Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

Inhibin-Positive "Cholangioblastic" Variant of Intrahepatic Cholangiocarcinoma: Report of 3 New Patients With Review of the Literature.

Cholangiocarcinoma is the second most common primary liver malignant neoplasm. It usually affects older individuals in their seventh decade of life with no gender predilection. Recently, a distinct subtype of cholangiocarcinoma has emerged with 2 proposed names: "cholangioblastic" and "solid tubulocystic." This variant predominantly occurs in younger women who lack the common risk factors for patients diagnosed with cholangiocarcinomas, such as older age and chronic liver disease or cirrhosis. We describe 3 new patients with a cholangioblastic variant of intrahepatic cholangiocarcinoma. At the time of diagnosis, the patients were aged 19-, 46-, and 28-year-old; 2 females and 1 male (the 46-year-old). None of our patients had a history of chronic liver disease or known predisposing factors for liver tumors. Tumor size ranged from 2.3 to 23 cm in greatest dimension. Histological examination of these tumors demonstrated reproducible morphology characterized by trabecular, nested, and multicystic patterns with micro and macro follicles filled with eosinophilic material. The immunohistochemical profile showed that the tumor cells were positive for keratin 7, inhibin, synaptophysin, and albumin in situ hybridization, while negative for HepPar1, arginase, and INSM1. All tumors lacked conventional intrahepatic cholangiocarcinoma/adenocarcinoma morphology. We also review the literature and emphasize that neuroendocrine tumors should be recognized as a major diagnostic pitfall of this variant.

Gastrointestinal, hepatic, and pancreatobiliary involvement by plasma cell neoplasms: clinicopathologic correlations in a retrospective cohort of 116 cases.

AIMS: Plasma cell neoplasms (PCNs) may involve the gastrointestinal (GI) tract in two forms: plasmacytoma (PC), an isolated lesion that lacks marrow involvement, and extramedullary myeloma (EMM). However, previous literature on PCNs involving the GI tract, liver, and pancreas is limited. We evaluated the clinicopathologic features of the largest series of GI PCNs to date. METHODS AND RESULTS: Six institutional archives were searched for GI, liver, and pancreas cases involved with PCNs. Medical records were reviewed for clinical and imaging features. Histopathologic features evaluated included involved organ, tumor grade, and marrow involvement. Overall, 116 cases from 102 patients were identified. The tumors most presented as incidental findings (29%). The liver was most involved (47%), and masses/polyps (29%) or ulcers (21%) were the most common findings. Most cases had high-grade morphology (55%). The majority (74%) of GI PCNs were classified as EMM due to the presence of marrow involvement at some point during the disease course, occurring within a year of marrow diagnosis in 46% of patients. PC was classified in 26% of patients due to the lack of marrow involvement. Most (70%) patients died from disease within 10 years (median 14.1) of diagnosis and more than half (58%) died within 6 months. CONCLUSION: PC and EMM involving the GI tract, liver, and pancreas have a wide range of clinicopathologic presentations. Tumors may occur virtually anywhere in the GI tract or abdomen and may precede the diagnosis of marrow involvement. Both GI PC and EMM are associated with a poor prognosis.

Natural history of gastrointestinal schwannomas.

