been well characterized to date. METHODS AND RESULTS: For 35 carcinomas with adenomatous-like areas (cytological and surface architectural appearance that would be insufficient to warrant a diagnosis of adenocarcinoma if evaluated on biopsy), we recorded staging information, molecular data, clinical outcome, whether precursor adenoma was present and whether previous biopsy had been diagnosed as malignant. Despite advanced T-category in 23 (66%) tumours, only seven (20%) had nodal metastases, and only five patients (15%) developed distant metastases. Fifteen cases (43%) had been diagnosed as adenoma on biopsy. Twenty-one resections (60%) showed no residual associated adenoma, including nine called adenoma on biopsy. Median follow-up was 44 months. Four patients (12%) died of disease; 22 were alive at last follow-up. KRAS mutation was seen in 14 of 24 (58%) and four of 17 (24%) were microsatellite-unstable. Patients had significantly improved survival compared to a cohort of patients with conventional well-differentiated CRC after controlling for age and stage (P = 0.011). CONCLUSIONS: Adenoma-like adenocarcinoma is an uncommon variant of CRC with a low rate of metastasis and good prognosis. Biopsy diagnosis of this lesion may be challenging.

The transcriptional corepressor MTGR1 regulates intestinal secretory lineage allocation.

Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation.

Gastric Cancer, Version 3.2016, NCCN Clinical Practice Guidelines in Oncology.

Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of death from cancer in the world. Several advances have been made in the staging procedures, imaging techniques, and treatment approaches. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Gastric Cancer provide an evidence- and consensus-based treatment approach for the management of patients with gastric cancer. This manuscript discusses the recommendations outlined in the NCCN Guidelines for staging, assessment of HER2 overexpression, systemic therapy for locally advanced or metastatic disease, and best supportive care for the prevention and management of symptoms due to advanced disease.

Activation of the epidermal growth factor receptor in macrophages regulates cytokine production and experimental colitis.

Macrophages regulate innate immunity to maintain intestinal homeostasis and play pathological roles in intestinal inflammation. Activation of the epidermal growth factor receptor (EGFR) promotes cellular proliferation, differentiation, survival, and wound closure in several cell types. However, the impact of EGFR in macrophages remains unclear. This study was to investigate whether EGFR activation in macrophages regulates cytokine production and intestinal inflammation. We found that EGFR was activated in colonic macrophages in mice with dextran sulfate sodium (DSS)-induced colitis and in patients with ulcerative colitis. DSS-induced acute colitis was ameliorated, and recovery from colitis was promoted in Egfr(fl/fl)LysM-Cre mice with myeloid cell-specific deletion of EGFR, compared with LysM-Cre mice. DSS treatment increased IL-10 and TNF levels during the acute phase of colitis, and increased IL-10 but reduced TNF levels during the recovery phase in Egfr(fl/fl)LysM-Cre mice. An anti-IL-10 neutralizing Ab abolished these effects of macrophage-specific EGFR deletion on DSS-induced colitis in Egfr(fl/fl)LysM-Cre mice. LPS stimulated EGFR activation and inhibition of EGFR kinase activity enhanced LPS-stimulated NF-κB activation in RAW 264.7 macrophages. Furthermore, induction of IL-10 production by EGFR kinase-blocked RAW 264.7 cells, in response to LPS plus IFN-γ, correlated with decreased TNF production. Thus, although selective deletion of EGFR in macrophages leads to increases in both pro- and anti-inflammatory cytokines in response to inflammatory stimuli, the increase in the IL-10 level plays a role in suppressing proinflammatory cytokine production, resulting in protection of mice from intestinal inflammation. These results reveal an integrated response of macrophages regulated by EGFR in intestinal inflammatory disorders.