A preliminary report from the recent phase 3 trial of benralizumab, a monoclonal antibody that binds to interleukin-5 receptor alpha (IL5Rα), in patients with EoE revealed that medication use led to tissue eosinophil eradication but did not meet the clinical endpoint of symptom resolution. Here, we characterized the clinical, endoscopic, histologic, and transcriptional changes in patients with active EoE following benralizumab treatment. We retrospectively examined patients with EoE treated with benralizumab at the University of Utah (n = 11) and reviewed reported clinical symptoms, circulating and tissue eosinophilia, and endoscopic and histologic scores. Gene expression profiles from available esophageal tissue from benralizumab-treated patients were compared to those from patients with remission EoE (n = 5), active EoE (n = 10), and controls (n = 22). Benralizumab treatment resulted in partial symptom improvement and significant reduction in tissue eosinophilia, and endoscopic and histologic disease scoring (P < 0.01). Histologic score reductions were driven by eosinophil feature scores, while scores for epithelial features (basal cell hyperplasia and dilated intercellular spaces) were similar to those in active EoE. The gene signatures in benralizumab-treated patients mimicked those of active EoE (e.g. upregulation of POSTN, CDH26, CCL26, and downregulation of DSG1). RNA profiles and pathways support histologic findings of impaired epithelial function that persists despite benralizumab treatment. In conclusion, despite eosinophil eradication, patients treated with benralizumab had persistent epithelial injury at the histologic and transcriptional level. In this cohort, benralizumab therapy failed to eradicate inflammation and epithelial dysfunction showing that interleukin-5 receptor alpha blockade monotherapy is insufficient to control EoE.

BACKGROUND & AIMS:Evidence for a genetic contribution to eosinophilic esophagitis (EoE) exists from family and genome-wide association studies. Extensive investigation into rare variants contributing to EoE has not been performed. The study's aim was to evaluate families with multiple cases of EoE by genomic and transcriptomic sequencing to identify genes predisposing to EoE. METHODS:Distant relative pairs (eg, cousins) in extended EoE families and other affected relatives were whole exome sequenced to identify rare, shared, potentially pathologic variants. RNA sequencing was performed in nuclear families with multiple EoE cases. We compared the overlap of genes from DNA and RNA sequencing for relevance to disease manifestations. RESULTS:Whole exome sequencing was performed in 50 familial cases in 21 EoE extended pedigrees. We observed 189 rare candidate predisposition variants in 181 genes with complete sharing among all affected family members within each pedigree. RNA sequencing was performed for 43 EoE cases in 18 nuclear families, including 6 relatives without EoE. We observed 698 total differentially expressed genes compared with controls. We identified 3 genes (MUC16, ADGRE1, and TENM3) with evidence of rare variant sharing among all affected family members and differential gene expression. We identified 36 other genes with partial sharing of rare variants among some affected family members and with differential gene expression. Several genes identified as prominent in EoE were also differentially expressed in unaffected relatives. CONCLUSIONS:Genes related to immune response, barrier dysfunction, and cell adhesion were identified in familial EoE cases and unaffected family members, supporting a genetic familial predisposition and a possible multihit background to disease pathophysiology.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Colonic SSLs are thought to predispose to ∼30% of colonic adenocarcinomas. This increased risk, compared to benign HPs, makes their distinction vitally important. However, no gold standard exists to differentiate them, and wide observer variability is reported. To better distinguish these polyps, we investigated 94 serrated polyps (53 SSLs and 41 HPs) using an easy-to-apply pathologic scoring system that combines, for the first time, three established distinguishing features: polyp morphology, location, and size. As an additional novel approach, polyp size was assessed by serrated biopsy number compared to endoscopic size. RNA expression profiling served as an additional biomarker. The considerable morphologic overlap across serrated polyps was quantitated for the first time. Interobserver variability was assessed by 8 expert gastrointestinal pathologists. By ROC analysis, polyp size by biopsy number performed best, followed by polyp location and morphology (areas under the curves [AUCs] = 85.9%, 81.2%, and 65.9%, respectively). Optimal discrimination combined all 3 features (AUC = 92.9%). For polyp size, the biopsy number proved superior to endoscopic size (AUC = 85.9% versus 55.2%, P = .001). Interobserver variability analysis yielded the highest reported Fleiss and Kappa statistics (0.879) and percent agreement (96.8%), showing great promise toward improved diagnosis. The proposed 3-criteria pathologic system, combining size by biopsy number, location, and morphology, yields an improved, easy-to-use, and highly reproducible diagnostic approach for differentiating SSLs and HPs.

