Serum Markers: What WhoSerum Markers: What WhoSerum Markers: What, Who, Serum Markers: What, Who, When and Why?When and Why?

Sunanda Kane, MD, MSPH, FACG, FACP, AGAFAssociate Professor of Medicine

Mayo Clinic

Crohn’s Disease:Crohn’s Disease:Microbial AntibodiesMicrobial Antibodies

ASCA Anti- Saccharomyces cerevisiae

Anti-I2 Protein from pseudomonas fluorescens

Anti-OmpC Escherichia coli outer membrane porin

Bir1 Flagellin Antibodies to CBir FlagellinBir1 Flagellin Antibodies to CBir Flagellin

Targan S. Gastroenterology 2005;128(7): 2020-8

Combining Markers Improves Sensitivity Combining Markers Improves Sensitivity but Not Overall Accuracybut Not Overall Accuracy

Relationships of Serum Responses in CD

Total = 68% T t l 84%Total = 68% Total = 84%

Adapted from Landers CJ, et al. Gastroenterology. 2002;123:689-699.

Antibodies to CBir1 Antibodies to CBir1 FlagellinFlagellin

• Antigen of the gut flora• Antigen of the gut flora• Induces a strong B cell and CD4+ T cell

response in colitic miceresponse in colitic mice• 50% of patients with Crohn’s disease

have serum reactivityhave serum reactivity• Little or no reactivity in UC, inflammatory

controls, and normal controls,

Targan S. Gastroenterology 2005;128(7): 2020-8

Immune ResponseImmune ResponseCD PatientsCD Patients

60

555655

40

50

s

30

40

of P

atie

nts

23

10

20% o

0ASCA

1Anti-OmpC

1Anti-CBir1

2pANCA

1

1. Adapted from Landers CJ, et al. Gastroenterology. 2002;123:689-699.2. Targan SR, et al. Gastroenterology. 2005;128:2020-2028.

AntiAnti--CBir1 Presence CBir1 Presence CD PatientsCD Patients

3.)

50%2ody

(O.D

% % %

50%

1Bir1

Ant

ibo

8% 14% 6%

0

1

Ant

i-CB

n=40 n=21 n=50 n=100

0Normal

ControlsInflammatory

Controls UC CD

P vs CD:% Positive <0.001 <0.02 <0.001 n/a

Level <0.001 <0.003 <0.001 n/a

Targan SR, et al. Gastroenterology. 2005;128:2020-2028.

AntiAnti--CBir1 Helps Distinguish CBir1 Helps Distinguish Between pANCA+ PatientsBetween pANCA+ Patients

3

)

44%P<0.001 (level)

2

Bir1

(O.D

. 11/25

4%1

Ant

i-CB

1/254%

0.623

0pANCA+

UCpANCA+

CD

0.255

Targan SR, et al. Gastroenterology. 2005;128:2020-2028.

Performance Comparison:Performance Comparison:Crohn’s DiseaseCrohn’s Disease

ASCA & ANCAASCA+ ASCA+ & pANCA-97%88%

49%61%

Sensitivity Specificity Sensitivity Specificity

In a clinical study, serum samples from 100 CD,101 UC and 2 li id /di h l i d 163 l l d i

Sensitivity Specificity Sensitivity Specificity

N=391 (51% IBD, 50% CD, 50% UC)

Quinton JF, et al. Gut. 1998;42:788-791.

27 colitides/diarrheal patients and 163 controls were analyzed using pANCA IFA and ASCA ELISA

Performance Comparison Ulcerative ColitisPerformance Comparison Ulcerative Colitis

ANCA+ pANCA+ & ASCApANCA+ pANCA+ & ASCA-97%

85%

65%57%

65%

Sensitivity Specificity Sensitivity Specificity

N=391 ( 51% IBD, 50% CD, 50% UC)

In a clinical study, serum samples from 100 CD,101 UC and

Sensitivity Specificity Sensitivity Specificity

Quinton JF, et al. Gut. 1998:42:788-791.

y27 colitides/diarrheal patients and 163 controls were analyzed using pANCA IFA and ASCA ELISA.

Prometheus IBD Serology 7Prometheus IBD Serology 7

IBD Predicted

PROMETHEUS IBD Serology 7Overall Performance

IBD CD UC

Sensitivity 93% 88% 93%

Test Result

IBD Predicted

IBD Not Predicted

Ulcerative Colitis PredictedPROMETHEUS Predictive Algorithm Description:• Utilizes Smart Diagnostic Algorithm (SDA) technology to

Specificity 95% 98% 97%

PPV 96% 96% 89%

NPV 90% 93% 98%

Ulcerative Colitis Predicted

Crohn’s Disease Predicted

• Utilizes Smart Diagnostic Algorithm (SDA) technology tocharecterize complex relationships between markers to producea diagnosic prediction with greater accuracy than simple comparison of assay results to a reference range.

• Developed (n=1813; 36% CD, 24% UC, 20% IBS, 20% normal) and validated (n=500; 38% CD, 21% UC, 41% normal) using serology results for samples with a known diagnosis

ASCA lgA ASCA lgG Anti-OmpC Anti-CBir1

Neutrophi-Specific Nuclear Auto Antibodies (NSNA) (IBO Specific pANCA)

Auto Antibody IFA Perinuclear DNAse

Assay Information

AssayASCA lgA

ELISAASCA lgG

ELISAAnti OmpC

lgA ElisaAnti CBir1

ELISAAntibody ELISA

IFA Perinuclear Pattern Sensitivity

Assay Value : 109.4 EU/ml 113.8 EU/ml 26.0 EU/ml 50.2 EU/ml <12.1 EU/ml NOT Detected NOT Detected

Note: Test result determined by the PROMETHEUS Predictive Algorithm without direct consideration of assay values relative to reference values. However, interpretation of prognostic information should be made based on relative differences between assay values and reference values

Reference Values <20.0 EU/ml <40.0 EU/ml <16.5 EU/ml <21.0 EU/ml < 12.1 EU/ml Not Detected Not Detected

Progression of CD and Response to Progression of CD and Response to Microbial Antigens Scottish PopulationMicrobial Antigens Scottish Population

Number of Positive Antibodies* (%)0 1 2 3 P Value OR (3:0)

Disease Progression 24 52 73 87 <0.001 20

Surgery 32 57 52 89 <0.001 17

*Antibodies tested: ASCA, anti-OmpC, anti-I2

Adapted from Arnott ID, et al. Am J Gastroenterol. 2004;99:2376-2384.

