Genetics, Immunology andbiomarkers in clinical practice

Peter Laszlo Lakatos1st Department of Medicine

Semmelweis University BudapestHungary

Why and where during the course of IBD are markers needed?

•Diagnosis and differential diagnosis?

•Prognosis and risk for complications?

•Assessement of disease activity?

•Optimazing drug therapy and side effects?

•Risk for post-operative recurrence?

Genetics

114 IBD patients (82, 71.9% with CD)-76.8% (86 of 112) would undergo testing for diagnostic confirmation-81.3% (91 of 112) for prognostic value-88.4% (99 of 112) for therapeutic decision making-85.0% (96 of 113) for advancement of medical knowledge- patients with a first-degree relative with IBD (raw score on self-willingness 4.62 vs 4.36; P = 0.026)

Konda V Inflamm Bowel Dis 2006;12:497-502.

Do patients want to be tested?

Classification of IBD…fiction?

IBD

Crohn’s

diease

Ulcerativecolitis

UC-like CD

IC

IBD… a complex phenotype

DizygoticMonozygotic

3.8%44.4%80Tysk et al

0%50%102Orholm et al

6.5%20%143Thompson et al

Concordance for diseaseN twinsReference

How to suspect genetic component?

Locus1(causal allele or genotype)

Monogenic versus Polygenic disease

Phenotype Phenotype

Locus2 Locus3 Locus3

Type II DM

CAPN10 HLA Susceptibility

Strong relationshipNo factor necessary/sufficient

to cause disease, allincrease individual’s risk

Effects of human functional genetic variation

Susceptibility… …neutral… …protective

IL23R

DLG5_e26

CARD15

OCTN 1 and 2

DLG5 R30Q

ATG16L1 T300A

NOD2/CARD15 (16q12)Caspase Recruitment Domain Family member 15

Arg702Trp

SNP8Gly908Arg

SNP12

Leu1007fsinsC

3020insC

SNP13

CARD 2

220127

NBD

273 577

LRR

1020744 1040

CARD 1

1 12428

Protein/proteininteractions

ProteinOligomerisation

Recognition PAMPs

NOD2/CARD15 NOD2/CARD15 mutationsmutations

+

αα--defensindefensinlysosimlysosim

Pattern Pattern recognition recognition receptorreceptorss((PRRsPRRs))

NOD2/CARD15 mutation prevalence

0

20

40

60

80

100

Hugot

Ahmad

Cuthbe

rtAbre

uVerm

eire

Esters

Buning

Inoue

Leon

gHeli

oArno

ttLa

katos

%

controls CD

CARD15 Mutations and relative risk for CD

29.3

-

12.9

9.8

13.6

14.4

2.4

2.8

1.7

Ahmad et al.

Esters et al.

Lakatos et al.

24.822.23.0Hampe et al.

-42.12.6Cuthbert et al.

-17.61.5Ogura et al.

44.038.03.0Hugot et al.

Compound Heterozygote

HomozygoteHeterozygote

2-6/1.000 2-8%

Ahmad et al, Gastroenterology 2002, 122: 867-874

NOD2/CARD15 and disease phenotype

PAR% of population attributable risk

Increase (proportional) in disease in individuals with mutation in comparison with those without mutation

Ileal CD Colonic CDNOD2HLAOther

Lala S et al; Gastroenterology 2003; 125: 47-57

Ogura et al; Gut 2003; 52: 1591-1597

NOD2 expression in Paneth cells

Defensins and NOD2

Wehkamp J Gut 2004; 53: 1658-64Kobayashi et al Science 2005;307:731-734

Wehkamp PNAS 2005

Fellermann K et al; Am J Hum Genet. 2006;79:439-48

0

20

40

60

80

<4 4 >4

Gene copy number

case

s (%

)

0

20

40

60

80

<4 4 >4

Gene copy number

case

s (%

)

0

20

40

60

80

<4 4 >4

Gene copy number

case

s (%

)

0

20

40

60

80

<4 4 >4

Gene copy number

case

s (%

)

