Challenges in Inter-rater Agreement on Lamina Propria Fibrosis in Esophageal Biopsies.

BACKGROUND: Mucosal biopsies in eosinophilic esophagitis (EoE) can exhibit lamina propria (LP) fibrosis, which may portend stenotic complications; however, the histologic diagnosis of LP fibrosis is subjective. We sought to assess and improve the consistency of LP fibrosis diagnosis among our pathologist group. METHODS: At a large pediatric hospital, 25 esophageal biopsy slides from 19 patients (16 with EoE) exhibiting a wide spectrum of LP area, artifacts, and fibrosis severity were scanned into whole-slide images. Staff pediatric pathologists (n = 8) separate from the authors classified each biopsy by LP adequacy and fibrosis severity 1 month before and after completion of an educational tutorial. Consensus was defined as >70% agreement. RESULTS: At baseline, 16/25 (64%) cases reached consensus for no fibrosis (n = 3), fibrosis (n = 7), or inadequate LP (n = 6); agreement was fair (α = 0.34). Post-tutorial, 13/25 (52%) cases reached consensus for no fibrosis (n = 2), fibrosis (n = 7), or inadequate LP (n = 4); agreement was again fair (α = 0.33). There was moderate agreement in grading of fibrosis severity (α = 0.54). CONCLUSION: We document only fair-to-moderate agreement in the diagnosis of esophageal LP fibrosis and adequacy in a large pediatric pathologist group despite targeted education, highlighting a challenge in incorporating this feature into EoE research and clinical decision-making.

A Case Series on the Use of Dupilumab for Treatment of Refractory Eosinophilic Gastrointestinal Disorders.

The incidence and prevalence of eosinophilic esophagitis (EoE), eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), and eosinophilic colitis (EoC) are increasing ( 1 ). These conditions will inevitably become more widely recognized and better understood. There is currently no Food and Drug Administration (FDA)-approved treatment for EoE, but there are standard-of-care treatments that are well established and widely used. In contrast, there is a paucity of data regarding standard-of-care treatment for non-EoE eosinophilic gastrointestinal disorders (EGID). We identified 3 patients that all achieved clinical and histopathologic remission on dupilumab, a monoclonal antibody that blocks the downstream signaling of interleukin (IL)-4 and IL-13. These patients had extra-esophageal forms of EGID with two patients failing to achieve remission on standard-of-care therapies and one patient experiencing significant side effects on swallowed budesonide therapy. The reduction in mucosal eosinophilia in several GI tract segments in these 3 patients highlights a new potential clinical indication for dupilumab in the treatment of pediatric EGID patients.

Calretinin Staining in Anorectal Line Biopsies Accurately Distinguished Hirschsprung Disease in a Retrospective Study.

INTRODUCTION: The absence of submucosal ganglion cells does not reliably distinguish Hirschsprung disease from non Hirschsprung disease in anorectal line biopsies. Calretinin staining might be helpful in these biopsies. To determine its value, we analyzed calretinin positive mucosal neurites in anorectal line biopsies. METHODS: Two pediatric pathologists, without access to patient data, evaluated calretinin positive mucosal neurites in anorectal line junctional mucosa in archival rectal biopsies contributed by 17 institutions. A separate investigator compiled patient information and sent data for statistical analysis. RESULTS: Biopsies with anorectal junctional mucosa from 115 patients were evaluated for calretinin positive mucosal neurites. 20/20 Hirschsprung disease biopsies were negative. 87/88 non Hirschsprung disease biopsies and 7/7 post pullthrough Hirschsprung disease neorectal biopsies were positive. Statistical analysis of the 108 non pullthrough biopsies yielded an accuracy of 99.1% (sensitivity 100%, specificity 98.9%). Age range was preterm to 16 years. Biopsy size was less than 1 mm to over 1 cm. CONCLUSIONS: Absence of calretinin positive mucosal neurites at the anorectal line was highly accurate in distinguishing Hirschsprung disease from non Hirschsprung disease cases in this blinded retrospective study. Calretinin staining is useful for interpreting biopsies from the physiologic hypoganglionic zone up to the anorectal line.