Association of ASCA and Early Surgery Association of ASCA and Early Surgery Newly Diagnosed CD PatientsNewly Diagnosed CD Patients

Early Surgery( 35)

No Early Surgery ( 35)

P Value OR(n=35) (n=35)

ASCA IgA 63% 20% .001 8.5

ASCA IgG 40% 14% .027 5.5

ASCA IgA & IgG 37% 14% .043 5.0

Adapted from Forcione DG, et al. Gut. 2004;53:1117-1122.

High ASCA High ASCA IgAIgA & & IgGIgG in CD Associated With in CD Associated With More Aggressive Small Bowel DiseaseMore Aggressive Small Bowel Disease

100B

owel

ge

ry 86%P<0.0001

50

75

With

Sm

all

uirin

g S

urg

57%pANCA+ (≥40) & ASCA-All OthersASCA IgG & IgA + (≥50)

25

Pat

ient

s W

ease

Req

u

29%and ANCA-

0CD Subgroup

% o

f PD

ise

7 99 14

Adapted from Vasiliauskas EA, et al. Gut. 2000;47:487-496.*Other Investigators: Sands, Dassopoulos, RiisSimilar results

n=7 n=99 n=14

Frequency of Complications Increases With Number Frequency of Complications Increases With Number of Serologic Markers Children With CDof Serologic Markers Children With CD

/Stenotic OnlyNonperforating Nonstenotic Perianal Perforating Only Internal Perforating/Stenotic Only

S l i k8090

100

avio

r (%

)

Nonperforating Nonstenotic Perianal Perforating Only Internal Perforating

11.0*

Serologic markers:ASCA Anti-OmpC Anti-CBir1 Anti-I250

607080

isea

se B

eh

1.9* 2.3*5.5*

Anti I2

10203040

uenc

y of

Di

n=40 n=60 n=42 n=29 n=12

010

0 1 2 3 4

No. of Immune Responses

Freq

u

Dubinsky M, et al. Am J Gastroenterol. 2006;101:360-367.

(P trend=0.002)*Odds ratios

No. of Immune Responses

AntiAnti--microbial responsiveness is microbial responsiveness is increased in NOD2 mutant carriersincreased in NOD2 mutant carriers

• Mutations in Nod2 may decrease innate immune clearance of bacteria leading y gto secondary increase in adaptive immune responses

• Analyzed 732 Crohn’s disease patients, 220 healthy relatives, 200 controls• Nod2 mutant carriers have higher titers of anti-microbial antibodies:

CBir1 I2 ASCA OmpC

10 46

11.26

11.0

11.5

CBir1, I2, ASCA, OmpC

p-trend = 0.002

sum

9.72

10.46

9 5

10.0

10.5

n qu

artil

e

8.5

9.0

9.5

WT 1 t 2 t

Mea

n=499 n=194 n=39

WT 1 mut 2 mutNOD2/CARD15 Variant status

One way ANOVADevlin S, et al. DDW 2006, Los Angeles. Abstract #442

Potential Roles for IBD SerologyPotential Roles for IBD Serology

• Help identify IBD in patients with unclear diagnosis (but chronic inflammation already established)Help assess the need for endoscopy in patients who• Help assess the need for endoscopy in patients who are suspected of having IBD (pediatric population)

• Help differentiate between CD and UC• Help differentiate between CD and UC• Help improve the accuracy of diagnosis prior to

surgery (eg, colectomy, IPAA)surgery (eg, colectomy, IPAA)• Help identify patients at risk for aggressive disease

behavior

Indeterminate Colitis:Indeterminate Colitis:Value of SerologyValue of Serology

100 80%

Leuven, Lille, Vienna 97ptDefinite Diagnosis 31pt70

8090

100 64%

Definite Diagnosis 31pt pANCA- / ASCA - 40pt

40506070

UC pANCA+/ASCA

102030

UC pANCA+/ASCA-

CD pANCA-/ASCA+

0

Joossens S. Gastroenterology 2002. 122(5): 1242-47Joossens S. Gastroenterology 2003. 124: A323

IBD IBD SerologiesSerologies: When to order?: When to order?

• IC / Colectomy is planned (UC vs CD,IC / Colectomy is planned (UC vs CD, pouchitis risk)

• Pediatric: low suspicion • Mainly extra-intestinal manifestations (is it IBD?)

– Ankylosing spondylitisA th iti– Arthritis

– Pyoderma gangenosum– UveitisUveitis

• Second opinion / questionable diagnosis• Prognostication: ready for prime time?g y p

– Expensive: will insurance cover?

IBD Serologies: SummaryIBD Serologies: Summary

• Identify IBD in cases of diagnostic uncertainty• Assess the need for invasive endoscopy in• Assess the need for invasive endoscopy in

patients who are suspected of having IBD• Differentiate between CD and UCDifferentiate between CD and UC• Identify patients at risk for aggressive disease

behaviorbehavior• Insurance / cost still an issue