Ileitis Colitis

UCIleocolitis

Copy-number variation of Human beta-defensin

1 2 3 4 5 6 7 8 9 10Chrom 4p14 4q31 4q35 9q33 1q41 4p14 Xp22 Xp22 3p21 4

Bacteriallipoprotein

MycoplasmalLipopetide

Viral dsRNA CpG DNA

motifsFlagellin

BacterialLPS

MycoplasmalLipopetide

Toll Like Receptors

Franchimont Gut 2004, Torok Clin Immunol 2004, Ouburg Gut 2005, Lakatos World J Gastro 2005, Braat J Mol Med 2005, Pierik M IBD 2006

IBD5 and OCTN (5q31-33)Novel Organic Cation Transporter

Rioux JD Nat Genet 2001; 29:223-28Peltekova Nat Genet 2004; 36: 471-5

C1672T and promoter G–207C

Risk haplotype TC

54% IBD versus 42% controls

OCTNNovel Organic Cation Transporter

• Membrane transporter for drugs and organic cations

• OCTN may also transport carnitine:– essential for metabolism of lipid– Role in transport of short chain fatty acids to mitochondria

for β-oxidation

Peltekova Nat Genet. 2004; 36: 471-5

• Rectal instillation of sodium 2-bromo-octanoate (inhibitor β-oxidation) in rats:• Weight loss and bloody diarrhea• ulcers, mucus cell depletion, vasodilation, increase of acute inflammatory cells

(Roediger et al Br J Exp 1986)

• Association of OCTN1 and RA (Tokuhiro et al Nat Genet 2003)

p=0.05

p=0.034

35

43,4 44

53,5

0

10

20

30

40

50

60

% patients

Perianal disease Fistulising disease

No TC risk haplotypeTC Homozygous

TC risk haplotype associated with fistulising disease OR 1.474 (95% CI 1.028-2.114); p=0.035

(Forward Wald log regression SPSS 12.0)

OCTN TC risk haplotype

p=0.05

p=0.034

35

43,4 44

53,5

0

10

20

30

40

50

60

% patients

Perianal disease Fistulising disease

No TC risk haplotypeTC Homozygous

OCTN TC risk haplotype?

Or non stricturing/non penetrating- colonic –no surgery??

OCTN TC risk haplotype??

DLG5 (10q23)Discs Large Homolog 5

DUF622 PDZ PDZ PDZ PDZ SH3 GK

N C

member of MAGUK (Membrane Associated Guanylate Kinase) family of scaffolding proteins

• involved in intracellular signal transduction • important in maintaining epithelial structure and integrity of barrier

G113A (R30Q)Haplotype D-tagging SNP

DLG5_e26(One of 8) Haplotype A-tagging SNPs

Stoll M et al Nat Genet. 2004; 36: 476-80

Stoll M et al Nat Genet. 2004; 36: 476-80

DLG5 R30Q

25

17

0

10

20

30

%

CD (n=538) Controls (n=548)

p=0.001

OR=1.6

Daly M et al Eur J Hum Genet 2005;

0.5 1 1.5 2 2.5

Canada/Italy

UK

Canada/Italy

Estimated OR for R30Q

OR=1.25

Not confirmed in other studies:

Lakatos PL IBD 2006, Noble CL Gut 2005, Torok HP Gut 2005, Vermeire S Gastroenterol 2005, Waller S DDW 2005

From linkage analyses usingmicrosatellites…

…to SNP association genome scans

NOD1 (CARD4) (7p14)IL23R (1p31)- the 300.000 SNP story

Duerr RH Science 2006

IL23R (1p31)

Arg381Gln

1,9

7

012345678

CD controls

All

freq

%

Confirmed in other studies:

Cummings Inflamm Bowel Dis 2007 + rs7517847 (OR 0.65, 95%CI: 0.56-0.75)

Tremelling Gastroenterology 2007

ATG16L1 (2q)Autophagy-related 16-like 1 gene

OR G allele: 1.35-1.45, GG gen: 1.71-1.77

Confirmed in other studies:

Rioux Nat Genet 2007, Prescott NJ Gastroenterology 2007

Cummings Inflamm Bowel Dis 2007 no association to NOD2/IL23,IBD5 & phenotype

Genetic markers studied: TUCAN (CARD8), NOD1, NOD2, IBD5, TNFSF15

Gene chip for the diagnosis of IBD? Genetics in combination with environmental factors

McGowern Gastroenterology 2006

Predicting respone to medical therapy: A role for pharmacogenetics?