SummaryBackgroundEosinophilic oesophagitis (EoE) is associated with elevated IgG4 in oesophageal tissue and serum. Previously, we showed brush‐collected oesophageal secretions of EoE patients contained food antigen‐specific antibodies IgA and IgG4. It is unknown whether other food‐specific antibodies are present along the surface of the oesophagus in EoE.AimTo identify whether immunoglobulins other than IgG4 and food‐specific antibodies are elevated along the oesophageal mucosal surface in oesophageal secretions in EoE patientsMethodsConcentrations of total IgA, IgG1, IgG2, IgG3, IgG4, IgM and IgE were measured in oesophageal secretions from patients with active (n = 19) and inactive EoE (n = 9), and non‐EoE controls (n = 10). Food‐specific antibodies were measured using beads coupled to protein components from dairy, wheat and egg. Total immunoglobulin and cytokine and chemokine concentrations were measured in serum, saliva and oesophageal secretions of four patients with active EoE.ResultsOesophageal secretions have a unique immune profile. Patients with active EoE had elevated IgG2, IgG4 and IgM concentrations in oesophageal secretions compared to those with inactive EoE. Food‐specific IgG1, IgG2, IgG4 and IgM were significantly increased in patients with active EoE compared to inactive EoE and non‐EoE patients. Furthermore, active patients with a known dairy trigger display higher dairy‐specific IgG1, IgG2, IgG4, IgM, IgA and IgE.ConclusionsThere is a distinct localised profile of immunoglobulins and food‐specific antibodies found within oesophageal secretions in EoE. These findings expand our knowledge about the currently identified immune responses in EoE and suggest possible roles for multiple immunoglobulins and food‐specific antibodies in the pathophysiology of EoE.

The immunologic mechanisms promoting eosinophilic granulomatosis with polyangiitis (EGPA) are unclear. To characterize the mechanisms underlying pulmonary EGPA, we examined and compared EGPA paraffin-embedded lung biopsies with normal lung biopsies, using immunostaining, RNA sequencing, and RT-PCR. The results revealed novel type 2 as well as immuneregulatory features. These features included basophils and increased mast cell contents; increased immunostaining for tumor necrosis factor ligand superfamily member 14; sparse mast cell degranulation; numerous forkhead box protein P3 (FoxP3) + regulatory T cells and IgG4 plasma cells; and abundant arachidonate 15-lipoxygenase and 25-hydroxyvitamin D-1 alpha hydroxylase, mitochondrial. Significantly decreased 15-hydroxyprostaglandin dehydrogenase [NAD((+))], which degrades eicosanoids, was observed in EGPA samples. In addition, there was significantly increased mRNA for chemokine (C-C motif) ligands 18 and 13 and major collagen genes, IgG4-rich immune complexes coating alveolar macrophages, and increased immunostaining for phosphorylated mothers against decapentaplegic homolog 2/SMAD2, suggesting transforming growth factor-beta activation. These findings suggest a novel self-promoting mechanism of activation of alveolar macrophages by arachidonate 15-lipoxygenaseederived eicosanoids to express chemokines that recruit a combined type 2/immunoregulatory immune response, which produces these eicosanoids. These results suggest that the pulmonary EGPA immune response resembles the immune response to a tissue-invasive parasite infection.

