Advances in the etiology of chronic pancreatitis European Postgraduate School in Gastroenterology Prague, April 2010 550 Jahre Universität Greifswald Medizinische Klinik der königlichen Universität Greifswald 1456 1859 Markus M. Lerch Department of Medicine A Ernst-Moritz-Arndt Universität Greifswald

Chronic Pancreatitis - Etiology Reasons to discriminate between different etiologies: Specific treatment options Inherent co-morbidities Different cancer surveillance strategies Alcohol Idiopathic Metabolic Anatomical Hereditary Autoimmune

Layer et al, Gastroenterology 1994;107:1481- 87 Chronic Pancreatitis – Alcohol Cessation Non-Alcoholic CP Incidence of calcifications Propabililty in % Alcoholic-CP Time after onset of symptoms Layer et al, Gastroenterology 1994;107:1481- 87

Chronic Pancreatitis - Alcohol Cessation Strum et al, 1971 Stop Alcohol Gastard et al, 1973 Leger et al, 1974 Prinz et al, 1974 Bornman et al, 1980 Marks et al, 1980 Miyaka et al, 1987 Hayakawa et al, 1989 Strum et al, 1995 20 40 60 80 100 Patients with pain [%]

Chronic Pancreatitis – Cessation of Smoking Maisoneuve P, Gut 2005; 54: 510-514 100 80 60 40 20 Log rank p<0.0001 Smokers OR 2.0 [CI 1.1-3.8] Cumulative Incidence of calcifications, % Non-Smokers 0 2 4 6 8 10 12 14 16 years after diagnosis of chronic alcoholic pancreatitis

Hereditary Pancreatitis Hereditary pancreatitis is clinically indistinguishable from other forms and varities of pancreatitis. 14 year old girl with chronic pancreatitis and R122H-mutation 48 year old women with chronic pancreatitis and R122H-mutation

Hereditary pancreatitis in Germany 1952, first description of hereditary pancreatitis (autosomal dominant trait). 1996 Discovery of the first mutation associated with hereditary pancreatitis in the cationic trypsinogen gene (PRSS1) by Whitcomb et al. Greifswald Münster Weiss F.U. Am J Gastroenterol. 2008;103:2585-8. Haplotype-Analysis of 10 unrelated families with a R122H mutation from an area of 100 km resulted in 7 different haplotypes. This precludes a Founder-Effect Block A (17 kb) Block B (62 kb) Chr. 7: 142,092-142,183 kb PRSS1 PRSS2

Cationic trypsin D22G K23R R116C K92N E79K G83E R122H/C V123M P36R N29I/T D100H L104P C139F Activation site Cationic trypsin

Clinical course of Hereditary Pancreatitis Cumulative incidence of diabetes, % age, years 10 20 30 40 50 60 70 80 90 100 male female 100 90 80 70 60 Alcoholic CP Cumulative age at onset, % 50 40 30 20 10 10 20 30 40 50 60 age, years R122H negativ N29I A16V Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2: 252-261

cumulative pancreatic Pancreatic Cancer in Hereditary Pancreatitis 50-70% increased risk for pancreatic cancer in patients with hereditary pancreatitis. 40% cumulative risk until 70. years of age. Elimination/Treatment of causal factors: Smoking, Alcohol Hyperlipidemia, Hypercalcemia, Gallstones, Duct stricture, Drugs and Medications cumulative pancreatic cancer risk, % age at diagnosis of pancreatic cancer 80 70 60 50 40 30 20 Non-smokers smokers p < 0.01 60 50 40 30 20 10 20 40 60 age, years Howes N, Lerch MM et al Clinical Gastroenterology and Hepatology 2004; 2: 252-261; Lowenfels AB, JAMA 2001; 286: 169-170.