• TNFa, TNFR, NOD2, or NAT1-2not associated to response to steroids, azathioprine, mesalasine/sulphasalazineor infliximab

• MDR1:response to AZA and steroid dependency?

• IL10 1082AA: steroid dependency?

• DLG5 R30Q: resistance to steroids?

• FAS 843C/T and caspase9 93C/T: response to IFX? (concomittant AZA)

• TPMT: azathioprine!

So…genotype is also complex

Genetics start to unravel the complex phenotype of IBD

IBD9IBD5

IBD4

IBD3

IBD2

IBD1

IBD7

IBD6

DLG5

CARD15

OCTN

NOD1

IL23R

Genetics in clinical practice

NOD2/CARD15 is until now the most important NOD2/CARD15 is until now the most important genetic factor implicated in the disease susceptibility togenetic factor implicated in the disease susceptibility toCDCDConfConfiirmsrms the hypothesis that the hypothesis that dysregulateddysregulated response of response of the mucosal immune system toward microbial antigens the mucosal immune system toward microbial antigens may lead to the development of may lead to the development of CDCDClinical utilization:Clinical utilization:

Genetic testing of NOD2 may be an additional confirmatory Genetic testing of NOD2 may be an additional confirmatory factor in making the diagnosis and predicting disease factor in making the diagnosis and predicting disease phenotypephenotype to some extent (e.g. early onset , ileal involvement, to some extent (e.g. early onset , ileal involvement, stenostenossinging disease, increased need for surgery?)disease, increased need for surgery?)

‘‘‘‘The conclusions with respect to The conclusions with respect to the the NOD2/CARD15NOD2/CARD15

experience in inflammatory bowel experience in inflammatory bowel disease have led todisease have led to

recommendations with respect to recommendations with respect to clinical classificationclinical classification ofof

DiseaseDisease””

Biomarkers: for the diagnosis

Serologic markers in IBD

++ (15-30%)PancreasPAB

-+ (20-40%)glycan (Chitobioside)ACCA

-+ (20-50%)flagellincBir1

glycan (Laminaribioside)

Pseudomonas fluorescens

Outer Membrane porin

Neutrophils

Mannose of Saccharomycescerevisiae

Directed against

+ (40-60%)+ (UC-like CD)

pANCA

-+ (40-60%)ASCA

-+ (20-40%)I2

++ (20-40%)Omp (C)

-+ (20-40%)ALCA

UCCDAntibody

Mow S et al, Gastroenterology 2004; Targan SR et al, Gastroenterology 2005

A panel of markers may improve sensitivityand approach diagnostic utility

Dotan I et al, Gastroenterology2006; 131: 366-78

↔ Reduced specificity

↔ Considerably expensive

↔ Not widely available

S Joossens et al, Gastroenterology, 2002, 122: 1242-1247

n CD UC IC

ASCA+/ pANCA- 26 (26.8) 8 (30.8) 2 (7.7) 16 (61.5)

ASCA-/pANCA+ 20 (20.6) 4 (20) 7 (35) 9 (45)

ASCA+/ pANCA+ 4 (4.1) 2 (50) 1 (25) 1 (25)

ASCA-/pANCA- 47 (48.5) 3 (6.4) 4 (8.4) 40 (85.1)

Total 97 (100) 17 (17.5) 14 (14.4) 66 (68.1)

P<0.001

(Leuven n=30, Lille n=37, Vienna n=30)

Serologic markers in patients withcolitis type unclassified (IBDU)

Dubinsky M et al Am J Gastroenterol 2001

ASCA and ANCA ELISA

ASCA ELISA - pANCA 3-steps

Serologic markers may prioritize diagnostic work up in children

Dubinsky M et al Am J Gastroenterol 2001

↓

4/128 would have receivedunnecessary investigations

↑

Delayeddiagnosis

Novel algorythmic analysis of serology data for predicting IBD

Lichtenstein Gastroenterology 2007;132: A175 (S1106)

•Sophisticated computer-aided system developed from a sequential analysis of serologic assay results using two stage statistical classifiers including a neural network

