Pancreatitis and Pancreatic Cancer David C WhitcombDavid C Whitcomb MD Ph.D. David C Whitcomb Professor of Medicine, Cell Biology & Physiology and Human Genetics Chief, Division of Gastroenterology, Hepatology and Nutrition Director, Center for Genomic Sciences University of Pittsburgh David C WhitcombDavid C Whitcomb MD Ph.D. David C Whitcomb Professor of Medicine, Cell Biology & Physiology and Human Genetics Chief, Division of Gastroenterology, Hepatology and Nutrition Director, Center for Genomic Sciences University of Pittsburgh

Outline 1. Origins of Pancreatitis Premature trypsinogen activation 2. Genetics of Acute and Chronic Pancreatitis TrypsinogenSPINK1CFTR 3. Pancreatic Cancer Links with pancreatitis

Pancreas Anatomy

Histology Duct Islet Acinus fat

Pancreatitis Pancreas - an organ that makes bicarbonate to nutralize gastric acid, enzymes to digest the contents of a meal and insulin to signal the body to store ingested nutrients. Pancreas - an organ that makes bicarbonate to nutralize gastric acid, enzymes to digest the contents of a meal and insulin to signal the body to store ingested nutrients. Acute Pancreatitis - An acute, potentially life-threatening condition presenting with severe abdominal pain in which the pancreas appears to digest itself. It is usually caused by gallstones, alcohol or is idiopathic. Acute Pancreatitis - An acute, potentially life-threatening condition presenting with severe abdominal pain in which the pancreas appears to digest itself. It is usually caused by gallstones, alcohol or is idiopathic. Chronic Pancreatitis - an irreversible scarring of the pancreas with permanent loss of pancreatic function that typically causes unrelenting abdominal pain. Chronic Pancreatitis - an irreversible scarring of the pancreas with permanent loss of pancreatic function that typically causes unrelenting abdominal pain. Hereditary Pancreatitis - a unusual form of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 times normal. Hereditary Pancreatitis - a unusual form of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 times normal. Pancreas

CT of Pancreatic Necrosis

CT of Severe Chronic Pancreatitis

Chronic Pancreatitis: 1995 “chronic pancreatitis remains an enigmatic process of uncertain pathogenesis, unpredictable clinical course, and unclear treatment” Medical Progress: Chronic Pancreatitis. Volume 332(22) 1 Jun 1995 pp Michael L Steer, Irving Waxman, Steven Freedman

Origins of Pancreatitis Pre-1896:Pancreatitis is an infection Pre-1896:Pancreatitis is an infection 1896:Pancreatitis is pancreas autodigestion 1896:Pancreatitis is pancreas autodigestion 1996:Hereditary Pancreatitis is caused by mutations in the cationic trypsinogen gene (PRSS1) 1996:Hereditary Pancreatitis is caused by mutations in the cationic trypsinogen gene (PRSS1)

Hereditary Pancreatitis Hereditary pancreatitis (HP) is an unusual form of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 times normal. Hereditary pancreatitis (HP) is an unusual form of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 times normal. Although HP is only responsible for 2-3% of all cases of chronic pancreatitis, study of this disease has revolutionized our understanding of pancreatic diseases Although HP is only responsible for 2-3% of all cases of chronic pancreatitis, study of this disease has revolutionized our understanding of pancreatic diseases Pancreas

Dear Dr.Whitcomb, Hi, the Doctors think I have hereditary pancreatitis because my dad has it too. I don't really know much about it except that it hurts in your back, sides and stomachs areas. (speaking from experience) I've been in the hospital once for it because it was the worst attack I've had. If you could send me more information on it, it would be greatly appreciated. By the way I'm 12, just in case it matters. My dad's 42. Have a nice day Dr.Whitcomb

HP Kindred without 7q35 linkage A large family was identified through self-referral Seventy-one members Six affected Two obligate carriers