Esophageal Fibrosis in Eosinophilic Gastrointestinal Diseases.

OBJECTIVES: Eosinophilic esophagitis (EoE), the most common eosinophilic gastrointestinal disease (EGID), is associated with lamina propria (LP) fibrosis. The relationship of EoE to other EGIDs is still unclear. We frequently observe cases of concurrent esophageal eosinophilia and extra-esophageal mucosal eosinophilia. The purpose of this study was to compare clinical, endoscopic, and histologic features, as well as the prevalence of esophageal LP fibrosis in children with EGID and concurrent esophageal eosinophilia to children with EoE. We also examine the current practices of pathologists in evaluating fibrosis. METHODS: We reviewed esophageal biopsies from index cases of EoE (N = 38), EGID with significant esophageal eosinophilia (≥15 eos/hpf) (EGID-SEE, N = 38), EGID with mild esophageal eosinophilia (1-14 eos/hpf) (EGID-MEE, N = 12), and EGID with no esophageal eosinophilia (EGID-NEE, N = 12) for LP presence, adequacy, and fibrosis. RESULTS: EoE and EGID-SEE cases share similar demographics, esophageal endoscopic features, and symptoms. A majority of EGID-SEE cases (71%) had adequate LP for the evaluation of fibrosis, similar to EoE cases (87%). The prevalence of esophageal fibrosis in EoE (79%) and EGID-SEE (55%) cases were similar, whereas no fibrosis was detected in the EGID-MEE and EGID-NEE cases. The fibrosis was patchy and often detected in the distal esophagus. Fourteen cases were reclassified from their original clinical diagnosis as having fibrosis by the study pathologists. CONCLUSIONS: Cases of EGID-SEE have overlapping features with EoE, suggesting that all EGIDs are part of a disease continuum. A consensus for the evaluation of LP fibrosis is needed.

Epithelial Indoleamine 2,3-Dioxygenase 1 Modulates Aryl Hydrocarbon Receptor and Notch Signaling to Increase Differentiation of Secretory Cells and Alter Mucus-Associated Microbiota.

BACKGROUND & AIMS: Inflammation, injury, and infection up-regulate expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in the intestinal epithelium. We studied the effects of cell-specific IDO1 expression in the epithelium at baseline and during intestinal inflammation in mice. METHODS: We generated transgenic mice that overexpress fluorescence-tagged IDO1 in the intestinal epithelium under control of the villin promoter (IDO1-TG). We generated intestinal epithelial spheroids from mice with full-length Ido1 (controls), disruption of Ido1 (knockout mice), and IDO1-TG and analyzed them for stem cell and differentiation markers by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. Some mice were gavaged with enteropathogenic Escherichia coli (E2348/69) to induce infectious ileitis, and ileum contents were quantified by polymerase chain reaction. Separate sets of mice were given dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid to induce colitis; intestinal tissues were analyzed by histology. We utilized published data sets GSE75214 and GDS2642 of RNA expression data from ilea of healthy individuals undergoing screening colonoscopies (controls) and patients with Crohn's disease. RESULTS: Histologic analysis of small intestine tissues from IDO1-TG mice revealed increases in secretory cells. Enteroids derived from IDO1-TG intestine had increased markers of stem, goblet, Paneth, enteroendocrine, and tuft cells, compared with control enteroids, with a concomitant decrease in markers of absorptive cells. IDO1 interacted non-enzymatically with the aryl hydrocarbon receptor to inhibit activation of NOTCH1. Intestinal mucus layers from IDO1-TG mice were 2-fold thicker than mucus layers from control mice, with increased proportions of Akkermansia muciniphila and Mucispirillum schaedleri. Compared to controls, IDO1-TG mice demonstrated an 85% reduction in ileal bacteria (P = .03) when challenged with enteropathogenic E coli, and were protected from immune infiltration, crypt dropout, and ulcers following administration of dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid. In ilea of Crohn's disease patients, increased expression of IDO1 correlated with increased levels of MUC2, LYZ1, and aryl hydrocarbon receptor, but reduced levels of SLC2A5. CONCLUSIONS: In mice, expression of IDO1 in the intestinal epithelial promotes secretory cell differentiation and mucus production; levels of IDO1 are positively correlated with secretory cell markers in ilea of healthy individuals and Crohn's disease patients. We propose that IDO1 contributes to intestinal homeostasis.

