Pancreatitis and Pancreatic Cancer David C WhitcombDavid C Whitcomb MD Ph.D. David C Whitcomb Professor of Medicine, Cell Biology & Physiology and Human.

Pancreatitis and Pancreatic CancerPancreatitis and Pancreatic Cancer

David C WhitcombDavid C Whitcomb MD Ph.D. MD Ph.D.Professor of Medicine, Cell Biology & Physiology Professor of Medicine, Cell Biology & Physiology

and Human Geneticsand Human Genetics

Chief, Division of Gastroenterology, Hepatology Chief, Division of Gastroenterology, Hepatology and Nutritionand Nutrition

Director, Center for Genomic SciencesDirector, Center for Genomic Sciences

University University ofof Pittsburgh Pittsburgh

David C WhitcombDavid C Whitcomb MD Ph.D. MD Ph.D.Professor of Medicine, Cell Biology & Physiology Professor of Medicine, Cell Biology & Physiology

and Human Geneticsand Human Genetics

Chief, Division of Gastroenterology, Hepatology Chief, Division of Gastroenterology, Hepatology and Nutritionand Nutrition

Director, Center for Genomic SciencesDirector, Center for Genomic Sciences

University University ofof Pittsburgh Pittsburgh

OutlineOutline

1.1. Origins of PancreatitisOrigins of PancreatitisPremature trypsinogen activationPremature trypsinogen activation

2. 2. Genetics of Acute and Chronic PancreatitisGenetics of Acute and Chronic PancreatitisTrypsinogenTrypsinogen

SPINK1SPINK1

CFTRCFTR

3.3. Pancreatic CancerPancreatic CancerLinks with pancreatitisLinks with pancreatitis

Pancreas AnatomyPancreas Anatomy

HistologyHistology

DuctDuct

IsletIslet

AcinusAcinusfatfat

PancreatitisPancreatitis

PancreasPancreas - - an organ that makes an organ that makes bicarbonatebicarbonate to nutralize gastric acid, to nutralize gastric acid, enzymesenzymes to digest the contents of a meal and to digest the contents of a meal and insulininsulin to signal the body to to signal the body to store ingested nutrients.store ingested nutrients.

Acute PancreatitisAcute Pancreatitis - An acute, potentially life-threatening condition - An acute, potentially life-threatening condition presenting with severe abdominal pain in which the pancreas appears to presenting with severe abdominal pain in which the pancreas appears to digest itself. It is usually caused by gallstones, alcohol or is idiopathic.digest itself. It is usually caused by gallstones, alcohol or is idiopathic.

Chronic Pancreatitis Chronic Pancreatitis - an irreversible scarring of the pancreas with - an irreversible scarring of the pancreas with permanent loss of pancreatic function that typically causes unrelenting permanent loss of pancreatic function that typically causes unrelenting abdominal pain.abdominal pain.

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

PanPancreacreass

CT of Pancreatic NecrosisCT of Pancreatic Necrosis

CT of Severe Chronic PancreatitisCT of Severe Chronic Pancreatitis

Chronic Pancreatitis: 1995Chronic 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 1482-1490

Michael L Steer, Irving Waxman, Steven Freedman

Origins of Pancreatitis Origins of Pancreatitis

Pre-1896Pre-1896:: Pancreatitis is an infectionPancreatitis is an infection

1896:1896: Pancreatitis is pancreas autodigestionPancreatitis is pancreas autodigestion 1996:1996: Hereditary Pancreatitis is caused by Hereditary Pancreatitis is caused by

mutations in the mutations in the cationic cationic trypsinogentrypsinogen gene (PRSS1)gene (PRSS1)

Hereditary PancreatitisHereditary Pancreatitis

Hereditary pancreatitis Hereditary pancreatitis (HP) is an unusual form (HP) is an unusual form of acute and chronic pancreatitis that runs in of acute and chronic pancreatitis that runs in families. The risk of pancreatic cancer is >50 families. The risk of pancreatic cancer is >50 times normal.times normal.

Although HP is only responsible for 2-3% of all Although HP is only responsible for 2-3% of all cases of chronic pancreatitis, study of this disease cases of chronic pancreatitis, study of this disease has revolutionized our understanding of has revolutionized our understanding of pancreatic diseasespancreatic diseases

PaPancrencreasas

E-mailE-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 7q35 linkage HP Kindred without 7q35 linkage

A large family was identified through self-referralA large family was identified through self-referral

Seventy-one membersSeventy-one membersSix affectedSix affectedTwo obligate carriersTwo obligate carriers

Linkage of HP to Chromosome 7q35Linkage of HP to Chromosome 7q35

D7S

505

D7S

661

D7S

684

D7S

523

D7S

461

D7S

495

D7S

483

D7S

550

D7S

559-5

3

0

LO

D S

core

s

HP GENE

5

Whitcomb et al. GASTROENTEROLOGY 110:1975, 1996Whitcomb et al. GASTROENTEROLOGY 110:1975, 1996