Linkage of HP to Chromosome 7q35 D7S505 D7S661D7S684 D7S523 D7S461D7S495 D7S483 D7S550D7S LOD Scores HP GENE 5 Whitcomb et al. GASTROENTEROLOGY 110:1975, (P value) Markers on chromosome 7 HereditaryPancreatitisGene CysticFibrosisGene Chromosome 7 TCRB

Discovery of the Pancreatitis Gene DNA 1. Family 2. Genetic Mapping Chromosome 7 Hereditary Pancreatitis gene ccaccaccagtcaggcac actctaccaccATGAA TCCACTCCTGAT CCTTACCTTTGT G G/A CAGCTGC TCgtgagtatcatgccct gcctcaggccccaaccac ccccccgttcctggccga 3. Mutation Mutation in the trypsinogen DNA sequence Recruitment 4. Mechanism Functional significance determined Trypsinogen Whitcomb et al 1996

Physiology of Trypsin Enterokinase TrypsinogenChymotrypsinogenProelastaseProcarboxypeptidaseProenzymes TrypsinChymotrypsinElastaseCarboxypeptidaseEnzymesTrypsinChymotrypsinElastaseCarboxypeptidaseEnzymes TRYPSIN Solid Food

Fail-safe Trypsin Inactivation Trypsinogen Trypsin (wt) + - (Autoactivation) Proenzymes Enzymes Whitcomb et al, Nature Genetics Trypsin Trypsin - - PSTI AutodigestionPancreatitis R122 Trypsin H122 ? PSTI

Trypsinogen Alignment & Mutations Exon 1 signal peptide CODON | | | CT MNPLLILTFVAAAL ATMNLLLILTFVAAAV MTMNPFLILAFVGAAV ChyMAFLWLLSCWALLGTTF Exon 2 TAP CODON | | | | | | | | CTA APFDDDDK IVGGYNCEENSVPYQVSLN SGYHFCGGSLINEQWVVSAGHCYKS ATA APFDDDDK IVGGYICEENSVPYQVSLN SGYHFCGGSLISEQWVVSAGHCYKS MTA VPFDDDDK IVGGYTCEENSLPYQVSLN SGSHFCGGSLISEQWVVSAAHCYKT ChyG CGVPAIHPVLSGLSR IVNGEDAVPGSWPWQVSLQDKTGFHFCGGSLISEDWVVTAAHCGVR | | || | | | | Chy# Exon 3 CODON | | | | | | | | | | | | CTRIQ VRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPT APPA TGTKCLISGWGNTASSGA ATRIQ VRLGEHNIEVLEGNEQFINAAKIIRHPKYNSRTLDNDILLIKLSSPAVINSRVSAISLPT APPA AGTESLISGWGNTLSSGA MTRIQ VRLGEHNIKVLEGNEQFINAAKIIRHPKYNRDTLDNDIMLIKLSSPAVINARVSTISLPT APPA AGTECLISGWGNTLSFGA Chy TSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNNDITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKTK YNAN | | | | | | | | | | | Chy# APFDDDDK IVGGYNCEENSVPYQVSLN Whitcomb MCNA 2000 Whitcomb 1996 Gorry 1997 Ferec 1999 Witt 1999 Teich 2000 Pfutzet 1999 (+CFTR R117H)

PRSS1 Mutations - Overview Two mutations are both common and disease- causing: PRSS1 R122H and N29I (new numbers) Two mutations are both common and disease- causing: PRSS1 R122H and N29I (new numbers) Individuals with either of the two mutations have about an 80% chance of developing acute pancreatitis. Individuals with either of the two mutations have about an 80% chance of developing acute pancreatitis. Of those with acute pancreatitis, about half develop chronic pancreatitis Of those with acute pancreatitis, about half develop chronic pancreatitis 40% with chronic pancreatitis will develop pancreatic cancer by age 70 (smoking doubles risk) 40% with chronic pancreatitis will develop pancreatic cancer by age 70 (smoking doubles risk)