Characterization of a severe case of PIK3CA-related overgrowth at autopsy by droplet digital polymerase chain reaction and report of PIK3CA sequencing in 22 patients.

PIK3CA-related overgrowth spectrum (PROS) refers to a group of disorders of segmental overgrowth of a wide variety of tissues as well as venous and lymphatic malformations. Clinical and molecular diagnosis can be challenging due to phenotypic heterogeneity and difficulties detecting low-level mosaicism using standard methods. Here, we report a patient with a severe presentation of PIK3CA-related overgrowth with analysis of 27 posthumously collected tissues by droplet digital polymerase chain reaction (PCR) at autopsy. This patient had a complicated medical course, with coagulopathy, ischemic brain injury, and sepsis resulting in multi-organ failure and death at age 2 months despite sirolimus therapy. Five of the 27 tissues analyzed possessed a mosaic PIK3CA mutation (p.E545K), with mutation levels ranging from 3 to 20% across affected tissues. We found no correlation between tissue-specific disease severity and mutation levels, likely reflecting sampling limitations. We also tested a series of 22 individuals with somatic overgrowth and/or vascular-lymphatic malformations using a targeted next generation sequencing panel and found PIK3CA mutations in nine individuals, identifying three novel PIK3CA variants. This report expands the clinical and molecular spectrum of PROS, emphasizes that different molecular methods can be complimentary in the diagnosis of these disorders, and highlights the risk of coagulopathy in a subset of patients with PIK3CA-related overgrowth.

IDO1 metabolites activate ß-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice.

BACKGROUND & AIMS: Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Although IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon cancer cell lines. METHODS: C57Bl6 wild-type (control), IDO1-/-, Rag1-/-, and Rag1/IDO1 double-knockout mice were exposed to azoxymethane and dextran sodium sulfate to induce colitis and tumorigenesis. Colitis severity was assessed by measurements of disease activity, cytokine levels, and histologic analysis. In vitro experiments were conducted using HCT 116 and HT-29 human colon cancer cells. 1-methyl tryptophan and small interfering RNA were used to inhibit IDO1. Kynurenine pathway metabolites were used to simulate IDO1 activity. RESULTS: C57Bl6 mice given pharmacologic inhibitors of IDO1 and IDO1-/- mice had lower tumor burdens and reduced proliferation in the neoplastic epithelium after administration of dextran sodium sulfate and azoxymethane than control mice. These reductions also were observed in Rag1/IDO1 double-knockout mice compared with Rag1-/- mice (which lack mature adaptive immunity). In human colon cancer cells, blockade of IDO1 activity reduced nuclear and activated ß-catenin, transcription of its target genes (cyclin D1 and Axin2), and, ultimately, proliferation. Exogenous administration of IDO1 pathway metabolites kynurenine and quinolinic acid led to activation of ß-catenin and proliferation of human colon cancer cells, and increased tumor growth in mice. CONCLUSIONS: IDO1, which catabolizes tryptophan, promotes colitis-associated tumorigenesis in mice, independent of its ability to limit T-cell-mediated immune surveillance. The epithelial cell-autonomous survival advantage provided by IDO1 to colon epithelial cells indicate its potential as a therapeutic target.

Eosinophilic esophagitis, Barrett's esophagus and esophageal neoplasms in the pediatric patient: a narrative review.

