Disclosures Expression May Distinguish Expression May Distinguish - - PDF document

1

Differential Epithelial Gene Differential Epithelial Gene Expression May Distinguish Expression May Distinguish Eosinophilic Esophagitis from Eosinophilic Esophagitis from Gastroesophageal Reflux Gastroesophageal Reflux

NASPGHAN Annual Meeting NASPGHAN Annual Meeting Salt Lake City UT Salt Lake City UT Salt Lake City, UT Salt Lake City, UT October 19 October 19th

th, 2012

, 2012 Vincent A. Mukkada M.D. Vincent A. Mukkada M.D. Hasbro Children Hasbro Children’ ’s Hospital s Hospital Brown University Brown University Providence, RI Providence, RI

Disclosures

• I have had no relevant financial

relationships with the manufacturers of any commercial products and/or providers of commercial services discussed in this activity.

• I do not intend to discuss an

unapproved or investigative use of a commercial product or device.

Introduction Introduction

• Eosinophilic Esophagitis (EoE) is a

chronic inflammatory disease of the esophagus whose clinical, endoscopic, and histologic presentation can overlap significantly with Gastroesophageal Reflux Disease (GERD)

• Pathogenesis remains unclear, but

roles postulated for allergy and impaired mucosal barrier

Hypothesis/Aims Hypothesis/Aims

• Hypothesis

Hypothesis-

• Epithelial gene expression

Epithelial gene expression is differentially regulated in is differentially regulated in Eosinophilic Esophagitis Eosinophilic Esophagitis

• Aim 1

Aim 1-

• Identify patterns of epithelial

Identify patterns of epithelial y p p y p p gene expression to differentiate EoE gene expression to differentiate EoE from GERD and normal controls from GERD and normal controls

• Aim 2

Aim 2-

• Demonstrate differential

Demonstrate differential expression pattern by expression pattern by immunohistochemistry (IHC) immunohistochemistry (IHC)

Materials and Methods

• Retrospective study performed on

previously obtained biopsy material

• 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

Materials and Methods

Identification of Markers

Initial Screening Microarray-RNA from pediatric EoE cases pre/post topical steroid treatment

2

Materials and Methods

Identification of Markers

Initial Screening Microarray-RNA from pediatric EoE cases pre/post topical steroid treatment Choice of five markers of interest Choice of five markers of interest

Materials and Methods

Identification of Markers

Initial Screening Microarray-RNA from pediatric EoE cases pre/post topical steroid treatment Choice of five markers of interest Choice of five markers of interest Confirmation of Expression Patterns by RT-PCR

Materials and Methods

Identification of Markers

Initial Screening Microarray-RNA from pediatric EoE cases pre/post topical steroid treatment Choice of five markers of interest Choice of five markers of interest Confirmation of Expression Patterns by RT-PCR Use of Identified Markers for IHC

Materials and Methods Materials and Methods

Postulated Roles for Genes Studied Postulated Roles for Genes Studied

• ALOX15- lipoxygenase pathway, has role in

asthma pathogenesis

• TNFAIP6- associated with inflammation and

tissue remodeling Fil i l l i b i f ti f

• Filaggrin- plays role in barrier function of

skin and eczema pathogenesis

• SLURP-1- associated with differentiation of

keratinocytes and barrier function

• CRISP-3- possible role in innate immune

function

Materials and Methods

Immunohistochemistry

• Used these 5 markers for IHC staining
• n 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

Results Results

Genes Upregulated on Microarray and Genes Upregulated on Microarray and Validated by RT Validated by RT-

• PCR

PCR

3

Results Results

Genes Downregulated on Microarray and Genes Downregulated on Microarray and Validated by RT Validated by RT-

• PCR

PCR

Results Results

IHC: ALOX15 IHC: ALOX15

Normal Control EoE-Pre-treatment EoE-Post-treatment GERD

Results Results

IHC: TNFAIP6 IHC: TNFAIP6

Normal Control EoE-Pre-treatment EoE-Post-treatment GERD

Results Results

IHC: Filaggrin IHC: Filaggrin

Normal Control EoE-Pre-treatment EoE-Post-treatment GERD

Results Results

IHC Summary Results IHC Summary Results Upregulated Markers Upregulated Markers

Results Results

IHC Summary Results IHC Summary Results Downregulated Markers Downregulated Markers

4

Summary Summary

• We have identified a number of easily

We have identified a number of easily assessed markers differentially expressed assessed markers differentially expressed in EoE in EoE

• These markers can reverse with treatment

These markers can reverse with treatment These markers can reverse with treatment These markers can reverse with treatment and may allow assessment of therapy and may allow assessment of therapy

• These genes may also indicate roles for

These genes may also indicate roles for inflammation and impaired barrier inflammation and impaired barrier function in pathogenesis function in pathogenesis

Conclusions/Future Directions Conclusions/Future Directions

• Use of these markers may allow more

Use of these markers may allow more efficient differentiation of EoE from GERD efficient differentiation of EoE from GERD

• Next steps

Next steps-

• Evaluate equivocal EoE vs GERD

Evaluate equivocal EoE vs GERD q q

• Include cases of

Include cases of “ “PPI responsive PPI responsive eosinophilia eosinophilia” ”

• Evaluate more cases pre/post treatment

Evaluate more cases pre/post treatment

• Examine the role of these markers in adult

Examine the role of these markers in adult EoE EoE

Acknowledgements Acknowledgements

• Division of Pediatric Gastroenterology

Division of Pediatric Gastroenterology

• Collaborators in Pathology:

Collaborators in Pathology:

– Andres Matoso MD Andres Matoso MD – Shaolei Lu MD Shaolei Lu MD – Renee Monahan Renee Monahan – Kelly Cleveland Kelly Cleveland – Shamlal Mangray MD Shamlal Mangray MD – Nicholas Shillingford MD Nicholas Shillingford MD – Murray Resnick MD, PhD (Co Murray Resnick MD, PhD (Co-

• PI)

PI)

• Grant Funding:

Grant Funding:

– Brown University Department of Pediatrics Faculty Brown University Department of Pediatrics Faculty Research Grant Research Grant – Hearst Foundations Hearst Foundations

EoE (n=42) EoE-AT (n=7) GERD (n=15) Normal (n=17) Age (mean±S.D) 10.01±5.1 7 7.0±5.0 3 10.13±5.0 2 10.52±4.5 9 Sex (M:F) 32:10 5:2 7:8 10:7 Symptoms Abdominal pain (%) 21 (50) 2 (29) 11 (73) 11 (64) Vomiting (%) 18 (43) 0 (0) 12 (80) 3 (18) Dysphagia (%) 28 (66) 1 (14) 3 (20) 1 (6) Food impaction (%) 12 (29) 0 (0) 0 (0) 0 (0) Heartburn (%) 14 (33) 0 (0) 4 (27) 3 (18) Failure to thrive (%) 8 (19) 1 (14) 2 (13) 1 (6) Endoscopy Normal (%) 1 (2) 1 (14) 8 (53) 14 (82) Erythema (%) 24 (57) 2 (29) 4 (27) 0 (0) Rings (%) 3 (7) 1 (14) 0 (0) 1 (6) Ridging (%) 22 (52) 1 (14) 1 (6) 1 (6) Furrows 29 (69) 4 (57) 1 (6) 0 (0) 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 55 38±24 0 57±0 6 6±2 79 0±0 Eosinophils/HPF (mean±S.D) 55.38±24. 95 0.57±0. 97 6.6±2.79 0±0 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)