Pancreatitis and Pancreatic Cancer David C Whitcomb MD

Pancreatitis and Pancreatic Cancer David C Whitcomb MD Ph. D. 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 Trypsinogen SPINK 1 CFTR 3. Pancreatic Cancer Links with pancreatitis

Pancreas Anatomy

Histology Duct Islet fat Acinus

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. 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. Hereditary Pancreatitis - a unusual form of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 times normal.

CT of Pancreatic Necrosis

CT of Severe Chronic Pancreatitis

Chronic Pancreatitis: 1995 Medical Progress: Chronic Pancreatitis. Volume 332(22) 1 Jun 1995 pp 1482 -1490 Michael L Steer, Irving Waxman, Steven Freedman “chronic pancreatitis remains an enigmatic process of uncertain pathogenesis, unpredictable clinical course, and unclear treatment”

Origins of Pancreatitis Pre-1896: Pancreatitis is an infection 1896: Pancreatitis is pancreas autodigestion 1996: Hereditary Pancreatitis is caused by mutations in the cationic trypsinogen gene (PRSS 1)

Hereditary Pancreatitis Pancreas 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

E-mail 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 7 q 35 linkage A large family was identified through self-referral Seventy-one members Six affected Two obligate carriers

Linkage of HP to Chromosome 7 q 35 Whitcomb et al. GASTROENTEROLOGY 110: 1975, 1996 (P value) D 7 S 559 D 7 S 550 D 7 S 661 D 7 S 505 D 7 S 483 HP GENE D 7 S 495 D 7 S 684 Hereditary Pancreatitis -5 Gene D 7 S 461 TCRB 0. 00001 0. 1 5 4 3 2 1 0 D 7 S 523 Cystic Fibrosis Gene LOD Scores Chromosome 7 Markers on chromosome 7

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

Physiology of Trypsin Enterokinase TRYPSIN Trypsinogen Chymotrypsinogen Proelastase Procarboxypeptidase Proenzymes Solid Food Trypsin Chymotrypsin Elastase Carboxypeptidase Enzymes

Fail-safe Trypsin Inactivation Trypsinogen + - -- (Autoactivation) Trypsin (wt) - R 122 H 122 PSTI - Proenzymes Whitcomb et al, Nature Genetics 1996 ? Trypsin Enzymes Autodigestion Pancreatitis

Trypsinogen Alignment & Mutations Whitcomb MCNA 2000 Exon 1 signal peptide CODON 1 10 14 | | | CT MNPLLILTFVAAAL AT MNLLLILTFVAAAV MT MNPFLILAFVGAAV Chy MAFLWLLSCWALLGTTF Witt 1999 Teich 2000 Ferec 1999 Gorry 1997 APFDDDDK IVGGYNCEENSVPYQVSLN Pfutzet 1999 (+CFTR R 117 H) Exon 2 TAP CODON 15 16 23 29 40 50 60 67 | | | | A APFDDDDK IVGGYNCEENSVPYQVSLN SGYHFCGGSLINEQWVVSAGHCYKS A APFDDDDK IVGGYICEENSVPYQVSLN SGYHFCGGSLISEQWVVSAGHCYKS A VPFDDDDK IVGGYTCEENSLPYQVSLN SGSHFCGGSLISEQWVVSAAHCYKT G CGVPAIHPVLSGLSR IVNGEDAVPGSWPWQVSLQDKTGFHFCGGSLISEDWVVTAAHCGVR | | | | | 1 10 2021 30 40 50 57 CT AT MT Chy# Exon 3 CODON Whitcomb 1996 68 70 80 90 100 110 122 130 140 152 || | | || CT RIQ VRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPT APPA TGTKCLISGWGNTASSGA AT RIQ VRLGEHNIEVLEGNEQFINAAKIIRHPKYNSRTLDNDILLIKLSSPAVINSRVSAISLPT APPA AGTESLISGWGNTLSSGA MT RIQ VRLGEHNIKVLEGNEQFINAAKIIRHPKYNRDTLDNDIMLIKLSSPAVINARVSTISLPT APPA AGTECLISGWGNTLSFGA Chy TSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNNDITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKT

PRSS 1 Mutations - Overview Two mutations are both common and diseasecausing: PRSS 1 R 122 H and N 29 I (new numbers) 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 40% with chronic pancreatitis will develop pancreatic cancer by age 70 (smoking doubles risk)

SPINK 1/PSTI Alignment & Mutations Pfutzer et al, Gastroenterology, 2000 Comparison of human , porcine and rat , SPINK 1/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 SPINK 1 (PSTI) is 71% Exon 1 Codon # obs var | human Porcine rat Exon 2 1 10 18 | P | MKVTGIFLLSALALLSLS MKVAIIFLLSALALLNLA Peptide # 24 29 | | * GNTGADSLGRE * TSPQRE GNTTAKVIGKK | | 1 6 Exon 3 Codon # obs var human porcine rat 30 34 40 50 55 60 65 | S |* E S | | AKCYNELNGCTKIYDPVCGTDGNTYPNECVLCFENR * * ATCTSEVSGCPKIYNPVCGTDGITYSNECVLCSENK ANCPNTLIGCPRDYDPVCGTDGKTYANECILCFENR | | || Peptide # 7 11 20 30 40 42 Exon 4 Codon # 66 70 79 obs var | | | human KRQTSILIQKSGPC porcine KRQTPVLIQKSGPC rat KFGTSIRIQRRGLC | | | Peptide # 43 50 56 Changes human to porcine

SPINK 1 / PSTI N 34 S Mutation Pfutzer et al, Gastroenterology, 2000 Superposition of the porcine SPINK 1 structure (blue) on the human (modified for chymotrypsin specificity) SPINK 1 structure (red). Model by Andrew Brunskil & William F. Furey

SPINK 1 Genotype-Phenotype Pfutzer et al, Gastroenterology, 2000 Cumulative Incidence of Pancreatitis 100 90 80 SPINK 1 N 34 S ICP SPINK 1 N 34 S / N 34 S SPINK 1 N 34 S / P 55 S 70 Affected (% total) 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Age of Symptom Onset (years)

SPINK 1 Mutations - Overview SPINK 1/PSTI mutations are common in the population (~2%) SPINK 1/PSTI are clearly associated with ICP (~25%). The mutation associated risk is low (<1%). Modeling and familial clustering suggest that SPINK 1 mutations are disease modifying. SPINK 1/PSTI mutations may lower the threshold for pancreatitis from other genetic or environmental factors, but by themselves are not disease causing The N 34 S mutations has a world-wide distribution.

SPINK 1 Mutations - Overview SPINK 1/PSTI mutations are common in the population (~2%) SPINK 1/PSTI are clearly associated with ICP (~25%). The mutation associated risk is low (<1%). Modeling and familial clustering suggest that SPINK 1 mutations are disease modifying. SPINK 1/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 A. B. Lowenfels, P. Maisonneuve, D. C. Whitcomb, and the International Hereditary Pancreatitis Study Group 150 Age (years) Relative Risk of Pancreatic Cancer 100 50 0 GP HP HP + Smoked 80 70 60 50 40 30 20 10 0 Age at diagnosis of pancreatic cancer Never Smoked Ever Smoked