Background and study aims Data are lacking on the natural history of gastrointestinal tract schwannomas. We aimed to study the natural history of all gastrointestinal schwannomas including location, diagnosis, management, and long-term outcomes. Patients and methods Patients with a pathological diagnosis of gastrointestinal schwannoma between January 2000 and March 2020 were identified. Data on baseline demographics, presentations, associated malignancies, malignant transformation, treatment, and recurrence were collected. Results Our cohort consisted of 44 patients with a mean age of 58.6 years, with 63.6 % women and 84.1 % White. The stomach (38.6 %) was the most common location followed by the colorectum (31.8 %). Only 22.7 % of patients were symptomatic and 22.0 % had a personal history of other malignancies. Tissue diagnosis was obtained via endoscopy in 47.7 % and from surgical pathology in 52.3 %. On histology, 65.9 % of the tumors were solid, 11.4 % had mixed features, and 2.3 % had necrosis. SP100 was tested in all but one patient and was positive in all. Mean Ki-67 in 12 patients with tumors measuring ≥ 2 cm was 3.0 % indicating a low proliferation rate. Of the patients, 77.3 % had surgery and 18.2 % underwent endoscopic resection. At a mean follow-up of 5.0 ±â€Š4.31 years, there was no malignant transformation, recurrence or mortality associated with gastrointestinal schwannomas. Conclusions Gastrointestinal schwannomas are diagnosed in the fifth to sixth decade with predominance in women and Whites. They are benign, mostly asymptomatic, and diagnosed incidentally. Asymptomatic gastrointestinal schwannomas including lesions ≥ 2 cm in size do not appear to need further monitoring or intervention. Patients with them should be counseled to remain up to date with routine screening guidelines pertaining to the colon, breast, and lung cancer due to the high incidence of concomitant malignancy.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

p300 Serine 89: A Critical Signaling Integrator and Its Effects on Intestinal Homeostasis and Repair.

Differential usage of Kat3 coactivators, CBP and p300, by ß-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by ß-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.

Allaying uncertainty in diagnosing buried Barrett's esophagus.

Subsquamous intestinal metaplasia (SSIM) in the setting of Barrett's esophagus (BE) is a technically challenging diagnosis. While the risk for progression of BE involving the surface mucosa is well documented, the potential risk for development of advanced neoplasia associated with SSIM has been controversial. This study aimed to determine the effects of specimen adequacy, presence of dysplasia, and interobserver agreement for SSIM interpretation. Adult patients (n = 28) who underwent endoscopic therapy for BE with high-grade dysplasia or intramucosal carcinoma (HGD/IMC) between October 2005 and June 2013 were included. Initial evaluation (n = 140 slides) by an experienced gastrointestinal pathologist was followed by an interobserver study by 8 pathologists. Forty-seven (34%) slides had insufficient subsquamous tissue to assess for SSIM. SSIM was found in 19% of all slides and 29% of slides with sufficient subsquamous tissue. At least one slide had SSIM in 54% to 64% of patients. Subsquamous low grade dysplasia (LGD) was found in 4 (15%) slides with SSIM and subsquamous HGD/IMC was found in 5 (19%) slides with SSIM. At the patient level, 8 (53%) had no dysplasia, 4 (27%) had LGD and 3 (20%) had HGD/IMC. Overall agreement for SSIM by slide was 92% to 94% (κ = 0.73 to κ = 0.82, moderate to strong agreement), and by patient was 82% to 94% (κ = 0.65 to κ = 0.87, moderate to strong agreement). This study confirms the need for assessing specimen adequacy and assessing the prevalence of SSIM and is the first to assess interobserver agreement for SSIM and dysplasia within SSIM.

An unusual presentation of gastric mucosa-associated lymphoid tissue (MALT) lymphoma following blunt abdominal trauma.

A 79-year-old woman presented to the emergency department following a motor vehicle collision. As part of her workup she underwent a CT scan which identified a large mass containing calcifications centred around the gastric antrum, and while being assessed she produced 500 mL of haematemesis. An endoscopy revealed an area of friable mucosa the nature of which was uncertain, and multiple biopsies revealed amyloid deposition and active Helicobacter pylori gastritis. Following review of imaging and pathology, a diagnosis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma was established. She was treated with quadruple therapy for the H. pylori and at 6-month follow-up she is asymptomatic with repeat endoscopy revealing healing of the ulceration and no biopsy evidence of H. pylori or MALT.

Concurrent Adult-Onset Diffuse ß-Cell Nesidioblastosis and Pancreatic Neuroendocrine Tumor: A Case Report and Review of the Literature.