Indications for Genetic Hereditary Pancreatitis testing  Recurrent (2 or more) episodes of acute Pancreatitis without identifyable cause or etiology or  Idiopathic chronic Pancreatitis - especially in children and young adults under the age of 25 years  Pancreatitis in a patients with a positive family history of Pancreatitis (one or more first or second degree relatives) I. Ellis, M.M. Lerch, D.C. Whitcomb: Pancreatology 2001:1:405-415

Pathophysiological role of two mutant trypsinogens: Decreased autoactivation PRSS1 PRSS2 100 80 Wild-Type 80 60 Wild-Type 60 Trypsin activity (%) Trypsin activity (%) 40 40 20 20 R122C G191R 0.5 1 1.5 2 5 10 15 20 25 30 Time (h) Time (min) pH 8.0, 5 mM Ca2+ Simon et al. J. Biol. Chem. 2002; 277: 5404-5410 Witt et al Nature Genetics 2006; 38: 668-73.

insertion of a new cleavage wt R122C reducing non-reducing 60 42 30 22 17 kDa Simon P. et al. J. Biol. Chem. 2002; 277: 5404-5410 SH I CH2 C H =COO- H3N+- PRSS1 Trypsinogen Wild type G191R 0 2 5 10 0 2 5 10 min Trypsin insertion of a new cleavage site EGGKD / ERGKD PRSS2 Witt et al Nature Genetics 2006; 38: 668-73. Cystein

The phenotype of two novel trypsinogen mutations II:1 II:4 II:2 62y II:3 54y III:6 12y III:5 17y III:4 24y III:3 25y III:2 30y III:1 31y II:5 52y II:6 II:7 45y I:1 87y 69y diab. Symbole o. A. Pancreatitis test for R122C carrier PRSS1 Simon P. et al. J. Biol. Chem. 2002; 277: 5404-5410 Screening of Exon 4 for G191R mutations: ICP/HP 17/1414 (1.2%) ACP 4/405 (1.0%) CONTROL 157/4581 (3.5%) PRSS2 Witt et al Nature Genetics 2006; 38: 668-73.

R116C Trypsin – Retention and ER Stress II III IV 63y 62y 48y 44y 40y 38y 36y 17y 15y 100 R116C Wild type 80 60 Trypsin Activity (%) 40 35 25 Homogenate Pellet Supernatant kDa wt R116C Expression in 293T cells 20 pH 8.0, 5 mM Ca2+ 0.5 1 1.5 2 time (h) 100 70 BiP (78 kDa) wt R116A R116C Kereszturi et al. Hum Mutation 2009; 30: 575-82

Cathepsin B-ind. activation PRSS-Mutations: „gain or loss of function“ ? Autoactivation Autolysis Cathepsin B-ind. activation A16V ? D19A ▲ D22G K23R N29I ◄► E79K ▼ R122C R122H XXX R116C G191R ▼ intracellular processing ▼ intracellular processing

Sporadic point mutations in the PRSS1- Gen in idiopathic chronic Pancreatitis In 5 of 50 Patients with idiopathic Pancreatitis (10%) mutations in the cationic Trypsinogen gene were found. 10% 65% 90% Affected Patients represented 35% of all patients under 25 years. Simon P, Weiss F.U. et al JAMA. 2002;288:2122 35%

Sporadic point mutations in the PRSS1- The French Cohort Rebours V Am J Gastroenterol 2008; 103: 111-119, Rebours AJG 2009; 104: 2312 Masson E et al Clin Gastroenterol Hepatol 2008; 6: 82-88 In 78 families with hereditary pancreatitis and 200 individuals (6673 patients years) PRSS1 mutations were detected in 68%. R122H: 78%, N29I: 12% and others 10%. Cumulative risk of pancreatic cancer was 11% at the age of 50 and 49% at the age of 75. Smoking and diabetes mellitus are the main risk factors. Trypsinogen copy number variations are present in 6% of idiopathic chronic pancreatitis cases but are unrelated to familial chronic or tropical calcifying pancreatitis.

Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis Rosendahl et al Nature Genetics 2008; 40: 78-82 Chymotrypsin C is a trypsin-degrading enzyme. Alterations in the CTRC Gene at position p.R254W and p.K247_R254del are present in 30 out of 901 (3.3%) pancreatitis individuals but only in 21 of 2804 controls (0.7%). Functional analysis showed impaired activity or reduced secretion indicating that loss of function alterations in CTRC predispose to pancreatitis.

