Disclosures Expression May Distinguish Expression May Distinguish - PDF document

Differential Epithelial Gene Differential Epithelial Gene Disclosures Expression May Distinguish Expression May Distinguish Eosinophilic Esophagitis from Eosinophilic Esophagitis from • I have had no relevant financial relationships with the manufacturers of Gastroesophageal Reflux Gastroesophageal Reflux any commercial products and/or NASPGHAN Annual Meeting NASPGHAN Annual Meeting providers of commercial services Salt Lake City, UT Salt Lake City, UT Salt Lake City UT Salt Lake City UT discussed in this activity. October 19 October 19 th th , 2012 , 2012 • I do not intend to discuss an Vincent A. Mukkada M.D. Vincent A. Mukkada M.D. unapproved or investigative use of a Hasbro Children Hasbro Children’ ’s Hospital s Hospital commercial product or device. Brown University Brown University Providence, RI Providence, RI Introduction Introduction Hypothesis/Aims Hypothesis/Aims • Eosinophilic Esophagitis (EoE) is a • Hypothesis Hypothesis- - Epithelial gene expression Epithelial gene expression chronic inflammatory disease of the is differentially regulated in is differentially regulated in esophagus whose clinical, endoscopic, Eosinophilic Esophagitis Eosinophilic Esophagitis and histologic presentation can overlap • Aim 1 Aim 1- - Identify patterns of epithelial Identify patterns of epithelial y p y p p p significantly with Gastroesophageal gene expression to differentiate EoE gene expression to differentiate EoE Reflux Disease (GERD) from GERD and normal controls from GERD and normal controls • Pathogenesis remains unclear, but • Aim 2 Aim 2- - Demonstrate differential Demonstrate differential roles postulated for allergy and expression pattern by expression pattern by impaired mucosal barrier immunohistochemistry (IHC) immunohistochemistry (IHC) Materials and Methods Materials and Methods Identification of Markers • Retrospective study performed on Initial Screening Microarray-RNA from pediatric EoE cases pre/post previously obtained biopsy material topical steroid treatment • All clinical history abstracted from chart review • Patients assigned to groups based on stringent criteria including 2011 consensus EoE guidelines • Study approved by Rhode Island Hospital IRB 1

Materials and Methods Materials and Methods Identification of Markers Identification of Markers Initial Screening Microarray-RNA Initial Screening Microarray-RNA from pediatric EoE cases pre/post from pediatric EoE cases pre/post topical steroid treatment topical steroid treatment Choice of five markers of interest Choice of five markers of interest Choice of five markers of interest Choice of five markers of interest Confirmation of Expression Patterns by RT-PCR Materials and Methods Materials and Methods Materials and Methods Identification of Markers Postulated Roles for Genes Studied Postulated Roles for Genes Studied • ALOX15- lipoxygenase pathway, has role in Initial Screening Microarray-RNA from pediatric EoE cases pre/post asthma pathogenesis topical steroid treatment • TNFAIP6- associated with inflammation and tissue remodeling Choice of five markers of interest Choice of five markers of interest • Filaggrin- plays role in barrier function of Fil i l l i b i f ti f skin and eczema pathogenesis • SLURP-1- associated with differentiation of Confirmation of Expression keratinocytes and barrier function Patterns by RT-PCR • CRISP-3- possible role in innate immune function Use of Identified Markers for IHC Results Results Materials and Methods Genes Upregulated on Microarray and Genes Upregulated on Microarray and Immunohistochemistry Validated by RT- Validated by RT -PCR PCR • Used these 5 markers for IHC staining on biopsies of pediatric patients with EoE (n=42) compared with GERD (n=15) and normal controls (n=15) and normal controls (n=15) – Had 7 patients with biopsies after successful topical steroid therapy • Staining evaluated quantitatively for intensity (0-3) and extent (0-3) – Score ≥ 3 was positive – Scored independently by 2 pathologists 2

Results Results Results Results Genes Downregulated on Microarray and Genes Downregulated on Microarray and IHC: ALOX15 IHC: ALOX15 Validated by RT- Validated by RT -PCR PCR Normal Control EoE-Pre-treatment GERD EoE-Post-treatment Results Results Results Results IHC: TNFAIP6 IHC: TNFAIP6 IHC: Filaggrin IHC: Filaggrin Normal Control EoE-Pre-treatment Normal Control EoE-Pre-treatment GERD EoE-Post-treatment GERD EoE-Post-treatment Results Results Results Results IHC Summary Results IHC Summary Results IHC Summary Results IHC Summary Results Upregulated Markers Upregulated Markers Downregulated Markers Downregulated Markers 3