Background: A significant proportion of colorectal cancers, as much as up to 30%, develop through the alternate serrated pathway. The precursor lesions in this pathway comprise of sessile serrated adenomas or polyps (SSA/SSPs) and hyperplastic polyps (HPs), both with a varying risk-profile associated with them for progression into colorectal cancer. Although both types of polyps can be identified histologically, the clinical challenge for gastroenterologists and pathologists remain for risk-prediction as to which of these polyps have a higher likelihood for subsequent development and progression into colorectal cancer. These data highlight the imperative need for the development of more specific and objective markers that can adequately differentiate between the two types of colonic polyps. Aim: The aim of our study was to determine a variety of molecular biomarkers that may distinguish SSA/Ps and HPs, and to establish biomarker profiles that associate with low vs. high-risk SSA/Ps. Methods: We conducted a retrospective study of all colonoscopies (n=12,085) performed at the Howard University Hospital between January 2010 to December 2015; of which 83% were conducted in patients of African American (AA) descent (n=10,027). Among AAs, pathology reports confirmed 4,070 patients with polyps, including 252 with SSA/Ps. Gene expression and mutation frequency profiles were analyzed in a total of 47 patients which included 62 specimens (29 SSPs, 26 HP, 3 tubular adenomas (TA) and 4 normal colonic tissues). From a panel of 51 candidate transcripts, we validated 4 RNA markers (MUC6, FSCN1, SEMG1, and TRNP1) using qRT-PCR. MSI and BRAF mutations were also analyzed. CIMP analysis was performed for the aberrant methylation of CACNA1G, IGF2, NEUROG1, RUNX, SOCS and MLH1. The frequency of gender, age groups, anatomic location, clinical/pathological symptoms and reason for colonoscopy in SSA/P patients was analyzed. The median age range for SSA/P diagnosis was between 50 to 64 years. Results: MUC6, SEMG1, TRNP1, and FSCN1 expression was significantly higher in SSA/Ps vs. HPs (P < 0.05); with corresponding fold differences of 37.2 10.7, 5.8 and 2.5, respectively. BRAF mutations were found in 55.6% of SSA/Ps as opposed to 12.0% of HPs (P < 0.05). The frequency of CIMP was higher in SSA/Ps and correlated with BRAF mutation, while the degree of MSI was more prevalent in HP (P > 0.05). SSA/P lesions were distal (67%). Conclusion: Our results show that MUC6 and SEMG1 expression and BRAF mutation have the strongest correlation with SSA/Ps in comparison to HPs. In addition, SSA/Ps were predominantly distal in location. These are novel and distinguishing features compared to the published literature in non-AA populations and may help explain why MSI and CIMP, usually linked to proximal lesions, are not optimal molecular biomarkers in AA patients with such serrated lesions. Note: This abstract was not presented at the meeting. Citation Format: Hassan Ashktorab, Saman Azam, Taraneh Tarjoman, Priyanka Kanth, Edward Lee, Mehdi Nouraie, Nazli Atefi, Babak Shokrani, Adeyinka Laiyemo, Ajay Goel, Mark W. Hazel, Ruoxin Yao, Angela Snow, Deborah Neklason, Don Delker, Hassan Brim. Do molecular markers differentiate between sessile serrated adenoma/polyps and hyperplastic polyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3159.