•Prevalence of IBD in validation cohort (n=500): 59%•Overall accuracy of algorithm: 92%

939890NPV

988996PPV

989795Specificity

889393Sensitivity

CD (n=188)UC (n=105)IBD

Israeli Defense Corp serumrepository

32 persons with CD from whomserum before the diagnosis

ASCA in 10/32 (32%) before diagnosis

mean interval between ASCA detection and diagnosis 38 M

Israeli E, et al. Gut 2005

‘Subclinical’ disease: is serology preceeding disease?ASCA before onset of Crohn’s disease

Time before/after CD diagnosis (months)

0

70

80

90

100

-60 -48 -36 -24 -12 6 18

No

with

ASC

A p

ositi

ve (%

)

40

50

60

10

20

30

-54 -42 -30 -18 -6 0 12

Diagnosis

Is there a genetic susceptibility for the seroreactivity in IBD?

Antimicrobial peptidesASCA, anti-OmpC, Anti-

I2, cBir1 flagellin

NOD2/CARD15, NOD1, TLR4, ATG16L1, …

ASCA, I2, CBir1, OmpC

Devlin S et al, Gastroenterology 2007

9,7

10,4

11,3

9

9,5

10

10,5

11

11,5

12

No Variant 1 Variant 2 Variants

NOD2 Variant Status

Mea

n Q

uart

ile S

um

732 CD

p=0.002

Antimicrobial responses as consequence of geneticdefects in innate immune system?

9,7

10,8

9

9,5

10

10,5

11

11,5

12

No Variant Any Variant

NOD2 Variant Status

Mea

n Q

uart

ile S

um 220 Healthy relatives

p=0.02

9,7

10,7

9

9,5

10

10,5

11

11,5

12

No Variant Any Variant

NOD2 Variant Status

Mea

n Q

uart

ile S

um 200 Healthy controls

p=0.07

Antimicrobial responses due to genetic defects in innate immune system?

Henckaerts L al; submitted

51%

0%

25%

50%

75%

100%

CARD15 0

64%

CARD15 1

72%

CARD15 2

n = 238 n = 187 n = 68

p < 0.0001

0%

25%

50%

75%

100%

34%

TLR 4 0

25%

TLR4 1

9%

TLR4 2

n=230 n=28 n=1

p = 0.03

gASCA

ACCA

0102030405060708090

100

0 1 2

Papp M Inflamm Bowel Dis 2007

% p<0.0001

ASCA

CARD15

• n=82 adults with abdominal symptoms – St Mark’s hospital• All received clinical examination, rectal biopsy, ESR, CRP,

α1 glycoprotein

Shine et al; Clin Chim Acta 1985; 148: 105-9

0%50%100%increased CRP

Functional boweldisorder (n=41)

UC (n=22)

CD (n=19)

Serum markers of inflammation in IBD

Serum markers of inflammation in IBD• n=91 children - mean 11yr (3M-18yr)• Referred for endoscopy with symptoms of abdominal pain, diarrhea,

rectal bleeding, weight loss or mouth ulceration for minimum of 3 M• All underwent endoscopy, blood tests and SBFT

Beattie et al; Arch Dis Childhood 1995; 73: 354-5

*TBC (2), IC (3), lymphoid nodular hyperplasia (3)

284214424228

6006091

120012088

30231570608

168535881000

Hb <10 g/dLESR >25 mm/hourAlbumin <36 g/dLPlatelet >400 x 109/lCRP >5 mg/LAll normal

Others* (n=7)

Normal (n=37)

Polyps(n=8)

Ulcerativecolitis(n=13)

Crohn’s disease(n=26)

Investigation

Tibble et al, Gut 2000

Stool markers: calprotectin• Non-invasive 36 kDa calcium and zinc

binding protein (S100A8/A9)

• Neutrophil marker: 60% of cytosolicproteins in granulocytes

• Presence in faeces proportional to neutrophil infiltration in mucosa and shedding in the gut lumen

• Antibacterial, antifungal activity

• Resistant to degradation: stable for >1 week at room temp

• Non-specific (neoplasia, infections, polyps)