Nesidioblastosis is an uncommon cause of organic persistent hyperinsulinemic hypoglycemia in adults. We report a case of adult-onset diffuse ß-cell nesidioblastosis in a 49-year-old woman who was status-post Roux-en-Y gastric bypass and distal pancreatectomy for a well-differentiated pancreatic neuroendocrine tumor. While the neuroendocrine tumor was suspected to be an insulinoma, persistent hypoglycemia postoperatively suggested either incomplete resection or a second pancreatic neoplasm. Completion pancreatectomy revealed islet ß-cell hyperplasia and nuclear pleomorphism consistent with ß-cell nesidioblastosis. The patient's blood glucose levels normalized after completion pancreatectomy. While ß-cell nesidioblastosis and insulinomas can coexist in the same patient, pathologists should be aware of ß-cell nesidioblastosis as a potential cause for hyperinsulinemic hypoglycemia and should exclude it in patients who have not shown definitive clinical response after surgical excision of a pancreatic neuroendocrine tumor.

Programmed Cell Death-Ligand 1 (PD-L1) Expression in Anal Cancer.

OBJECTIVE: To evaluate the expression of programmed cell death-ligand 1 (PD-L1) in anal cancer. PATIENTS AND METHODS: In a retrospective cohort analysis, subjects with squamous cell carcinoma of the anal canal were tested for PD-L1 expression, then followed for recurrence and survival. Crude recurrence rates (CRRs), crude mortality rates (CMRs), and crude event rates (CERs) were assessed for PD-L1-dependent differences using Poisson regression. All 3 types of crude rate were expressed as the number that occurred per hundred person-years (hPY) of follow-up. RESULTS: Samples from 41 subjects were evaluated for PD-L1 expression; 23 (56%) were positive. Subjects with PD-L1-expressing versus PD-L1-negative tumors respectively had CRRs of 30.8 versus 12.1 recurrences/hPY (P=0.082), CMRs of 16.7 versus 12.0 deaths/hPY (P=0.47), and CERs of 39.2 versus 16.9 events/hPY (P=0.069). CONCLUSIONS: PD-L1 positivity was associated with worse CRR and CER, and marginally worse CMR. The effect on progression-free and overall survival needs to be validated in a study with a larger sample size.

Platform-independent gene expression signature differentiates sessile serrated adenomas/polyps and hyperplastic polyps of the colon.

BACKGROUND: Sessile serrated adenomas/polyps are distinguished from hyperplastic colonic polyps subjectively by their endoscopic appearance and histological morphology. However, hyperplastic and sessile serrated polyps can have overlapping morphological features resulting in sessile serrated polyps diagnosed as hyperplastic. While sessile serrated polyps can progress into colon cancer, hyperplastic polyps have virtually no risk for colon cancer. Objective measures, differentiating these types of polyps would improve cancer prevention and treatment outcome. METHODS: RNA-seq training data set and Affimetrix, Illumina testing data sets were obtained from Gene Expression Omnibus (GEO). RNA-seq single-end reads were filtered with FastX toolkit. Read mapping to the human genome, gene abundance estimation, and differential expression analysis were performed with Tophat-Cufflinks pipeline. Background correction, normalization, and probe summarization steps for Affimetrix arrays were performed using the robust multi-array method (RMA). For Illumina arrays, log2-scale expression data was obtained from GEO. Pathway analysis was implemented using Bioconductor package GSAR. To build a platform-independent molecular classifier that accurately differentiates sessile serrated and hyperplastic polyps we developed a new feature selection step. We also developed a simple procedure to classify new samples as either sessile serrated or hyperplastic with a class probability assigned to the decision, estimated using Cantelli's inequality. RESULTS: The classifier trained on RNA-seq data and tested on two independent microarray data sets resulted in zero and three errors. The classifier was further tested using quantitative real-time PCR expression levels of 45 blinded independent formalin-fixed paraffin-embedded specimens and was highly accurate. Pathway analyses have shown that sessile serrated polyps are distinguished from hyperplastic polyps and normal controls by: up-regulation of pathways implicated in proliferation, inflammation, cell-cell adhesion and down-regulation of serine threonine kinase signaling pathway; differential co-expression of pathways regulating cell division, protein trafficking and kinase activities. CONCLUSIONS: Most of the differentially expressed pathways are known as hallmarks of cancer and likely to explain why sessile serrated polyps are more prone to neoplastic transformation than hyperplastic. The new molecular classifier includes 13 genes and may facilitate objective differentiation between two polyps.