Abstract Introduction Eosinophilic esophagitis (EoE) is food-triggered immune esophageal disease characterized by dysphagia and food impaction. The pathophysiology of EoE is poorly understood. Prior eosinophilic esophagitis studies showed abundant IgG4 with IgG4+ plasma cells in esophageal subepithelial tissue. Methods Seven esophagectomies and one deep biopsy from patients with active EoE were compared. Seven uninvolved esophageal areas from surgical resections for tumor and autopsy were used as controls. Histology, immunostains, RNA in situ hybridization, and RNA-seq findings were compared with EoE epithelial biopsy RNA-seq. Results The superficial lamina propria (LP) of EoE patients has dense fibrosis, tertiary lymphoid tissue with abundant B cells, IgG4 plasma cells, and Foxp3+ regulatory T cells. RNA-seq confirms this, with abundant B cell related transcripts, IGHG4, organized lymphoid tissue marker CCL21, and regulatory T cell markers. IL-10 in situ hybridization is present in mononuclear cells, in and near lymphoid aggregate germinal centers. Edema and degranulating mast cells are absent. By RNAseq, the LP of both eosinophilic esophagitis patients and controls have abundant thrombospondin-1, which could activate TGFβ. Nuclear immunostaining for phosphoSMAD2 and phosphoSMAD3 (suggesting active TGFβ), is present the epithelium and the superficial lamina propria. Conclusion The EoE LP immune response has abundant IgG4 plasma cells, regulatory T cells, and a predominantly immunoregulatory transcriptome. Some LP immunoregulatory features are present even in controls. Further work is needed to determine whether the immunoregulatory infiltrate contributes to the dysphagia-inducing subepithelial fibrosis.

Purpose: Serrated polyposis syndrome (SPS) presents with multiple sessile serrated adenoma/ polyps (SSA/Ps) and confers a high risk for development of colon cancer.SSA/Ps have unique histology with dilated basal crypts and typically present with a mucus or stool cap noted on colonoscopy exam.The underlying serrated neoplasia pathway is not entirely understood.Organoid models of colon epithelium can be used to study various genetic changes and therapeutic targets.Patient-derived colon organoid models of SSA/Ps have been reported in only one prior publication.Developing an organoid model of SSA/Ps is challenging, and currently no in vitro model exists to study genetic mechanisms in these polyps.The aim of this study was to develop and begin to examine colon organoid models of SSA/Ps from patients with SPS.Methods: Five SSA/Ps from four patients with SPS were obtained for organoid culture.For comparison, two adenomatous polyps from a patient with Familial Adenomatous Polyposis (FAP) were used to generate organoid cultures.Ascending colon, sigmoid colon and rectal biopsies from these patients' normal-appearing, uninvolved epithelium also produced organoid cultures, along with normal colon tissues from two patients with no hereditary colon cancer predisposition.Organoids were grown and passaged using the standard protocol described by Sato et al for 5 to 15 passages.BRAF V600E mutation, CpG island methylator phenotype (CIMP) and histological analyses were performed on organoids from normal control colon, SSA/Ps and adenomas.Results: Two of five SSA/P organoids were positive for V600E mutation in BRAF and for CIMP.BRAF-mutant SSA/P organoids showed altered morphology, including reduced budding of crypt domains and less cell differentiation.BRAF-mutant SSA/P organoids also showed dilated crypt domains and were positive for Alcian Blue mucin stain inside the dilated crypt lumen (Figure 1).None of the uninvolved colon organoids showed BRAF mutation.FAP adenomas appeared to have similar but likely increased crypt budding than normal colon organoids.BRAF V600E wild type SSA/P and normal colon organoids showed similar morphology.Conclusion: We have developed an organoid model of SSA/Ps that demonstrates genomic aberrations frequently found in SSA/Ps.SSA/P organoids that are BRAF-mutant and CIMP-positive also show unique morphology including mucin-filled dilated crypt domains and reduced differentiation.These results confirm that genomic aberrations and increased mucin production frequently observed in SSA/Ps originate and can be reproduced from colon epithelial stem cells.

