diagnostic markers in ibd - gihealthfoundation.org

Gary R Lichtenstein, MD Professor of Medicine

Director, IBD Center

University of PA School of Medicine

Philadelphia, PA

Diagnostic Markers in IBD

Diagnosing and Classifying IBD

Can be Challenging

• Large degree of overlap in clinical symptoms with other GI diseases

• Clinical symptoms don’t always correlate with disease activity and severity of inflammation

• Clinical indices (e.g., CDAI, HBI) are subjective and don’t correlate well with endoscopic findings and serum markers of inflammation

• Endoscopic imaging can’t detect transmural inflammation or penetrating disease

• Pathology assessment is often nonspecific

Distinguishing UC from CD is

Not Always Straightforward

Distribution Continuous, symmetric, and

diffuse distribution

Distribution is often discontinuous and

asymmetric with skipped segments and normal

intervening mucosa

Depth of Inflammation Mucosal/submucosal inflammation Mucosal, submucosal, and/or transmural

inflammation

Site Colon affected exclusively May affect any part of GI tract

Rectal Involvement Almost always involves

the rectum

Relative rectal sparing may be present

Ulcerative Colitis Crohn’s Disease

Multiple Tools are Typically Used to

Establish a Diagnosis

Stool cultures

Colonoscopy, Endoscopy

Crohn’s

Disease

Ulcerative

Colitis

Fletcher JG et al. Gastroenterology. 2011;140:1795–1806.

Benefits and Risks of Imaging Tests in IBD

Imaging Test Benefits Risks

CT enterography

(CTE)

• Detection of clinically occult inflammatory and

penetrating disease

• Complementary to ileocolonoscopy

• Images entire abdomen/pelvis

• Temporal and spatial resolution > MRE

• Widely available technology and expertise

• Radiation

• Iodine dye (renal

insufficiency, allergy)

• Cannot detect

colonic neoplasia

MR enterography

(MRE)

• Detection of clinically occult

inflammatory and penetrating

disease

• Complementary to ileocolonoscopy

• Images entire abdomen/pelvis

• Lack of radiation

• Image quality may be

suboptimal in some

patients

• Cannot detect

colonic neoplasia

Iliocolonoscopy (Ico)

• Best test for detection of colonic inflammation

• Histologic sampling

• Synchronous neoplasia surveillance

• Can assess mucosal healing

• Perforation

• Limited bowel evaluation

• Cannot stage

penetrating disease

Chromeondoscopy • improved detection of intraepithelial neoplasia compared to

surveillance colonoscopy

Capsule Endoscopy • Most sensitive for detecting small bowel mucosal

inflammation (other than double balloon)

• Retention

Inflammatory Bowel Disease

Genetics Has Made Great Progress

Prior 2005 2006 2007 2008

TNFSF15 ATG16L1

IL23R

ZNF365

PTGER4

IRGM

MST1

ECM1 C11orf30

JAK2

CCR6

CDKAL1

IL12B

PTPN22

ORMDL3

STAT4

ICOSLG

10p11

8q24

7p12

6q21

1q32

STAT3

13q14

19p13

21q21

MHC

5q31

NOD2

NKX2-3

ITLN1

LRRK2/MUC19

Barrett JC et al. Nat Genet. 2008;40:955-962.

Genes Associated with IBD, CD, and UC

Thompson AI and Lees CW. Inflamm Bowel Dis. 2011;17:831-848.

ATG16L1 – The Autophagy Gene

• Constitutive process for regulating

intracellular homeostasis and removal

of intestinal microbes

• Critical pathway for the breakdown of

bacteria: E coli, Salmonella, Legionella,

M tuberculosis

• ATG16L1 mutations are associated

with CD

– P=2.33 X 10-7

Bacterial Clearing1-6

1. Parkes M et al. Nat Genet. 2007;9: 830-832; 2. Yamazaki K et al. J Hum Genet. 2007;52:575-583; 3. Prescott NJ et al. Gastroenterology.