Stearoyl-CoA Desaturase Promotes Liver Fibrosis and Tumor Development in Mice via a Wnt Positive-Signaling Loop by Stabilization of Low-Density Lipoprotein-Receptor-Related Proteins 5 and 6.

BACKGROUND & AIMS: Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS: Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS: SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector ß-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and ß-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS: In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-ß-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma.

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV-related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region-specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease-free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Primary Bile Duct Melanoma Causing Obstructive Jaundice.

Malignant melanoma is one of the few malignancies that are well known for unusual behavior. Primary malignant melanoma usually originates from squamous epithelium of skin, mucous membranes, retina, and uvea. Although melanoma can metastasize to any part of the body, including biliary tract, primary malignant melanoma of bile ducts is an extremely rare entity. We present a 52-year-old man who presented with 5-month epigastric pain and 15-pound weight loss, with 1-week duration of jaundice, nausea/vomiting, pale stools, and dark urine, blood work suggested cholestatic jaundice. Imaging revealed a large perihilar/peripancreatic mass involving the portal vein and hepatic artery, and intrahepatic biliary dilation. Biliary brushings revealed neoplastic cells strongly suggestive of malignant melanoma.

Quantitative analysis of ex vivo colorectal epithelium using an automated feature extraction algorithm for microendoscopy image data.

Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia.

Cystoisospora belli Infection of the Gallbladder in Immunocompetent Patients: A Clinicopathologic Review of 18 Cases.

Cystoisospora belli, previously known as Isospora belli, is an obligate intracellular coccidian parasite that is most often associated with gastrointestinal disease in immunocompromised patients. In this study, we detail the clinicopathologic features of 18 cases of Cystoisospora infection affecting the gallbladder in immunocompetent individuals and compare them with a control group. Each case was reviewed for cholecystitis (none, acute, chronic), epithelial disarray, presence of intraepithelial lymphocytes (none, rare [≤5 per 20 epithelial cells], present [>5 per 20 epithelial cells]), architectural distortion, intramucosal eosinophilia, and mural thickening/serositis. The mean age of patients with Cystoisospora infection was 33 years and the male to female ratio 1:4.3. Cholecystectomy was performed for biliary dyskinesia (n=7), abdominal pain (n=7), suspected cholelithiasis (n=5), and cholecystitis (n=3). In 2 cases, Cystoisospora was found in donor gallbladders resected at the time of liver transplantation. Each case was characterized by eosinophilic, oval or banana-shaped intraepithelial parasites within perinuclear parasitophorous vacuoles. Most cases showed epithelial disarray and minimal intraepithelial lymphocytosis. Of the 11 cases with an average follow-up of 15 months, none had evidence of disease related to Cystoisospora infection within the biliary tract or elsewhere in the gastrointestinal tract. We present the largest series of gallbladder cystoisosporiasis in immunocompetent patients to date. Cystoisospora infection is underrecognized in the gallbladders of immunocompetent patients, in part due to the subtle findings in routine cholecystectomy specimens. On the basis of the clinical follow-up, gallbladder cystoisosporiasis in immunocompetent individuals appears to be a self-limited infection.

Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy.

Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

Soy Protein Isolate Protects Against Ethanol-Mediated Tumor Progression in Diethylnitrosamine-Treated Male Mice.