The receptor for advanced glycation end products (RAGE), a cell membrane receptor, recognizes ligands produced by cigarette smoke (CS) and has been implicated in the pathogenesis of COPD. We demonstrate that deletion or pharmacologic inhibition of RAGE prevents development of CS-induced emphysema. To identify molecular pathways by which RAGE mediates smoking related lung injury we performed unbiased gene expression profiling of alveolar macrophages (AM) obtained from RAGE null and C57BL/6 WT mice exposed to CS for one week or four months. Pathway analysis of RNA expression identified a number of genes integral to the pathogenesis of COPD impacted by the absence of RAGE. Altered expression of antioxidant response genes and lung protein 4-HNE immunostaining suggest attenuated oxidative stress in the RAGE null mice despite comparable CS exposure and lung leukocyte burden as the WT mice. Reduced endoplasmic reticulum stress in response to CS exposure also was observed in the AM from RAGE null mice. These findings provide novel insight into the sources of oxidative stress, macrophage activation, and the pathogenesis of lung disease due to CS exposure.

OBJECTIVES: Sessile serrated adenoma/polyps (SSA/Ps) contribute up to 30% of all colon cancers. There is considerable histological overlap between SSA/Ps and hyperplastic polyps. Inadequate consensus exists among pathologists, and no molecular biomarkers exist to differentiate these lesions with high accuracy. Lack of reliable diagnosis adversely affects clinical care. We previously defined a novel 7-gene panel by RNA sequencing that differentiates SSA/Ps from hyperplastic polyps. Here, we use the 7-gene panel as a molecular approach to differentiate SSA/Ps and HPs with higher sensitivity and specificity in a large sample set from a tertiary health care center. METHODS: Reverse transcription quantitative polymerase chain reaction of the 7-gene panel was performed on 223 formalin-fixed, paraffin-embedded serrated polyp and normal colon samples. We compare the sensitivity and specificity of the 7-gene panel with the BRAF and KRAS mutation incidence in differentiating SSA/Ps and HPs. We also evaluate the clinical data of patients with SSA/Ps showing high and low expression of the gene panel. RESULTS: The 7-gene RNA expression panel differentiates SSA/Ps and HPs with 89.2% sensitivity and 88.4% specificity. The gene panel outperforms BRAF mutation in identification of SSA/Ps. Clinical data suggest that expression of the 7-gene panel correlates with the development of SSA/Ps in the future. DISCUSSION: This study describes a novel 7-gene panel that identifies SSA/Ps with improved accuracy. Our data show that RNA markers of SSA/Ps advance the distinction of serrated lesions and contribute to the study of the serrated pathway to colon cancer.

Although current American guidelines distinguish proton pump inhibitor-responsive oesophageal eosinophilia (PPI-REE) from eosinophilic oesophagitis (EoE), these entities are broadly similar. While two microarray studies showed that they have similar transcriptomes, more extensive RNA sequencing studies have not been done previously.To determine whether RNA sequencing identifies genetic markers distinguishing PPI-REE from EoE.We retrospectively examined 13 PPI-REE and 14 EoE biopsies, matched for tissue eosinophil content, and 14 normal controls. Patients and controls were not PPI-treated at the time of biopsy. We did RNA sequencing on formalin-fixed, paraffin-embedded tissue, with differential expression confirmation by quantitative polymerase chain reaction (PCR). We validated the use of formalin-fixed, paraffin-embedded vs RNAlater-preserved tissue, and compared our formalin-fixed, paraffin-embedded EoE results to a prior EoE study.By RNA sequencing, no genes were differentially expressed between the EoE and PPI-REE groups at the false discovery rate (FDR) ≤0.01 level. Compared to normal controls, 1996 genes were differentially expressed in the PPI-REE group and 1306 genes in the EoE group. By less stringent criteria, only MAPK8IP2 was differentially expressed between PPI-REE and EoE (FDR = 0.029, 2.2-fold less in EoE than in PPI-REE), with similar results by PCR. KCNJ2, which was differentially expressed in a prior study, was similar in the EoE and PPI-REE groups by both RNA sequencing and real-time PCR.Eosinophilic oesophagitis and PPI-REE have comparable transcriptomes, confirming that they are part of the same disease continuum.