Serum markers of inflammation in IBD

Tibble Gut 2000

Serum markers of inflammation in IBD

Von Roon AC AJG 2007

-5983 patients-For IBD: cut off 100 ug/g instead of 50 ug/g

-Sensitivity 95%-Specificity 91%-ROC AUC 95%

Faecal S100A12(Calgranulin C, EN-RAGE)

De Jong NS et al; Inflamm Bowel Dis 2006;12:566-72Sidler MA, Gastroenterology DDW 2007897981CRP (3mg/L)

919374ESR (15mm/h)

9567100Calprotectin(50 mg/kg)

999797S100A12 (10mg/kg)

ROC-AUC (%)

Specificity (%)Sensitivity (%)Children- member of the S100 family of calcium-binding proteins in granulocytes- exhibits proinflammatory functions, including potent chemotactic activity- ligand for the receptor for advanced glycation end products (RAGE)

Faecal lactoferrin

Walker TR et al; J Pediatr Gastroenterol Nutr. 2007; 44:414-22

• 76 kDa iron binding glycoprotein • Major component of secondary granules of PMN neutrophils• Released during inflammatory process – role in innate immunity

IBD vs healthy controls

Fagerberg UL et al J Pediatri Gastroenterol Nutr 2003; 37: 468-72

Fagerberg UL et al J Pediatr Gastroenterol Nutr. 2005; 40:450-5

Stool markers in pediatric IBD• N=36 children with symptoms and suspected inflammation of the colon

• Correlation between calprotectin in stools and endoscopic findings

n=14 n=22IBD in 20/22 children

Sens 95%, spec 93%

PPV 95%, NPV 93%

Are biomarkers helpful in predicting disease course andcomplications?

FibrostenosisSB surgery Perforating

SB diseasefibrostenosisperforatingSB surgeryNo NOD2

Mow S et al, Gastroenterology 2004; Targan SR et al, Gastroenterology 2005, Papp, Inflamm Bowel Dis 2007

Anti-flagellin

Fibrostenosisperforating

NOD2

Serological markers predict complicated disease?

83,2

71,7

56,0

42,0

0%

25%

50%

75%

100%

Group A (n=181)

Group B (n=200)

Group C (n=184)

Group D(n=173)

% w

ith

com

plic

ated

dis

ease

Score

< 1.5

Score

1.5 or 2.0

Score

2.5 or 3.0

Score

> 3.0

OR: 1.76p: .006

OR: 2.00p: .001

OR: 1.96p: .010

Serological markers predict complicated disease?

Ferrante M et al; Gut apr 24 2007 epubPapp M Inflamm Bowel Dis 2007Dubinsky Am J GE 2006

Adults: gASCA, AMCA, ALCA, ACCA, Omp

65 48 79 57 78 800

102030405060708090

100

0 1 2

Ilealnon-inflamm

%

Serology marker positivity

ASCA, Omp

OR: 1.92-4.28For 0 vs 2

Children: ASCA, Omp, I2, CBir1

Serological markers predict aggressive disease?

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

ASCA, OmpC, cBir1, I2

SatsangiSatsangi J J GGutut 2006;55:7492006;55:749--53.53.

‘‘the assessment of the role of serological markers at the present time, the Working Party determined that the use of these markers for diagnosis is not currently justified, given the limited sensitivity of available markers’’

The clinical utility of serological markers?

Assessment of disease activity?

Clinical disease and the CRP response

SLE, Scleroderma, Dermatomyositis, Ulcerative colitis, Leukemia, Graft-vs.-host disease

Modest or absent CRP response

Bacterial, Fungal, Mycobacterial, Viral (severe or systemic)Rheumatic fever, Erythema nodosum

Rheumatoid arthritis, Ankylosingspondylitis, psoriatic arthritis, systemic vasculitis, PMR, Crohn’s diseaseObesity, atherosclerosis

Myocardial infection, Tumourembolization, Acute pancreatitisSurgery, burns, fracturesLymphoma, carcinoma, sarcoma

Major CRP responseInfections

Hypersensitivity complication of infection

Inflammatory disease

Metabolic diseases

Tissue necrosis

TraumaNeoplasia

CRP in IBD

0102030405060708090

Mild Moderate Severe

Median CRP mg/L

CD (n=64)UC (n=50)