2007;132:1665-1671; 4. Rioux JD, et al. Nat Genet. 2007;39:596-604; 5. Hampe J et al. Nat Genet. 2007;39: 207-211; 6. Cummings JR et al. Inflamm

Bowel Dis. 2007:13-18

ECM1: The UC Susceptibility Gene

• Related to intestinal barrier function

• Plays a role in epithelial cell interactions

• Mutations are associated with increased mucosal layer permeability

• P=2.3 x 10-6 in UC

UC Susceptibility Gene

GWA=genome-wide association; MHC=major histocompatibility complex

1. Thompson AI and Lees CW. Inflamm Bowel Dis. 2011;17:831-848; 2. Fisher SA et al Nat Genet. 2008;40:710-712.

STAT3 – Proinflammatory Mediator

• Signal transduction and activator of transcription

• Activated STAT3 is necessary for the signaling of pro-inflammatory cytokines in IBD

• Plays a role in the biology of Th17 cells

• P=9.15x10-8 in CD

Proinflammatory Mediator1,2

1. Jarnicki A et al. Cell Division. 2010;5:14; 2. Barret JC et al. Nat Genet. 2008;40:955-962.

NKX2-3 – Transcription Factor

• Transcription factor implicated in cell type specification and maintenance of differentiation

• Expressed in endothelial cells and intestinal lamina propria mesenchymal cells

• Associated with both UC and CD

• CD P=.009; UC P=.001

Transcription Factor

Meggyesi A et al. World J Gastroenterol. 2010;16:5233-5240.

Genetic Markers

Marker General

Prevalence Role/Function Effect

ATG16L1

(associated

with CD)1-6

OR 1.45 • Elongates membranes that form

autophagosomes in autophagy

• May decrease ability to remove

intestinal microbes

• Increases production of

inflammatory cytokines from macrophages

ECM1

(associated

with UC)7,8

OR 1.3 – 1.4 • Interacts with basal membrane,

inhibits MMP9 proteins, and

activates signaling of

proinflammatory cytokine

pathway NF-kB

• Exhibits defective barrier function

• Causes thickening and scarring of mucus

membranes

NKX2.3

(associated with

CD and UC)9

OR 1.2 – 1.6 • Encodes transcription factor that

functions in cell specification and

maintenance of tissue differentiation

• Associated with defects in gut development,

abnormal tissue architecture, abnormalities

in migration and segregation of B and T

cells

STAT3

(associated with

CD and UC)10-11

OR 1.18 • Plays role in the Th17-dependent

autoimmune process

• Expansion and overactivity on Th17 helper

cell cytokines can lead to intestinal

inflammation

OR=odds ratio

1. Parkes M et al. Nat Genet. 2007;9: 830-832; 2. Yamazaki K et al. J Hum Genet. 2007;52:575-583; 3. Prescott NJ et al. Gastroenterology.

2007;132:1665-1671; 4. Rioux JD, et al. Nat Genet. 2007;39:596-604; 5. Hampe J et al. Nat Genet. 2007;39: 207-211; 6. Cummings JR et al. Inflamm

Bowel Dis. 2007:13-18; 7. Thompson AI and Lees CW. Inflamm Bowel Dis. 2011;17:831-848; 8. Fisher SA et al Nat Genet. 2008;40:710-712; 9.

Meggyesi A et al. World J Gastroenterol. 2010;16:5233-5240; 10. Jarnicki A et al. Cell Division. 2010;5:14; 11. Barret JC et al. Nat Genet.

2008;40:955-962.

Role of Angiogenesis in IBD

• Serum concentrations are significantly increased in active CD and UC patients compared to those with quiescent disease or healthy controls

• Serum levels correlate with disease activity

– Increased intramural blood flow

– Higher production rates in inflamed mucosa of active CD and UC patients vs normal mucosa of healthy controls

• Induces epithelial cell migration which can play a role in intestinal cell restitution

VEGF

Koutroubakis D et al. Inflamm Bowel Dis. 2006;12:515-523.

Role of Adhesion Molecules in IBD

• Unregulated in response to inflammation in both CD and UC patients

• Facilitate adhesion and infiltration of leukocytes into the inflammation site

• Correlate with disease activity in IBD patients

• Play an important role in colitis associated angiogenesis

ICAM-1, VCAM-1

Chidlow et al., Am J Physiol Gastrointest Liver Physiol 2007, 293: G5–G18

Inflammatory Markers Correlate

with Disease Activity

• Produced in the liver in response

to inflammation

• Can be used to differentiate IBD

from functional bowel diseases

• Stronger response in CD patients

vs UC patients and in colonic vs.