There are several esophageal disorders that can occur in the pediatric population. Eosinophilic esophagitis (EoE) is an eosinophil predominant inflammatory disease of the esophagus that was first characterized in the early 1900's. EoE is the most common pediatric esophageal inflammatory condition after gastroesophageal reflux disease (GERD). Longstanding GERD is a known risk factor for the development of Barrett's esophagus (BE) in both children and adults. BE is associated with the development of dysplasia and, if left undiagnosed, may progress to the development of esophageal adenocarcinoma (EAC). EAC and esophageal squamous cell carcinoma (ESCC) comprise the majority of childhood esophageal malignant neoplasms. The prevalence of EoE continues to rise within the pediatric population. On the other hand, both BE and esophageal neoplasms remain extremely rare in children. The relationship between a chronic inflammatory condition like EoE to BE and/or esophageal neoplasms remains unclear. The current research of these disease entities is prioritized to further understanding the disease pathogenesis and disease progression, exploring new diagnostic modalities, and developing novel treatments or less invasive therapeutic options. The focus of the following narrative review is to provide a summary of the current clinical practices, future research and their implications on these various esophageal disorders.

Hypoplasia of Extrahepatic Biliary Tree and Intrahepatic Cholangiolopathy in Cystic Fibrosis Imperfectly Mimic Biliary Atresia in 4 Infants With Cystic Fibrosis and Kasai Portoenterostomy.

Four male infants with cystic fibrosis and prolonged neonatal jaundice underwent Kasai procedure to relieve biliary obstruction due to apparent biliary atresia. The excised remnants had viscid mucus accumulation in hypoplastic gallbladders and distended peribiliary glands. Main hepatic ducts were narrow and/or malformed. Microscopic differences between the gallbladder and extrahepatic bile ducts in cystic fibrosis and sporadic biliary atresia were unequivocal, despite some histologic overlap; no erosive or fibro-obliterative lesions typical of biliary atresia were seen. Common in liver, biopsies were small duct cholangiopathy with intense focal cholangiolitis and massive accumulation of ceroid pigment within damaged cholangiocytes, and in portal macrophages, portal fibrosis, and unequivocal features of large duct obstruction were inconspicuous compared with biliary atresia. Plugs of bile in small ducts tended to be pale and strongly periodic acid-Schiff-reactive in cystic fibrosis. Distinguishing the liver lesion from that of biliary atresia is challenging but possible. Liver biopsies from 2 additional infants with cystic fibrosis and prolonged jaundice that spontaneously resolved showed a similar small duct cholangiopathy. Small gallbladders and extrahepatic ducts challenge surgical judgment as findings in liver biopsies challenge the pathologist. The decision to perform a Kasai procedure is reasonable when mimicry of biliary atresia is grossly complete. We hypothesize that a disorder of bile volume/flow during development and/or early infancy linked to the CFTR mutation alone or in combination with the stresses of neonatal intensive care causes destructive cholangiolitis and intrahepatic reduction of bile flow with secondary hypoplasia of extrahepatic biliary structures.

Colonic Adventitial Fibromuscular Dysplasia: A Nonspecific Arteriopathy Associated With Hirschsprung Disease and Other Obstructive Disorders.