Pancreatic secretory Trypsin Inhibitor (PSTI, SPINK-1) Witt et al, (Nat. Genet., 2000) Mutations in 23% of children with idiopathic chronic Pancreatitis  autosomal-recessive disorder Pfützer et al, (Gastroenterology, 2000) Mutations in idiopathic chronic Pancreatitis (25%), hereditary Pancreatitis and in the healthy population (2%).  Modifier - Gene, risk of pancreatitis < 1% Bhatia et al, Schneider et al, (Gastroenterology, 2002) Mutations in tropical calcifying pancreatitis (up to 44%) and in ‘Fibrocalculous Pancreatic Diabetes mellitus‘ (55%).  Risk factor for tropical Pancreatitis and Diabetes mellitus Model by A. Brunskil & W. F. Furey

SPINK1-Mutations in Patients with Hereditary Pancreatitis IV:1 8 IV:2 5 I:1 54 I:2 81 II:1 64 II:3 61 II:5 60 III:2 36 II:2 II:4 69 II:7 50 III:5 16 II:6 68 III:6 13 II:8 46 III:3 35 III:1 III:4 Symbols Empty symbol chronic Pancreatitis positiv for PRSS1 Mutation positiv for N34S Mutation Asymptomatic carrier wt I. II. I:2 45 II:1 I:1 I:1 I:2 87 II:2 59 II:3 II:4 62 III:4 32 II:1 50 III:1 28 III:2 26 III:3 30 III. SPINK1 Mutations (N34S) are found among Pancreatitis patients as well as among healthy carriers of Trypsinogen mutations. Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

SPINK1 Mutations in Hereditary Pancreatitis Cumulative Incidence of Pancreatitis 100 100 100 80 80 80 60 60 60 Phenotype, % of total 40 40 40 PRSS1, SPINK wt SPINK N34S mild Pancreatitis No Diabetes 20 20 20 severe Pancreatitis Diabetes 10 20 30 40 50 60 10 20 30 40 50 60 10 20 30 40 50 60 Age at disease onset, years SPINK1 Mutations have no influence on the severity of clinical disease course of hereditary pancreatitis Weiss F.U. et al. J. Med. Gen. 2003;40:e40,1-5

Association of CTSB Polymorphisms with Tropical Calcific Pancreatitis Hyderabad Calicut Total Patients/Controls Patients/Controls Patients/Controls (n=140) (n=155) (n=166) (n=175) (n=306) (n=330) Leu26Val 0.48 0.30 0.45 0.28 0.46* 0.29* * p= 0.013 Ser53Gly 0.10 0.06 0.09 0.04 0.09* 0.04* * p= 0.152 His110 His111 E64d N34S positive N34S negative Controls (n=134) (n=172) (n=330) Leu26Val 0.45 0.46 0.29 ethnic origin: Dravidian Mahurkar et al. Gut. 2006 55:1270-5.

CTSB Val26 mutation in German ICP Patients C/C C/G G/G pG (Mahurkar) Patients 64 23 31 10 0,398 (51/128) 0,46 Controls 100 30 44 26 0,480 (96/200) 0,29 X2 p-Value 0,428 0,578 0,117 0,147 0,013 OR 0,764 0,836 1,897 0,718 2,09 95% CI 0,372 - 1,569 0,424 - 1,648 0,792 - 4,621 0,45 - 1,15 1,55 - 2,81 Weiss et al. Gut. 2007 56(9):1322-3.