SPINK1/PSTI Alignment & Mutations Exon 1 Exon 2 Codon # obs var | | P | | | human MKVTGIFLLSALALLSLS *GNTGADSLGRE * Porcine TSPQRE ratMKVAIIFLLSALALLNLAGNTTAKVIGKK | | Peptide # 1 6 Exon 3 Codon # obs var| S |* E S | | humanAKCYNELNGCTKIYDPVCGTDGNTYPNECVLCFENR * * porcineATCTSEVSGCPKIYNPVCGTDGITYSNECVLCSENK ratANCPNTLIGCPRDYDPVCGTDGKTYANECILCFENR | | | Peptide # Exon 4 Codon # obs var | | | humanKRQTSILIQKSGPC porcineKRQTPVLIQKSGPC ratKFGTSIRIQRRGLC | | | Peptide # Pfutzer et al, Gastroenterology, 2000 Comparison of human, porcine and rat, SPINK1/PSTI. Obs var; observed variations in human protein sequence deduced from allele polymorphisms. *; the “bait” lysine (K) in human and porcine, and arginine (R ) in rat that projects into the specificity pocket of trypsin during trypsin inhibition. Homology between human and porcine SPINK1 (PSTI) is 71% Changes human to porcine

SPINK1 / PSTI N34S Mutation Superposition of the porcine SPINK1 structure (blue) on the human (modified for chymotrypsin specificity) SPINK1 structure (red). Superposition of the porcine SPINK1 structure (blue) on the human (modified for chymotrypsin specificity) SPINK1 structure (red). Model by Andrew Brunskil & William F. Furey Model by Andrew Brunskil & William F. Furey Pfutzer et al, Gastroenterology, 2000

SPINK1 Genotype-Phenotype SPINK1 N34S Cumulative Incidence of Pancreatitis Age of Symptom Onset (years) Affected (% total) SPINK1 N34S / N34S ICP SPINK1 N34S / P55S Pfutzer et al, Gastroenterology, 2000

SPINK1 Mutations - Overview SPINK1/PSTI mutations are common in the population (~2%) SPINK1/PSTI mutations are common in the population (~2%) SPINK1/PSTI are clearly associated with ICP (~25%). SPINK1/PSTI are clearly associated with ICP (~25%). The mutation associated risk is low (<1%). The mutation associated risk is low (<1%). Modeling and familial clustering suggest that SPINK1 mutations are disease modifying. Modeling and familial clustering suggest that SPINK1 mutations are disease modifying. SPINK1/PSTI mutations may lower the threshold for pancreatitis from other genetic or environmental factors, but by themselves are not disease causing SPINK1/PSTI mutations may lower the threshold for pancreatitis from other genetic or environmental factors, but by themselves are not disease causing The N34S mutations has a world-wide distribution. The N34S mutations has a world-wide distribution.

SPINK1 Mutations - Overview SPINK1/PSTI mutations are common in the population (~2%) SPINK1/PSTI mutations are common in the population (~2%) SPINK1/PSTI are clearly associated with ICP (~25%). SPINK1/PSTI are clearly associated with ICP (~25%). The mutation associated risk is low (<1%). The mutation associated risk is low (<1%). Modeling and familial clustering suggest that SPINK1 mutations are disease modifying. Modeling and familial clustering suggest that SPINK1 mutations are disease modifying. SPINK1/PSTI mutations may lower the threshold for pancreatitis from other genetic or environmental factors, but by themselves are not disease causing SPINK1/PSTI mutations may lower the threshold for pancreatitis from other genetic or environmental factors, but by themselves are not disease causing

Smoking & Risk of Cancer in HP EverSmoked Never Smoked Age at diagnosis of pancreatic cancer pancreatic cancer GPHP HP + Smoked Relative Risk Age (years) Risk of Pancreatic Cancer A.B. Lowenfels, P. Maisonneuve, D. C.Whitcomb, and the International Hereditary Pancreatitis Study Group