0.00001

0.001

0.1

(P v

alu

e)

0.01

0.00014

21

Markers on chromosome 7

HereditaryHereditaryPancreatitis Pancreatitis

GeneGene

Cystic Cystic FibrosisFibrosisGeneGene

Chromosome 7

TCRBTCRB

Discovery of the Pancreatitis GeneDiscovery of the Pancreatitis Gene

DNA

1. Family 2. Genetic Mapping

Chromosome 7

HereditaryPancreatitisgene

ccaccaccagtcaggcacactctaccaccATGAATCCACTCCTGATCCTTACCTTTGT

GG/ACAGCTGC TCgtgagtatcatgccctgcctcaggccccaaccacccccccgttcctggccga

3. Mutation

Mutation in the trypsinogenDNA sequenceRecruitment

4. Mechanism

Functional significancedetermined

Trypsinogen

Whitcomb et al 1996

Physiology of TrypsinPhysiology of Trypsin

EnterokinaseEnterokinase

TrypsinogenTrypsinogen

ChymotrypsinogenChymotrypsinogen

ProelastaseProelastase

ProcarboxypeptidaseProcarboxypeptidase

ProenzymesProenzymes

TrypsinTrypsin

ChymotrypsinChymotrypsin

ElastaseElastase

CarboxypeptidaseCarboxypeptidase

EnzymesEnzymes

TrypsinTrypsin

ChymotrypsinChymotrypsin

ElastaseElastase

CarboxypeptidaseCarboxypeptidase

EnzymesEnzymes

TRYPSIN Solid Food

Fail-safe Trypsin InactivationFail-safe Trypsin Inactivation

Trypsinogen

Trypsin (wt)Trypsin (wt)

+

-

(Autoactivation)

Proenzymes Enzymes

Whitcomb et al, Whitcomb et al, Nature Genetics Nature Genetics 19961996

-

-

TrypsinTrypsinTrypsinTrypsin

Trypsin

-

-

PSTIPSTI

AutodigestionAutodigestionPancreatitisPancreatitis

R122R122 TrypsinTrypsinTrypsinTrypsinH122

??

PSTIPSTI

Trypsinogen Alignment & MutationsTrypsinogen Alignment & Mutations

Exon 1 signal peptideCODON 1 10 14

| | |CT MNPLLILTFVAAALAT MNLLLILTFVAAAVMT MNPFLILAFVGAAVChy MAFLWLLSCWALLGTTF

Exon 2 TAP

CODON 15 16 23 29 40 50 60 67 | | | | | | | |CT A APFDDDDK IVGGYNCEENSVPYQVSLN SGYHFCGGSLINEQWVVSAGHCYKSAT A APFDDDDK IVGGYICEENSVPYQVSLN SGYHFCGGSLISEQWVVSAGHCYKSMT A VPFDDDDK IVGGYTCEENSLPYQVSLN SGSHFCGGSLISEQWVVSAAHCYKTChy G CGVPAIHPVLSGLSR IVNGEDAVPGSWPWQVSLQDKTGFHFCGGSLISEDWVVTAAHCGVR

| | || | | | | Chy# 1 10 2021 30 40 50 57

Exon 3 CODON 68 70 80 90 100 110 120 122 130 140 150 152 | | | | | | | | | | | |CT RIQ VRLGEHNIEVLEGNEQFINAAKIIRHPQYDRKTLNNDIMLIKLSSRAVINARVSTISLPT APPA TGTKCLISGWGNTASSGAAT RIQ VRLGEHNIEVLEGNEQFINAAKIIRHPKYNSRTLDNDILLIKLSSPAVINSRVSAISLPT APPA AGTESLISGWGNTLSSGAMT RIQ VRLGEHNIKVLEGNEQFINAAKIIRHPKYNRDTLDNDIMLIKLSSPAVINARVSTISLPT APPA AGTECLISGWGNTLSFGAChy TSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNNDITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKTKYNAN | | | | | | | | | | |

Chy# 62 70 80 90 100102 110 117 120 130 140

APFDDDDK IVGGYNCEENSVPYQVSLN

Whitcomb MCNA 2000

Whitcomb 1996Whitcomb 1996

Gorry 1997Gorry 1997Ferec 1999Ferec 1999Witt 1999Witt 1999 Teich 2000Teich 2000

PfutzetPfutzet 1999 1999 (+CFTR R117H) (+CFTR R117H)

PRSS1 Mutations - OverviewPRSS1 Mutations - Overview

Two mutations are both common and disease-causing: Two mutations are both common and disease-causing: PRSS1 R122H and N29I (new numbers) PRSS1 R122H and N29I (new numbers)

Individuals with either of the two mutations have Individuals with either of the two mutations have about an 80% chance of developing acute pancreatitis.about an 80% chance of developing acute pancreatitis.