Cathepsin B mutation Leu26Val in pancreatitis Mahurkar et al. Gut 2006 55:1270-5 N34S positive N34S negative Controls (n=134) (n=172) (n=330) Leu26Val 0.45 0.46 0.29 Population Ethnic origin (n) pG (Val26) CEPH Mixed Caucasian 92 0,320 Pooled_CEPH Caucasian 94 0,323 HapMap-CEU European 55 0,355 SC_95_C 45 0,367 HapMap-YRI Sub-Saharan African 52 0,394 TSC_42_C 41 0,430 JBIC-allele Japanese 732 0,493 HapMap-JPT Asian 44 0,523 HapMap-HCB 43 0,547 1198 0,452 ± 0,042 Weiss et al. Gut 2007 56:1322-3 see refSNP ID: rs12338 at http://www.ncbi.nlm.nih.gov /SNP/snp_ref.cgi?rs=12338

Chronic Pancreatitis and CFTR Mutations One third of patients (n=27) with idiopathic pancreatitis carry CFTR-Mutations (Risk x 80). J. Cohn et al. New Engl J Med 1998;339:653-58 CFTR Mutations represent a risk factor for chronic pancreatitis in patients without a history of alcohol abuse (19% of n = 60), but not for those with alcoholic pancreatitis (8.5% of n = 72).N. Sharer et al. New Engl J Med 1998;339:645-52 Kerem et al. Science 1989; 245: 1073-80 F 508 Mutation Weiss FU Gut 2005; 54: 1456-1460 CFTR allele frequency 12/66 Patients with CFTR Mutations, 8/66 Patients with T5 Allels

Prevalence of gene mutations in chronic pancreatitis Chymotrypsin C mutations 10% Idiopathic pancreatitis 45.5% Trypsinogen mutations 10% SPINK-1 mutations 15.2% T5 Allels 12.1% CFTR mutations 18.2%

Metabolic Chronic Pancreatitis – Causal treatment Hyperlipidemia (apoCII deficiency, lipoprotein lipase deficiency) Serum triglyercide levels > 1000 mg/dl Incidence extremely low Treatment which maintains TG below 500 mg/dl leads to resolution of symptoms. Toskes PP, Gastroenterol Clin North Am 1990; 19: 783-791. Hyperparathyroidism: Hyperthyreoidims leads to increased serum calcium levels, what is associated with an increased risk of pancreatitis. The incidence of chronic pancreatitis is between 1.5 - 7%. Early parathyreoidectomy leads to resolution of symptoms. Bess MA JAMA 1980; 243: 246-247; Russel CF Br. J. Surg. 1982; 69: 244-247;

Summary and Conclusion Hereditary chronic and idiopathic chronic pancreatitis are associated with mutations in the trypsinogen gene, the SPINK-1 Gene, the chymotrypsin C gene and the CFTR gene. More genes will surely be identified. The pathophysiological impact of these gene mutations are not completety understood and further experiments are warranted. Etiologies of pancreatitis already treatable (such as autoimmune pancreatitis etc. ) need to be distinguished from pancreatitis varieties that are not yet treatable – but may become so. In hereditary pancreatitis (trypsin mutations) smoking cessation may reduce the risk of pancreatic cancer. Other surveillance strategies are, however, urgently needed.

www.pancreas.de Julia Mayerle Uli Weiss Peter Simon Ali Aghdassi Gabriele Sauter www.pancreas.de

Chronic Pancreatitis - Alcohol Cessation 100 Stop Alcohol Alcohol 80 60 Output in % of initial 40 20 n= 18 vs. 14 pt ∆ 4-11 years p<0.01 Bicarbonate Lipase Chymotrypsin Gullo et al, Gastroenterology 1988;95:1063-68

Diagnosis and Screening for Hereditary Pancreatitis Today, restriction enzyme digest with BstU I represents the most extensive initial screening test for hereditary pancreatitis. A C G C C N G C G T G T C T type type Control R122H R122C Control R122H R122C Wild Wild Sequence in Exon 3 Arg RSS1 wt AAC-GCC-CGC-GTG-T Cys R-122-C AAC-GCC-TGC-GTG-T Afl III BstU I P. Simon, F.U. Weiss et al. J Biol Chem. 2002;277:5404-10

Natural Course of Alcoholic Chronic Pancreatitis - Stage 1 (early) Stage 2 Stage 3 Pain Serum Enzyme Elevation Exocrine Pancreatic Function 5 10 15 years Modified according to R Ammann, Schweiz Rundsch Med Prax. 1970; 59: 792-5.