Background Smooth muscle differentiation ("adventitial fibromuscular dysplasia," AFD) was purported as specific to arteries in the transition zone of Hirschsprung disease (HSCR) patients. We investigated AFD in an HSCR population and controls and consider the pathogenesis and significance of the vascular pathology. Design Vascular histology in sections from colonic HSCR resections (n = 55) was compared with age- and site-matched controls with (n = 19) and without (n = 28) non-HSCR obstructive conditions. Vascular pathology was mapped, and correlations were sought between the vascular findings and bowel distension, inflammation, neuromuscular anatomy, preoperative clinical variables, and postsurgical complications. Results One of 2 forms of AFD was identified in 42% (23/55) of the HSCR resections: the previously described "mature" form with adventitial bundles of differentiated smooth muscle cells (7/23, all submucosal) and a newly described "immature" AFD characterized by densely packed myofibroblasts in the arterial adventitia (16/23, 3 submucosal, 3 serosal, and 10 both). Adventitial inflammation and/or medial necrosis was present in the immature form (6/16). Mature submucosal AFD was present in 2/28 (7.1%) nonobstructive and 5/19 (26%) obstructive non-HSCR controls ( P = .10). Immature AFD was only found in less than 1-month-olds, and mature AFD only in older patients, including the 7 affected controls. AFD did not correlate with sex, syndromic status, length of the aganglionic segment, or postoperative complications. AFD was present in grossly dilated (17/23) and narrowed (10/23) regions and in the aganglionic (2/23), ganglionic (14/23), or both (7/23) segments. In several cases, AFD existed proximal to the histological transition zone. Conclusion AFD occurs in HSCR and other obstructive conditions but is significantly less common in the colons of patients with no history of dysmotility. The pathology likely progresses from a reversible accumulation of myofibroblasts in neonates to a stable population of mature smooth muscle cells. The distribution of vascular lesions does not correlate with neuropathological findings and suggests a nonspecific form of vascular injury, possibly related to bowel distension. AFD in HSCR resections has not been shown to be clinically significant and should not influence management.

Adenoid Stones - "Adenoliths".

Stones made of bacterial aggregates can be found in chronically inflamed lymphoid tissue such as hypertrophied tonsils. Although it is common to find tonsilloliths in cryptic tonsils, it is rare to find stones in adenoid tissue. Here we present an interesting case of a patient who underwent adenoidectomy for adenoid hypertrophy, recurrent malaise and upper respiratory infections. Intraoperatively we found numerous bright green stones in the crypts of the adenoid tissue, reminiscent of tonsilloliths in tonsillar crypts. Pathology revealed polymicrobial bacterial aggregates surrounded by neutrophils. Our findings suggest that the pathophysiology is similar to that of tonsillolith formation. Thus, we should at least consider the presence of adenoid stones and consider adenoidectomy for symptoms often attributed to tonsilloliths. We have coined the term "adenoliths" to describe this interesting finding and present it as a potential source of recurrent infection.

Modeling colitis-associated cancer with azoxymethane (AOM) and dextran sulfate sodium (DSS).

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement. Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu. While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different. This distinction argues the need for specific animal models of CAC. Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors. Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]). The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks. Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.

Serum analysis of tryptophan catabolism pathway: correlation with Crohn's disease activity.

BACKGROUND: Indoleamine 2,3 dioxygenase-1 (IDO1) is a tryptophan catabolizing enzyme with immunotolerance-promoting functions. We sought to determine if increased gut expression of IDO1 in Crohn's disease (CD) would result in detectable changes in serum levels of tryptophan and the initial IDO1 pathway catabolite, kynurenine. METHODS: Individuals were prospectively enrolled through the Washington University Digestive Diseases Research Center. The Montreal Classification was used for disease phenotyping. Disease severity was categorized by the Physician's Global Assessment. Serum tryptophan and kynurenine were measured by high-pressure liquid chromatography. IDO1 immunohistochemical staining was performed on formalin-fixed tissue blocks. RESULTS: In all, 25 CD patients and 11 controls were enrolled. Eight CD patients had serum collected at two different timepoints and levels of disease activity compared. Strong IDO1 expression exists in both the lamina propria and epithelium during active CD compared to controls. Suppressed serum tryptophan levels and an elevated kynurenine/tryptophan (K/T) ratio were found in individuals with active CD as compared to those in remission or the control population. K/T ratios correlated positively with disease activity as well as with C-reactive protein and erythrocyte sedimentation rate. In the subgroup of CD patients with two serum measurements, tryptophan levels were elevated while kynurenine levels and the K/T ratio lowered as the disease activity lessened. CONCLUSIONS: IDO1 expression in CD is associated with lower serum tryptophan and an elevated K/T ratio. These levels may serve as a reasonable objective marker of gut mucosal immune activation and as a surrogate for CD activity.