small intestinal disease

• CRP levels at the time of

diagnosis are related to the

extent of disease

• 20%-30% of patients cannot

mount a CRP response

• Produced in the liver in response

to inflammation

• Physiologic levels are ~10x

higher than that of CRP

– Allows for easier detection

of slight elevations

• Similarly elevated in both CD

and UC

CRP SAA

CRP=C-reactive protein; SAA=serum amyloid A

Inflammatory Bowel Disease

Test Development

Objectives:

• To identify a set of blood-based immune, genetic, and inflammatory biomarkers that could be used to improve the identification of IBD patients and differentiation between CD and UC over that in the existing IBD Serology 7 test

• Develop the next generation IBD diagnostic test using a representative and well-characterized cohort of IBD, GI disease and healthy control samples

Plevy SE et al. DDW 2012. Abstract no. 166

Well-characterized samples with known diagnoses from

8 academic and 42 community centers in North America

Training and Validation Cohorts

Disease Diagnosis Training Cohort

n (%)

Validation Cohort

n (%)

Crohn’s Disease 419 (39) 153 (35)

Ulcerative Colitis 227 (21) 101 (23)

GI Controls 314 (29) 123 (28)

Healthy Controls 123 (11) 60 (14)

Total 1083 (100) 437 (100)

Plevy SE et al. DDW 2012. Abstract no. 166

Serology, Genetics, and Inflammation Markers

s • ASCA-A

• ASCA-G

• anti-OmpC

• anti-CBir-1

• anti-A4-Fla2

• anti-FlaX

• ANCA

• pANCA-IFA

• pANCA DNAse

g • ATG16L1

• ECM1

• NKX2-3

• STAT3

i • ICAM1

• VCAM1

• VEGF

• CRP

• SAA

Plevy SE et al. DDW 2012. Abstract no. 166

ROC Curves for Individual Markers Marginal Discrimination: IBD vs. non-IBD

ROC=receiver-operator curve

Plevy SE et al. DDW 2012. Abstract no. 166

Higher Level of Discrimination:

IBD vs Non-IBD

Plevy SE et al. DDW 2012. Abstract no. 166

IBD sgi Performs Better than IBD 7

in Differentiating CD vs UC

In a head-to-head

comparison, IBD sgi

demonstrated improved

overall performance over

IBD Serology 7 in

differentiating CD vs UC

Absolute

Improvement

Relative

improvement

CD /

UC (AUC)

15% 19%

Plevy SE et al. DDW 2012. Abstract no. 166335

Overall Diagnostic Performance IBD sgi vs. IBD Serology 7

In a head-to-head comparison of diagnostic performance,

IBD sgi demonstrated absolute and relative improvements

over IBD Serology 7

IBD sgi IBD Serology 7 Absolute

Improvement

Relative

Improvement

IBD vs. Non-IBD

(AUC) 0.87 0.80 7% 9%

CD / UC

(AUC) 0.93 0.78 15% 19%

Plevy SE et al. DDW 2012. Abstract no. 166

IBD sgi Performance Characteristics

74%

89%

98%

90%

81% 84%

0%

20%

40%

60%

80%

100%

IBD CD UC

Sensitivity Specificity

Plevy SE et al. DDW 2012. Abstract no. 166

Diagnosis Predictive

Values High Pre-test Probability

IBD

PPV 98%

NPV 37%

CD

PPV 96%

NPV 56%

UC

PPV 97%

NPV 87%

Low Pre-test Probability

56%

95%

45%

98%

52%

100%

Test Performance by Pre-test Probability

Med. Pre-test Probability

88%

77%

82%

88%

86%

97%

NPV=negative predictive value; PPV=positive predictive value

Plevy SE et al. DDW 2012. Abstract no. 166

Potential Benefits of an Accurate IBD Diagnosis

Early disease (mild-moderate

disease)

Advanced disease

(moderate-severe disease)

Prevention of

complicated disease

Selection of appropriate

management strategies

Conclusions

• The pathogenesis of IBD reflects dysregulated immunologic responses to diverse antigens (yeast, bacteria, flagellins, auto-antibodies) in a genetically susceptible person resulting in inflammation and tissue injury

• A random Forest model that analyzes the complex interactions between the 3 classes of markers (serology, genetics, inflammation) is more accurate at detecting patterns that differentiate IBD vs non-IBD and CD vs UC patients than using a single class of markers (serology)