Fagan EA et al. Eur J Clin Invest 1982; 12: 351-359

prospective

Correlation of CRP with clinical, radiographic and endoscopic activity in IBD

UC (n=43)

OR (95% CI)Logistic regressionIncreased CRP associated with:

NSNS

p=0.029NS

4.5 (1.1-18)4.1 (1.6-11)10 (1.0-97)

NS

Clinical activityEndoscopic activitySevere inflammation on BiopsyAbnormal Radiographic findings

CD (n=104)

Solem CA et al; Inflamm Bowel Dis 2005; 11:8:707-712

25%54%CRP elevated

75%46%CRP normal

Ileocolonoscopy does not show active CD (n=32)

Ileocolonoscopy shows active CD (n=72)

CRP or ESR ?

Fagan EA et al. Eur J Clin Invest 1982; 12: 351-359

Mild Moderate0

20

40

60

80

Severe

100

120

140

160 CRP

SevereMild Moderate

0

20

40

60

80

100 ESR

CRP or ESR ?

Consigny et al Inflamm Bowel Dis 2006;12:551

10.5 (2.3-48.1)CRP >20 mg/L only

9.9 (3.3-29.7)Both6.1 (1.9-18.9)ESR >15 mm only

RR of relapse (95%CI)

•71 CD patients in medical remission

•CRP>20mg/L and ESR >15 mm/h were selected as markers predictiveof relapse

•A binary biological predictive score was derived: "negative" when both were lower thantheir limits, "positive" when otherwise

•Sensitivity 89% •specificity was 43%

Faecal calprotectin

Roseth A et al Digestion 1997; 58: 176-80Roseth A et al Scand J Gastroenterol 2004; 39: 1017-1020

Predicting relapse?

Faecal calprotectin and risk for relapse

Tibble Gastroenterology 2000

0

RR 10.6 (CD)

RR 13.4 (UC)

• 43 CD

• 37 UC

• In remission for 1-4 months

25 (58%) relapse over period of 12 months

19 (51%) relapsed over period of 12 months

Proportion of patients without

a relapse

Time (Months)

UC Calprotectin <50mg/L

CD Calprotectin <50 mg/L

UC Calprotectin >50 mg/L

CD Calprotectin >50 mg/L

3 6 9 120

0.5

1

0.25

0.75

Faecal calprotectin and risk for relapse

Costa F Gut 2005

• 38 CD

• 41 UC

15 (39%) relapse over period of 12 months

19 (46%) relapsed over period of 12 months

Risk of relapse after withdrawal of azathioprine

Lemann M et al Gastroenterology 2005; 128: 1812-1818

84 CD in remission>42 months with AZA

AZA n=40

Placebo n=43

% of clinical relapse at 18 months?

21.3%

7.9%

Calprotectin predicts endoscopic post-surgical recurrence in asymptomatic CD

Orlando Eur Rev Med Pharmacol 2006;10:17

•39/50 patients evaluated at 1 yrpost-op by colonoscopy

•19 pts had endoscopic recurrence

•Calprotectin>200mg/L improvedsensitivity when compared to US

•Detection of calprotectin>200mg/L at third month aftersurgery represents an indicationto endoscopic examination

7563>200

8547>250

5578>150

4578>100

2594>50

Specificity (%)Sensitivity (%)CalprotectinCut-off

Conclusions

• Genetic markers:– provide evidence that altered NOD2/CARD15 (or TLR4)-mediated bacterial

sensing of normal commensal flora and mucosal permeability changes may be key mechanisms in the pathogenesis of IBD

– low sensitivity limited diagnostic value (targeted chip?)– Associated with disease phenotype (e.g. CARD15 and ileal/stenosing,

OCTN and perianal, HD and colon)

• Serological markers:– loss of tolerance to bacterial antigens in IBD – Low sensitivity: little value in diagnosis (panel-algorithm of markers?) – Associated with complicated (ileal) disease behaviour

• Other laboratory and stool markers:– Useful tools and should be part of global management of IBD– CRP: Good correlation for CD with disease activity-predicting response-

relapse, correlation less for UC except for severe extensive colitis – Stoolmarkers: more specific to detect gut inflammation in the colon