Of those with acute pancreatitis, about half develop Of those with acute pancreatitis, about half develop chronic pancreatitischronic pancreatitis

40% with chronic pancreatitis will develop pancreatic 40% with chronic pancreatitis will develop pancreatic cancer by age 70 (smoking doubles risk)cancer by age 70 (smoking doubles risk)

SPINK1/PSTI Alignment & MutationsSPINK1/PSTI Alignment & Mutations

Exon 1 Exon 2Codon # 1 10 18 24 29 obs var | | P | | | human MKVTGIFLLSALALLSLS * GNTGADSLGRE *Porcine TSPQRE rat MKVAIIFLLSALALLNLA GNTTAKVIGKK

| | Peptide # 1 6

Exon 3Codon # 30 34 40 50 55 60 65 obs var | S |* E S | |human AKCYNELNGCTKIYDPVCGTDGNTYPNECVLCFENR * *porcine ATCTSEVSGCPKIYNPVCGTDGITYSNECVLCSENKrat ANCPNTLIGCPRDYDPVCGTDGKTYANECILCFENR

| | | | | |Peptide # 7 11 20 30 40 42

Exon 4Codon # 66 70 79 obs var | | |human KRQTSILIQKSGPCporcine KRQTPVLIQKSGPCrat KFGTSIRIQRRGLC

| | |Peptide # 43 50 56

Pfutzer et al, Gastroenterology, 2000Pfutzer et al, Gastroenterology, 2000

Comparison of Comparison of human , porcinehuman , porcine andand rat rat , SPINK1/PSTI. , SPINK1/PSTI. Obs var;Obs var; observed variations in human observed variations in human protein sequence deduced from protein sequence deduced from allele polymorphisms. allele polymorphisms.

*;*; the “bait” lysine (K) in human the “bait” lysine (K) in human and porcine, and arginine (R ) in and porcine, and arginine (R ) in rat that projects into the rat that projects into the specificity pocket of trypsin specificity pocket of trypsin during trypsin inhibition. during trypsin inhibition.

Homology between human and Homology between human and porcine SPINK1 (PSTI) is 71%porcine SPINK1 (PSTI) is 71%

Changes human Changes human to porcineto porcine

SPINK1 / PSTI N34S MutationSPINK1 / PSTI N34S Mutation

Superposition of the Superposition of the porcineporcine SPINK1 SPINK1 structure (blue) on structure (blue) on the the humanhuman (modified (modified for chymotrypsin for chymotrypsin specificity) SPINK1 specificity) SPINK1 structure (red). structure (red).

Model by Andrew Brunskil Model by Andrew Brunskil & William F. Furey & William F. Furey

Pfutzer et al, Gastroenterology, 2000Pfutzer et al, Gastroenterology, 2000

SPINK1 Genotype-PhenotypeSPINK1 Genotype-Phenotype

656055504540353025201510500

10

20

30

40

50

60

70

80

90

100

SPINK1 N34S

Cumulative Incidence of PancreatitisCumulative Incidence of Pancreatitis

Age of Symptom Onset (years)Age of Symptom Onset (years)

AffectedAffected(% total)(% total)

SPINK1 N34S / N34SICP

SPINK1 N34S / P55S

Pfutzer et al, Gastroenterology, 2000Pfutzer et al, Gastroenterology, 2000

SPINK1 Mutations - OverviewSPINK1 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 Modeling and familial clustering suggest that SPINK1

mutations are disease modifying.mutations are disease modifying. SPINK1/PSTI mutations may lower the threshold for SPINK1/PSTI mutations may lower the threshold for

pancreatitis from other genetic or environmental factors, but pancreatitis from other genetic or environmental factors, but by themselves are not disease causingby themselves are not disease causing

The N34S mutations has a world-wide distribution.The N34S mutations has a world-wide distribution.

SPINK1 Mutations - OverviewSPINK1 Mutations - Overview

SPINK1/PSTI mutations are common in the population SPINK1/PSTI mutations are common in the population (~2%)(~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 Modeling and familial clustering suggest that SPINK1

mutations are disease modifying.mutations are disease modifying. SPINK1/PSTI mutations may lower the threshold for SPINK1/PSTI mutations may lower the threshold for

pancreatitis from other genetic or environmental factors, pancreatitis from other genetic or environmental factors, but by themselves are not disease causingbut by themselves are not disease causing

Smoking & Risk of Cancer in HPSmoking & Risk of Cancer in HP

8080707060605050404030302020101000

EverEverSmokedSmoked

Never Never SmokedSmoked

Age at diagnosis ofAge at diagnosis of pancreatic cancerpancreatic cancer

GPGP HP HP HP + HP + Smoked Smoked

150150

100100

5050

00

Rel

ativ

e R

isk

Rel

ativ

e R

isk

Age

(ye

ars)

Age

(ye

ars)

Risk of Pancreatic CancerRisk of Pancreatic Cancer

A.B. Lowenfels, P. Maisonneuve, D. C.Whitcomb, and A.B. Lowenfels, P. Maisonneuve, D. C.Whitcomb, and the International Hereditary Pancreatitis Study Groupthe International Hereditary Pancreatitis Study Group