Genetics, Immunology and biomarkers in clinical practice. Peter Laszlo Lakatos 1st Department of Medicine Semmelweis University Budapest Hungary

Transcription

1 Genetics, Immunology and biomarkers in clinical practice Peter Laszlo Lakatos 1st Department of Medicine Semmelweis University Budapest Hungary

2 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?

3 Genetics

4 Do patients want to be tested? Konda V Inflamm Bowel Dis 2006;12: 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)

5 Classification of IBD fiction? Crohn s diease UC-like CD IC IBD Ulcerative colitis

6 IBD a complex phenotype

7 How to suspect genetic component? Reference N twins Concordance for disease Monozygotic Dizygotic Tysk et al % 3.8% Thompson et al % 6.5% Orholm et al % 0%

8 Monogenic versus Polygenic disease Phenotype Type II DM Phenotype Strong relationship No factor necessary/sufficient to cause disease, all increase individual s risk Locus 1 (causal allele or genotype) Locus 2 Locus 3 Locus 3 CAPN10 HLA Susceptibility

9 Effects of human functional genetic variation Susceptibility neutral protective CARD15 OCTN 1 and 2 DLG5 R30Q ATG16L1 T300A IL23R DLG5_e26

10 NOD2/CARD15 (16q12) Caspase Recruitment Domain Family member 15 Protein/protein interactions CARD 1 CARD 2 Protein Oligomerisation NBD Recognition PAMPs LRR Arg702Trp SNP8 Gly908Arg SNP12 Leu1007fsinsC 3020insC SNP13

11 α-defensin lysosim Pattern recognition receptors (PRRs) + NOD2/CARD15 mutations

12 NOD2/CARD15 mutation prevalence Ahmad Cuthbert Abreu Vermeire Esters Buning Inoue Leong Helio Arnott Lakatos % Hugot controls CD

13 CARD15 Mutations and relative risk for CD Heterozygote Homozygote Compound Heterozygote Hugot et al Ogura et al Cuthbert et al Hampe et al Ahmad et al Esters et al Lakatos et al / %

14 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 CD NOD2 HLA Other Ahmad et al, Gastroenterology 2002, 122:

15 NOD2 expression in Paneth cells Lala S et al; Gastroenterology 2003; 125: Ogura et al; Gut 2003; 52:

16 Defensins and NOD2 Wehkamp PNAS 2005 Kobayashi et al Science 2005;307: Wehkamp J Gut 2004; 53:

17 Copy-number variation of Human beta-defensin 80 Ileitis 80 Colitis cases (%) 40 cases (%) <4 4 >4 0 <4 4 >4 80 Gene copy number 80 Gene copy number 60 Ileocolitis 60 UC cases (%) 40 cases (%) <4 4 >4 Gene copy number 0 <4 4 >4 Gene copy number Fellermann K et al; Am J Hum Genet. 2006;79:439-48

18 Toll Like Receptors Mycoplasmal Lipopetide Bacterial lipoprotein Viral ds RNA Bacterial LPS Flagellin Mycoplasmal Lipopetide CpG DNA motifs Chrom 4p14 4q31 4q35 9q33 1q41 4p14 Xp22 Xp22 3p21 4 Franchimont Gut 2004, Torok Clin Immunol 2004, Ouburg Gut 2005, Lakatos World J Gastro 2005, Braat J Mol Med 2005, Pierik M IBD 2006

19 IBD5 and OCTN (5q31-33) Novel Organic Cation Transporter Risk haplotype TC 54% IBD versus 42% controls C1672T and promoter G 207C Rioux JD Nat Genet 2001; 29: Peltekova Nat Genet 2004; 36: 471-5

20 OCTN Novel 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: 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)

21 OCTN TC risk haplotype p= p= , ,5 % patients No TC risk haplotype TC Homozygous 10 0 Perianal disease Fistulising disease TC risk haplotype associated with fistulising disease OR (95% CI ); p=0.035 (Forward Wald log regression SPSS 12.0)

22 OCTN TC risk haplotype? 60 p=0.05 p= , ,4 44 % patients No TC risk haplotype TC Homozygous Or non stricturing/non 0 penetrating- colonic no surgery?? Perianal disease Fistulising disease

23 OCTN TC risk haplotype??

24 DLG5 (10q23) Discs Large Homolog 5 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 DUF622 PDZ PDZ PDZ PDZ SH3 GK N C G113A (R30Q) Haplotype D-tagging SNP DLG5_e26 (One of 8) Haplotype A-tagging SNPs Stoll M et al Nat Genet. 2004; 36:

25 DLG5 R30Q p=0.001 OR=1.6 OR=1.25 Canada/Italy UK % 10 Canada/Italy 0 CD (n=538) Controls (n=548) Estimated OR for R30Q Stoll M et al Nat Genet. 2004; 36: Daly M et al Eur J Hum Genet 2005; 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

26 From linkage analyses using microsatellites to SNP association genome scans

27 IL23R NOD1 (1p31)-(CARD4) the (7p14) SNP story Duerr RH Science 2006

28 IL23R (1p31) Arg381Gln All freq % ,9 CD 7 controls Confirmed in other studies: Cummings Inflamm Bowel Dis rs (OR 0.65, 95%CI: ) Tremelling Gastroenterology 2007

29 ATG16L1 (2q) Autophagy-related 16-like 1 gene Confirmed in other studies: Rioux Nat Genet 2007, Prescott NJ Gastroenterology 2007 Cummings Inflamm Bowel Dis 2007 no association to NOD2/IL23,IBD5 & phenotype OR G allele: , GG gen:

30 Gene chip for the diagnosis of IBD? Genetics in combination with environmental factors Genetic markers studied: TUCAN (CARD8), NOD1, NOD2, IBD5, TNFSF15 McGowern Gastroenterology 2006

31 Predicting respone to medical therapy: A role for pharmacogenetics? TNFa, TNFR, NOD2, or NAT1-2 not associated to response to steroids, azathioprine, mesalasine/sulphasalazine or infliximab MDR1: response to AZA and steroid dependency? IL AA: steroid dependency? DLG5 R30Q: resistance to steroids? FAS 843C/T and caspase9 93C/T: response to IFX? (concomittant AZA) TPMT: azathioprine!

32 So genotype is also complex

33 Genetics start to unravel the complex phenotype of IBD IL23R IBD7 IBD9 OCTN IBD5 IBD3 NOD1 DLG5 IBD2 IBD4 CARD15 IBD1 IBD6

34 Genetics in clinical practice The conclusions with respect to NOD2/CARD15 is until now the most important genetic factor implicated in the disease susceptibility to CD the NOD2/CARD15 experience Confirms the hypothesis that dysregulated response of in the mucosal immune inflammatory system bowel toward microbial antigens may lead to disease the development have of CD led to Clinical utilization: recommendations with respect to Genetic testing of NOD2 may be an additional confirmatory factor in making the diagnosis and predicting disease phenotype clinical to some extent classification (e.g. early onset, ileal involvement, of stenosing disease, increased need for surgery?) Disease

35 Biomarkers: for the diagnosis

36 Serologic markers in IBD Antibody Directed against CD UC ASCA Mannose of Saccharomyces cerevisiae + (40-60%) - panca Neutrophils + + (40-60%) (UC-like CD) PAB Pancreas + (15-30%) + Omp (C) Outer Membrane porin + (20-40%) + I2 Pseudomonas fluorescens + (20-40%) - cbir1 flagellin + (20-50%) - ACCA glycan (Chitobioside) + (20-40%) - ALCA glycan (Laminaribioside) + (20-40%) -

37 A panel of markers may improve sensitivity and approach diagnostic utility Mow S et al, Gastroenterology 2004; Targan SR et al, Gastroenterology 2005 Dotan I et al, Gastroenterology 2006; 131: Reduced specificity Considerably expensive Not widely available

38 Serologic markers in patients with colitis type unclassified (IBDU) 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) P<0.001 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) (Leuven n=30, Lille n=37, Vienna n=30) S Joossens et al, Gastroenterology, 2002, 122:

39 Serologic markers may prioritize diagnostic work up in children ASCA and ANCA ELISA ASCA ELISA - panca 3-steps Dubinsky M et al Am J Gastroenterol 2001

40 4/128 would have received unnecessary investigations Dubinsky M et al Am J Gastroenterol 2001 Delayed diagnosis

41 Novel algorythmic analysis of serology data for predicting IBD 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% IBD UC (n=105) CD (n=188) Sensitivity Specificity PPV NPV Lichtenstein Gastroenterology 2007;132: A175 (S1106)

42 Subclinical disease: is serology preceeding disease? ASCA before onset of Crohn s disease Israeli Defense Corp serum repository Diagnosis 32 persons with CD from whom serum before the diagnosis ASCA in 10/32 (32%) before diagnosis mean interval between ASCA detection and diagnosis 38 M No with ASCA positive (%) Time before/after CD diagnosis (months) Israeli E, et al. Gut 2005