Summary Summary Conclusions/Future Directions Conclusions/Future Directions • Use of these markers may allow more Use of these markers may allow more • We have identified a number of easily We have identified a number of easily efficient differentiation of EoE from GERD efficient differentiation of EoE from GERD assessed markers differentially expressed assessed markers differentially expressed in EoE in EoE • Next steps Next steps- - • These markers can reverse with treatment These markers can reverse with treatment These markers can reverse with treatment These markers can reverse with treatment • Evaluate equivocal EoE vs GERD Evaluate equivocal EoE vs GERD q q and may allow assessment of therapy and may allow assessment of therapy • Include cases of Include cases of “ “PPI responsive PPI responsive eosinophilia eosinophilia” ” • These genes may also indicate roles for These genes may also indicate roles for • Evaluate more cases pre/post treatment Evaluate more cases pre/post treatment inflammation and impaired barrier inflammation and impaired barrier • Examine the role of these markers in adult Examine the role of these markers in adult function in pathogenesis function in pathogenesis EoE EoE Acknowledgements Acknowledgements EoE EoE-AT GERD Normal (n=42) (n=7) (n=15) (n=17) Age (mean±S.D) 10.01±5.1 7.0±5.0 10.13±5.0 10.52±4.5 7 3 2 9 • Division of Pediatric Gastroenterology Division of Pediatric Gastroenterology Sex (M:F) 32:10 5:2 7:8 10:7 Symptoms • Collaborators in Pathology: Collaborators in Pathology: Abdominal pain (%) 21 (50) 2 (29) 11 (73) 11 (64) – Andres Matoso MD Andres Matoso MD Vomiting (%) 18 (43) 0 (0) 12 (80) 3 (18) – Shaolei Lu MD Shaolei Lu MD Dysphagia (%) 28 (66) 1 (14) 3 (20) 1 (6) – Renee Monahan Renee Monahan Food impaction (%) 12 (29) 0 (0) 0 (0) 0 (0) – Kelly Cleveland Kelly Cleveland Heartburn (%) 14 (33) 0 (0) 4 (27) 3 (18) – Shamlal Mangray MD Shamlal Mangray MD Failure to thrive (%) 8 (19) 1 (14) 2 (13) 1 (6) Endoscopy – Nicholas Shillingford MD Nicholas Shillingford MD Normal (%) 1 (2) 1 (14) 8 (53) 14 (82) – Murray Resnick MD, PhD (Co Murray Resnick MD, PhD (Co- -PI) PI) Erythema (%) 24 (57) 2 (29) 4 (27) 0 (0) • Grant Funding: Grant Funding: Rings (%) 3 (7) 1 (14) 0 (0) 1 (6) – Brown University Department of Pediatrics Faculty Brown University Department of Pediatrics Faculty Ridging (%) 22 (52) 1 (14) 1 (6) 1 (6) Research Grant Research Grant Furrows 29 (69) 4 (57) 1 (6) 0 (0) – Hearst Foundations Hearst Foundations White plaques 29 (69) 2 (29) 0 (0) 0 (0) Erosion 5 (12) 0 (0) 2 (13) 0 (0) Allergies Food allergy (%) 22 (52) 6 (85) 1 (6) 1 (6) Asthma (%) 14 (33) 2 (29) 0 (0) 1 (6) Rhinitis or dermatitis (%) 16 (38) 2 (29) 1 (6) 1 (6) Histopathology Eosinophils/HPF Eosinophils/HPF 55.38±24. 55 38±24 0.57±0. 0 57±0 6 6±2 79 6.6±2.79 0±0 0±0 (mean±S.D) 95 97 Basal cell hyperplasia 42 (100) 0 (0) 15 (100) 0 (0) Papillary elongation 32 (76) 2 (29) 8 (53) 0 (0) Microabscesses 7 (17) 0 (0) 0 (0) 0 (0) Degranulation 36 (85) 0 (0) 2 (13) 0 (0) 4