In this study, diethylnitrosamine-treated male mice were assigned to three groups: (i) a 35% high fat ethanol liquid diet (EtOH) with casein as the protein source, (ii) the same EtOH liquid diet with soy protein isolate as the sole protein source (EtOH/SPI), (iii) and a chow group. EtOH feeding continued for 16 weeks. As expected, EtOH increased the incidence and multiplicity of basophilic lesions and adenomas compared with the chow group, P < 0.05. Soy protein replacement of casein in the EtOH diet significantly reduced adenoma progression when compared with the EtOH and EtOH/SPI group (P < 0.05). Tumor reduction in the EtOH/SPI group corresponded to reduced liver injury associated with decreased hepatic Tnfα and Cd14 antigen (Cd14) expression and decreased nuclear accumulation of NF-κB1 protein compared with the EtOH group (P < 0.05). Detection of sphingolipids using high-resolution matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging mass spectrometry revealed increased accumulation of long acyl chain ceramide species, and sphingosine-1-phosphate (S1P) in the EtOH group that were significantly reduced in the EtOH/SPI group. Chronic EtOH feeding also increased mRNA expression of ß-catenin transcriptional targets, including cyclin D1 (Ccnd1), matrix metallopeptidase 7 (Mmp7), and glutamine synthetase (Glns), which were reduced in the EtOH/SPI group (P < 0.05). We conclude that soy prevents tumorigenesis by reducing proinflammatory and oxidative environment resulting from EtOH-induced hepatic injury, and by reducing hepatocyte proliferation through inhibition of ß-catenin signaling. These mechanisms may involve changes in sphingolipid signaling. Cancer Prev Res; 9(6); 466-75. ©2016 AACR.

Gastric Proteins MUC5AC and TFF1 as Potential Diagnostic Markers of Colonic Sessile Serrated Adenomas/Polyps.

OBJECTIVES: A subset of colon cancers originates from sessile serrated adenomas/polyps (SSA/Ps). Our goal was to identify markers for SSA/Ps that could aid in distinguishing them from hyperplastic polyps (HPs). METHODS: We performed immunostaining for gastric proteins MUC5AC and TFF1 in formalin-fixed, paraffin-embedded (FFPE) samples of HPs (n = 47), SSA/Ps (n = 37), and normal colon (n = 30). RESULTS: Control mucosa expressed only trace amounts of MUC5AC and TFF1. HPs exhibited an 11.3- and 11.4-fold increase in MUC5AC and TFF1 expression confined to the upper segments of the crypts near the luminal surface of the polyps. SSA/Ps displayed on average 1.6-fold (MUC5AC, P < .008) and 1.4-fold (TFF1, P < .03) higher signal intensity for these markers than HPs, with a dramatic coexpression of MUC5AC and TFF1 typically occupying the entire length of the crypt. Immunoperoxidase results were similar to immunofluorescence staining for both MUC5AC and TFF1. CONCLUSIONS: Our results suggest that the analysis of expression of MUC5AC and TFF1 may be useful for differentiating SSA/Ps from HPs. We also suggest the possibility that crypt morphology may be at least partly due to overproduction of highly viscous gastric mucins and that these proteins may play a role in the serrated pathway to colon carcinogenesis.

Overexpression of p53 predicts colorectal neoplasia risk in patients with inflammatory bowel disease and mucosa changes indefinite for dysplasia.

BACKGROUND AND AIMS: We previously demonstrated a significant colorectal neoplasia risk in inflammatory bowel disease (IBD) patients with mucosal changes indefinite for dysplasia (IND) and the potential diagnostic utility of p53 and cytokeratin 7 immunohistochemistry in IBD-associated neoplasia. The primary aim of this exploratory study was to determine the predictive value of the two markers for neoplasia risk in the IBD-IND population. METHODS: We identified 44 eligible cases with IBD and IND in colon biopsy from our pathology database. We semi-quantified the expression of p53 and cytokeratin 7 in the colon biopsies by immunohistochemistry and correlated their expression, demographic information, and clinical features with colorectal neoplasia outcome. RESULTS: The mean age of the cohort was 46.6 ± 15.1 years, with 25 (56.8%) being male. The median follow-up was 101 months (range: 6-247) after IND diagnosis. Among these 44 patients, 11 (25%) progressed to neoplasia (low-grade dysplasia = 6; high-grade dysplasia = 2; cancer 3) at a median follow-up of 66 months (range: 19-145). Univariate analysis demonstrated that age and p53 overexpression were associated with progression to neoplasia. CONCLUSIONS: Twenty-five percent of patients with IBD and IND developed colorectal dysplasia or cancer. Overexpression of p53 and age are associated with neoplastic progression.