43 Is there a genetic susceptibility for the seroreactivity in IBD? NOD2/CARD15, NOD1, TLR4, ATG16L1, Antimicrobial peptides ASCA, anti-ompc, Anti- I2, cbir1 flagellin

44 Antimicrobial responses as consequence of genetic defects in innate immune system? ASCA, I2, CBir1, OmpC Mean Quartile Sum 12 11, ,5 10 9,5 p= ,7 10,4 11,3 Mean Quartile Sum 12 11, ,5 10 9, Healthy controls p= ,7 9,7 No Variant Any Variant NOD2 Variant Status 9 No Variant 1 Variant 2 Variants NOD2 Variant Status 732 CD Mean Quartile Sum 12 11, ,5 10 9,5 220 Healthy relatives p=0.02 9,7 10,8 Devlin S et al, Gastroenterology No Variant NOD2 Variant Status Any Variant

45 Antimicrobial responses due to genetic defects in innate immune system? gasca ACCA 100% 75% 50% 25% 0% 100% 75% 50% 25% 0% p < % 51% CARD15 0 CARD % n = 238 n = 187 n = 68 34% TLR 4 0 p = % TLR4 1 CARD15 2 9% n=230 n=28 n=1 TLR4 2 ASCA % p< CARD15 Henckaerts L al; submitted Papp M Inflamm Bowel Dis 2007

46 Serum markers of inflammation in IBD n=82 adults with abdominal symptoms St Mark s hospital All received clinical examination, rectal biopsy, ESR, CRP, α1 glycoprotein CD (n=19) UC (n=22) Functional bowel disorder (n=41) increased CRP 100% 50% 0% Shine et al; Clin Chim Acta 1985; 148: 105-9

47 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 Investigation Crohn s disease (n=26) Ulcerative colitis (n=13) Polyps (n=8) Normal (n=37) Others* (n=7) Hb <10 g/dl ESR >25 mm/hour Albumin <36 g/dl Platelet >400 x 10 9 /l CRP >5 mg/l All normal *TBC (2), IC (3), lymphoid nodular hyperplasia (3) Beattie et al; Arch Dis Childhood 1995; 73: 354-5

48 Stool markers: calprotectin Non-invasive 36 kda calcium and zinc binding protein (S100A8/A9) Neutrophil marker: 60% of cytosolic proteins 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) Tibble et al, Gut 2000

49 Serum markers of inflammation in IBD Tibble Gut 2000

50 Serum markers of inflammation in IBD patients -For IBD: cut off 100 ug/g instead of 50 ug/g -Sensitivity 95% -Specificity 91% -ROC AUC 95% Von Roon AC AJG 2007

51 Faecal S100A12 (Calgranulin C, EN-RAGE) Children S100A12 (10mg/kg) Calprotectin (50 mg/kg) Sensitivity (%) Specificity (%) ROC-AUC (%) member of the S100 family of calciumbinding proteins in granulocytes - exhibits proinflammatory functions, including potent chemotactic activity - ligand for the receptor for advanced glycation end products (RAGE) ESR (15mm/h) CRP (3mg/L) De Jong NS et al; Inflamm Bowel Dis 2006;12: Sidler MA, Gastroenterology DDW 2007

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

53 Stool markers in pediatric IBD N=36 children with symptoms and suspected inflammation of the colon Correlation between calprotectin in stools and endoscopic findings Sens 95%, spec 93% PPV 95%, NPV 93% n=14 n=22 IBD in 20/22 children Fagerberg UL et al J Pediatri Gastroenterol Nutr 2003; 37: Fagerberg UL et al J Pediatr Gastroenterol Nutr. 2005; 40:450-5

54 Are biomarkers helpful in predicting disease course and complications?

55 Serological markers predict complicated disease? Fibrostenosis SB surgery Perforating Anti-flagellin SB disease fibrostenosis perforating SB surgery No NOD2 Fibrostenosis perforating NOD2 Mow S et al, Gastroenterology 2004; Targan SR et al, Gastroenterology 2005, Papp, Inflamm Bowel Dis 2007

56 Ferrante M et al; Gut apr epub Papp M Inflamm Bowel Dis 2007 Dubinsky Am J GE 2006 Serological markers predict complicated disease? Adults: gasca, AMCA, ALCA, ACCA, Omp % with complicated disease 100% 75% 50% 25% 0% 42,0 Group A (n=181) Score <1.5 OR: 1.76 p: ,0 Group B (n=200) Score 1.5 or 2.0 OR: 2.00 p: ,7 Group C (n=184) Score 2.5 or 3.0 OR: 1.96 p: ,2 Group D (n=173) Score >3.0 Children: ASCA, Omp, I2, CBir1 % Ileal non-inflamm ASCA, Omp OR: For 0 vs Serology marker positivity

57 Serological markers predict aggressive disease? ASCA, OmpC, cbir1, I2 Dubinsky M et al; Am J Gastroenterol 2006; 101:

58 The clinical utility of serological markers? 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 Satsangi J Gut 2006;55:

59 Assessment of disease activity?

60 Clinical disease and the CRP response Major CRP response Infections Hypersensitivity complication of infection Bacterial, Fungal, Mycobacterial, Viral (severe or systemic) Rheumatic fever, Erythema nodosum Inflammatory disease Metabolic diseases Tissue necrosis Trauma Neoplasia Modest or absent CRP response Rheumatoid arthritis, Ankylosing spondylitis, psoriatic arthritis, systemic vasculitis, PMR, Crohn s disease Obesity, atherosclerosis Myocardial infection, Tumour embolization, Acute pancreatitis Surgery, burns, fractures Lymphoma, carcinoma, sarcoma SLE, Scleroderma, Dermatomyositis, Ulcerative colitis, Leukemia, Graftvs.-host disease

61 CRP in IBD prospective Median CRP mg/l Mild Moderate Severe CD (n=64) UC (n=50) Fagan EA et al. Eur J Clin Invest 1982; 12:

62 Correlation of CRP with clinical, radiographic and endoscopic activity in IBD Logistic regression Increased CRP associated with: OR (95% CI) CD (n=104) UC (n=43) Clinical activity Endoscopic activity Severe inflammation on Biopsy Abnormal Radiographic findings 4.5 (1.1-18) 4.1 (1.6-11) 10 (1.0-97) NS NS NS p=0.029 NS CRP normal CRP elevated Ileocolonoscopy shows active CD (n=72) 46% 54% Ileocolonoscopy does not show active CD (n=32) 75% 25% Solem CA et al; Inflamm Bowel Dis 2005; 11:8:

63 CRP or ESR? 160 CRP 100 ESR Mild Moderate Severe 0 Mild Moderate Severe Fagan EA et al. Eur J Clin Invest 1982; 12:

64 CRP or ESR? 71 CD patients in medical remission CRP>20mg/L and ESR >15 mm/h were selected as markers predictive of relapse A binary biological predictive score was derived: "negative" when both were lower than their limits, "positive" when otherwise Sensitivity 89% specificity was 43% RR of relapse (95%CI) CRP >20 mg/l only ESR >15 mm only Both 10.5 ( ) 6.1 ( ) 9.9 ( ) Consigny et al Inflamm Bowel Dis 2006;12:551

65 Faecal calprotectin Roseth A et al Digestion 1997; 58: Roseth A et al Scand J Gastroenterol 2004; 39:

66 Predicting relapse?

67 Faecal calprotectin and risk for relapse 43CD 25 (58%) relapse over period of 12 months 37UC 19 (51%) relapsed over period of 12 months In remission for 1-4 months Proportion of patients without a relapse UC Calprotectin <50mg/L CD Calprotectin <50 mg/l UC Calprotectin >50 mg/l CD Calprotectin >50 mg/l RR 10.6 (CD) RR 13.4 (UC) Time (Months) Tibble Gastroenterology 2000

68 Costa F Gut 2005 Faecal calprotectin and risk for relapse 38CD 41UC 15 (39%) relapse over period of 12 months 19 (46%) relapsed over period of 12 months

69 Risk of relapse after withdrawal of azathioprine 84 CD in remission >42 months with AZA AZA n=40 Placebo n=43 7.9% % of clinical relapse at 18 months? 21.3% Lemann M et al Gastroenterology 2005; 128:

70 Calprotectin predicts endoscopic postsurgical recurrence in asymptomatic CD 39/50 patients evaluated at 1 yr post-op by colonoscopy 19 pts had endoscopic recurrence Calprotectin Cut-off >50 Sensitivity (%) 94 Specificity (%) 25 > Calprotectin>200mg/L improved sensitivity when compared to US > Detection of calprotectin >200mg/L at third month after surgery represents an indication to endoscopic examination >200 > Orlando Eur Rev Med Pharmacol 2006;10:17

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